Encyclopedia of Cancer (4 Volume Set)

Encyclopedia of Cancer (2nd edition) M ANFRED S CHWAB (Ed.) Encyclopedia of Cancer (2nd edition) With 979 Figures* ...

Author: Manfred Schwab

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Encyclopedia of Cancer (2nd edition)

M ANFRED S CHWAB (Ed.)

Encyclopedia of Cancer (2nd edition)

With 979 Figures* and 210 Tables

*For color figures please see our Electronic Reference on www.springerlink.com

Editor: Manfred Schwab Professor for Genetics Director Division of Tumour Genetics (B030) German Cancer Research Center (DKFZ) Im Neuenheimer Feld 280 69120 Heidelberg Germany

A C.I.P. Catalog record for this book is available from the Library of Congress ISBN: 978-3-540-36847-2 This publication is available also as: Electronic publication under ISBN 978-3-540-47648-1 and Print and electronic bundle under ISBN 978-3-540-47649-8 Library of Congress Control Number 2008921484 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg New York 2008 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. THIS PARAGRAPH FOR MEDICAL TITLES ONLY: Product liability: The publishers cannot guarantee the accuracy of any information about the application of operative techniques and medications contained in this book. In every individual case the user must check such information by consulting the relevant literature. Springer is part of Springer Science+Business Media springer.com Printed on acid-free paper SPIN: 150231 2109 — 5 4 3 2 1 0

Preface To The First Edition

Cancer, although a dreadful disease, is at the same time a fascinating biological phenotype. Around 1980, cancer was first attributed to malfunctioning genes and, subsequently, cancer research has become a major area of scientific research supporting the foundations of modern biology to a great extent. To unravel the human genome sequence was one of those extraordinary tasks, which has largely been fuelled by cancer research, and many of the fascinating insights into the genetic circuits that regulate developmental processes have also emerged from research on cancer. Diverse biological disciplines such as cytogenetics, virology, cell biology, classical and molecular genetics, epidemiology, biochemistry, together with the clinical sciences, have closed ranks in their search of how cancer develops and to find remedies to stop the abnormal growth that is characteristic of cancerous cells. In the attempt to establish how, why and when cancer occurs, a plethora of genetic pathways and regulatory circuits have been discovered that are necessary to maintain general cellular functions such as proliferation, differentiation and migration. Alterations of this fine-tuned network of cascades and interactions, due to endogenous failure or to exogenous challenges by environmental factors, may disable any member of such regulatory pathways. This could, for example, induce the death of the affected cell, may mark it for cancerous development or may immediately provide it with a growth advantage within a particular tissue. Recent developments have seen the merger of basic and clinical science. Of the former, particularly genetics has provided instrumental and analytical tools with which to assess the role of environmental factors in cancer, to refine and enable diagnosis prior to the development of symptoms and to evaluate the prognosis of patients. Hopefully, even better strategies for causal therapy will become available in the future. Merging the basic and clinical science disciplines towards the common goal of fighting cancer, calls for a comprehensive reference source to serve both as a tool to close the language gap between clinical and basic science investigators and as an information platform for the student and the informed layperson alike. Obviously this was an extremely ambitious goal, and the immense progress in the field cannot always be portrayed in line with the latest developments. The aim of the Encyclopedia is to provide the reader with an entrance point to a particular topic. It should be of value to both basic and clinical scientists working in the field of cancer research. Additionally both students and lecturers in the life sciences should benefit highly from this database. I therefore hope that this Encyclopedia will become an essential complement to existing science resources. The attempts to identify the mechanisms underlying cancer development and progression have produced a wealth of facts, and no single individual is capable of addressing the immense breadth of the field with undisputed authority. Hence, the ‘Encyclopedic Reference of Cancer’ is the work of many authors, all of whom are experts in their fields and reputable members of the international scientific community. Each author contributed a large number of keyword definitions and in-depth essays and in so doing it was possible to cover the broad field of cancer-related topics within a single publication. Obviously this approach entails a form of presentation, in which the author has the freedom to set priorities and to promote an individual point of view. This is most obvious when it comes to nomenclature, particularly that of genes and proteins. Although the editorial intention was to apply the nomenclature of the Human Genome Organisation (HUGO), the more vigorous execution of this attempt has been left to future endeavours. In the early phase of planning the Encyclopedia, exploratory contacts to potential authors produced an overwhelmingly positive response. The subsequent contact with almost 300 contributory authors was a marvellous experience, and I am extremely grateful for their excellent and constructive cooperation. An important element in the preparation of the Encyclopedia has been the competent secretarial assistance of Hiltrud Wilbertz of the Springer-Verlag and of Ingrid Cederlund and Cornelia Kirchner of the DKFZ. With great attention to detail they helped to keep track of the technical aspects in the preparation of the manuscript. It was a pleasure to work with the Springer crew, including Dr. Rolf Lange as the Editorial Director (Medicine) and Dr. Thomas Mager, Senior Editor for Encyclopedias and Dictionaries. In particular I wish to thank Dr. Walter Reuss, who untiringly has mastered all aspects and problems associated with the management of the numerous manuscripts that were received from authors of the international scientific community. It has been satisfying and at times comforting to see how he made illustration files come alive. Thanks also to Dr. Claudia Lange who, being herself a knowledgeable cell biologist, has worked as the scientific editor. Her commitment and interest have substantially improved this Encyclopedia.

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Preface To The First Edition

As a final word, I would like to stress that although substantial efforts have been made to compose factually correct and well understandable presentations, there may be places where a definition is incomplete or a phrase in an essay is flawed. All contributors to this Encyclopedia will be extremely happy to receive possible corrections, or revisions, in order for them to be included in any future editions of the ‘Encyclopedic Reference of Cancer’. Heidelberg, September 2001 MANFRED SCHWAB

Preface To The Second Edition

Given the overwhelming success of the First Edition of the Cancer Encyclopedia, which appeared in 2001, and the amazing development in the different fields of cancer research, it has been decided to publish a second fully revised and expanded edition, following the principle concept of the first edition that has proven so successful. Recent developments are seeing a dynamic merging of basic and clinical science, with translational research increasingly becoming a new paradigm in cancer research. The merging of different basic and clinical science disciplines towards the common goal of fighting against cancer has long ago called for the establishment of a comprehensive reference source both as a tool to close the language gap between clinical and basic science investigators and as a platform of information for advanced students and informed laymen alike. It is intended to be a resource for all interested in information beyond their specific own expertise. While the First Edition had featured contributions from approximately 300 scientists/clinicians in one Volume, the Second Edition includes more than 1000 contributors in 4 Volumes with an A–Z format of more than 7000 entries. It provides definitions of common acronyms and short definitions of both related terms and processes in the form of keyword entries. A major information source are detailed essays that provide comprehensive information on syndromes, genes and molecules, and processes and methods. Each essay is well-structured, with extensive cross-referencing between entries. Essays represent original contributions by the corresponding authors, all distinguished scientists in their own field, Editorial input has been carefully restricted to formal aspects. A panel of Field Editors, each an eminent international expert for the corresponding field, has served to ensure the presentation of timely and authoritative Encyclopedia entries. These new traits are likely to meet the expectance that a wide community has towards a cancer reference works. An important element in the preparation of the Encyclopedia has been the competent support by the Springer crew, Dr. Michaela Bilic, Saskia Ellis and, lately, Jana Simniok. I am extremely grateful for their excellent and pleasant cooperation. The Cancer Encyclopedia, Second Edition, will be accessible both in print and online versions. Clinicians, research scientists and advanced students will find this an amazing resource and a highly informative reference to cancer. Heidelberg, March 15, 2008 MANFRED SCHWAB

Editor in Chief

Manfred Schwab Tumor Genetics German Cancer Research Center, DKFZ Heidelberg Germany [email protected]

Field Editors

S TEFAN B ARTH Department of Pharmaceutical Product Development, Fraunhofer-Institute for Molecular Biology and Applied Ecology Aachen Germany [email protected] PAOLO B OFFETTA Lifestyle, Environment and Cancer Group International Agency for Research on Cancer Lyon France [email protected] G RAHAM A. C OLDITZ Washington University in St. Louis St. Louis, MO USA [email protected]

Houston, TX USA [email protected] J OSEPH R. L ANDOLPH Departments of Molecular Microbiology and Immunology, and Pathology; USC/Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences, School of Pharmacy Health Sciences Campus University of Southern California Los Angeles, CA USA [email protected] H ENRY LYNCH Preventive Medicine and Public Health Hereditary Cancer Institute Creighton University Omaha, NE USA [email protected]

R OY J. D UHÉ Department of Pharmacology and Toxicology University of Mississippi Medical Center Jackson, MS USA [email protected]

PAUL G. M URRAY CRUK Institute for Cancer Studies The Medical School University of Birmingham Birmingham UK [email protected]

K ENT H UNTER Metastasis Susceptibility Section Laboratory of Cancer Biology & Genetics CCR/NCI/NIH Bethesda, MD USA [email protected]

S AUL S USTER The Ohio State University Columbus, OH USA [email protected]

J ESPER J URLANDER Department of Hematology and the Leukemia Laboratory Rigshospitalet Copenhagen Denmark [email protected] R AKESH K UMAR Molecular and Cellular Oncology The University of Texas MD Anderson Cancer Center

A NDREW T HORBURN University of Colorado at Denver and Health Sciences Center Aurora, CO USA [email protected] M ARTIN T OBI University of Pennsylvania School of Medicine Chief GI Section Philadelphia VAMC Philadelphia, PA USA [email protected]

List of Contributors

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List of Contributors V ESA A ALTONEN Department of Anatomy Institute of Biomedicine University of Turku Turku Finland [email protected]

FARRUKH A FAQ Department of Dermatology University of Wisconsin Medical Sciences Center Madison, WI USA [email protected]

C ORY A BATE -S HEN Center for Advanced Biotechnology and Medicine UMDMJ – Robert Wood Johnson Medical School Piscataway, NJ USA [email protected]

C HAPLA A GARWAL Department of Pharmaceutical Sciences, School of Pharmacy University of Colorado Health Sciences Center Denver, CO USA [email protected]

P HILLIP H. A BBOSH Department of Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, IN USA [email protected] F RITZ A BERGER Department of Molecular Biology University of Salzburg Salzburg, Austria [email protected] H INRICH A BKEN Tumor Genetics, Clinic I Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne Cologne, Germany [email protected] A MAL M. A BU -G HOSH Departments of Pediatric Hematology and Oncology Lombardi Comprehensive Cancer Center Georgetown University Washington DC, NW USA [email protected] R OSITA A CCARDI Infections and Cancer Biology Group International Agency for Research on Cancer Lyon France [email protected] F ILIPPO A CCONCIA Molecular and Cellular Oncology The University of Texas M. D. Anderson Cancer Center Houston, TX USA IFOM The FIRC Institute for Molecular Oncology Milan Italy

R AJESH A GARWAL Department of Pharmaceutical Sciences, School of Pharmacy University of Colorado Health Sciences Center Denver, CO USA [email protected] PATRIZIA A GOSTINIS Catholic University of Leuven Leuven Belgium [email protected] T ERJE A HLQUIST Department of Cancer Prevention Rikshospitalet-Radiumhospitalet Medical Centre Oslo Norway [email protected] S HAHID A HMED Saskatoon Cancer Center University of Saskatchewan Saskatoon, SK Canada [email protected] J OOHONG A HNN Department of Life Science Gwangju Institute of Science and Technology Buk-Gu, Gwangju Korea [email protected] C EM A KIN University of Michigan Ann Arbor, MI USA [email protected] E NRIQUE DE A LAVA Centro de Investigación del Cáncer-IBMCC Salamanca Spain [email protected]

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A MI A LBIHN Department of Microbiology Tumor and Cell Biology (MTC) Karolinska Institutet Stockholm Sweden [email protected] A DRIANA A LBINI IRCCS Multimedica Milano Italy [email protected] J ÉRÔME A LEXANDRE Faculté de Médecine Paris – Descartes UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul Paris France [email protected] S HADAN A LI Karmanos Cancer Institute Wayne State University Detroit, MI USA [email protected] M ALCOLM R. A LISON Centre for Diabetes and Metabolic Medicine Queen Mary’s School of Medicine and Dentistry Institute of Cell and Molecular Science London UK [email protected] C ATHERINE A LIX -PANABIERES University Medical Center, Lapeyronie Hospital Montpellier France [email protected] A LISON L. A LLAN Cancer Research Laboratories, London Regional Cancer Program; and Departments of Oncology and Anatomy and Cell Biology, Schulich School of Medicine and Dentistry University of Western Ontario, London, ON Canada [email protected] PAOLA A LLAVENA Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected] A NGEL A LONSO Deutsches Krebsforschungszentrum Heidelberg Germany [email protected]

G IANFRANCO A LPINI Departments of Medicine and Systems Biology and Translational Medicine, Central Texas Veterans Health Care System, Scott & White Hospital and Texas A&M University System Health Science Center Temple TX USA [email protected]

M ARIE -C LOTILDE A LVES -G UERRA The Wistar Institute Molecular and Cellular Oncogenesis Program Philadelphia, PA USA [email protected] P IERRE Å MAN LLCR, Department of pathology, Institute of Biomedicine Sahlgrenska Academy Goteborg University Gothenburg Sweden [email protected] K UROSH A MERI University of Stanford School of Medicine Department of Surgery/Surgical Oncology Stanford, CA USA [email protected] M OUNIRA A MOR -G UÉRET Institut Curie – UMR 2027 CNRS Orsay Cedex France [email protected] K ENNETH C. A NDERSON Department of Medical Oncology The Jerome Lipper Multiple Myeloma Center Dana-Farber Cancer Institute Harward Medical School Boston, MA USA [email protected] P ETER A NGEL Deutsches Krebsforschungszentrum Division of Signal Transduction and Growth Control Heidelberg Germany [email protected] A NDREA A NICHINI Department of Experimental Oncology Fondazione IRCCS Istituto Nazionale per lo Studio e la Cura dei Tumori Milan Italy [email protected]


P ETER D. A PLAN Genetics Branch, Center for Cancer Research National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected]

E LIAS S. J. A RNÉR Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden [email protected]

N ATALIA A PTSIAURI Hospital Universitario Virgen de las Nieves Granada Spain [email protected]

S TEFANO ATERINI Department of Experimental Pathology and Oncology University of Firenze Firenze Italy [email protected]

R AMI A QEILAN Molecular Virology, Immunology and Medical Genetics The Ohio State University, Comprehensive Cancer Center Columbus, OH USA [email protected]

M ARC A UMERCIER Institute of Biology of Lille, Pasteur Institute of Lille Universities of Lille 1 and Lille 2 Lille France [email protected]

T SUTOMU A RAKI Departments of Obstetrics and Gynecology Nippon Medical School Kawasaki and Tokyo Japan [email protected]

R ICCARDO A UTORINO Clinica Urologica Seconda Università degli Studi Napoli Italy [email protected]

S ANCHIA A RANDA School of Nursing The University of Melbourne Carlton, VIC Australia [email protected]

M ATIAS A. AVILA Division of Hepatology and Gene Therapy, CIMA University of Navarra Pamplona Spain [email protected]

D IEGO A RANGO Molecular Biology and Biochemistry Research Center (CIBBIM) Valle Hebron Hospital Research Institute Barcelona Spain [email protected]

H AVA AVRAHAM Division of Experimental Medicine Beth Israel Deaconess Medical Center Harvard Institutes of Medicine Boston, MA USA [email protected]

D AVID J. A RATEN NYU School of Medicine NYU Cancer Institute, and the New York VA Medical Center New York, NY USA [email protected]

S HALOM AVRAHAM Division of Experimental Medicine Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine Boston, MA USA [email protected]

VALENTINA A RCANGELI Department of Oncology Infermi Hospital Rimini Italy [email protected]

S ANJAY AWASTHI United States Longview Cancer Center Longview, TX USA [email protected]

G EMMA A RMENGOL Department of Pathology Vall d’Hebron University Hospital Barcelona, Spain [email protected]

Y OGESH C. AWASTHI Department of Human Biological Chemistry and Genetics University of Texas Medical Branch Galveston, TX USA [email protected]

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M ATHIJS B AENS Centre for Human Genetics, Molecular Genetics-Flanders Interuniversity Institute for Biotechnology University of Leuven Leuven Belgium [email protected] D EBASIS B AGCHI Department of Pharmacy Sciences Creighton University Medical Center Omaha, NE USA [email protected] X UE -TAO B AI State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital School of Medicine Shanghai Jiao Tong University Shanghai, People's Republic of China [email protected]

L AURENT B ALENCI INSERM Unité Mixte 873 Grenoble Cedex 09 France [email protected] S USHANTA K. B ANERJEE Division of Hematology and Oncology, School of Medicine Kansas University of Medical Center Kansas City, KS USA [email protected] M ICHAL B ANIYASH The Lautenberg Center for General and Tumor Immunology The Hebrew University-Hadassah Medical School Jerusalem Israel [email protected]

J ÜRGEN B AJORATH Department of Life Science Informatics B-IT University of Bonn, Bonn Germany [email protected]

R ACHEL B AR -S HAVIT Department of Oncology Hadassah-University Hospital Jerusalem Israel [email protected]

S TUART G. B AKER Biometry Research Group National Cancer Institute Bethesda, MD USA [email protected]

A DITYA B ARDIA Department of Internal Medicine Mayo Clinic College of Medicine Rochester, MN USA [email protected]

E LIZABETH K. B ALCER -K UBICZEK Department of Radiation Oncology University of Maryland School of Medicine Baltimore, MD USA [email protected]

R AFIJUL B ARI Vascular Biology Center, Cancer Institute, and Departments of Medicine and Molecular Sciences University of Tennessee Health Science Center Memphis, TN USA [email protected]

E NKE B ALDINI Department of Experimental Medicine University of Rome “Sapienza” Rome Italy [email protected] G RAHAM S. B ALDWIN Department of Surgery University of Melbourne Austin Health Melbourne, VIC Australia [email protected] S HERRI B ALE GeneDx, Rockville, MD USA [email protected]

D R N ICOLA L. P. B ARNES Department of Academic Surgery South Manchester University Hospital Manchester UK R OBERT B AROUKI INSERM UMR-S 747, Université René Descartes Paris Paris, France [email protected] JM B ARROS -D IOS Department of Preventive Medicine and Public Health University of Santiago de Compostela C/San Francisco Spain [email protected]


H ARRY B ARTELINK Department of Radiotherapy The Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital Amsterdam The Netherlands [email protected] S TEFAN B ARTH Department of Pharmaceutical Product Development Fraunhofer-Institute for Molecular Biology and Applied Ecology Aachen Germany [email protected] H ELMUT B ARTSCH Division of Toxicology and Cancer Risk Factors German Cancer Research Center (DKFZ) Heidelberg Germany [email protected] T HOMAS B ARZ Max-Panck-Institut für Psychiatrie München Germany [email protected] H OLGER B ASTIANS Philipps University Marburg Institute for Molecular Biology and Tumor Research (IMT) Marburg Germany [email protected] A. B ATISTATOU Ioannina University Medical School Ioannina Greece [email protected] F. B ATTEUX Faculté de Médecine Paris – Descartes UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul Paris France [email protected] J ACQUES B AUDIER INSERM Unité Mixte 873 Grenoble Cedex 09 France [email protected] PAUL B AUER Pfizer Research Technology Center Cambridge, MA USA [email protected]

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PAIGE J. B AUGHER Department of Cancer Biology Vanderbilt University Medical School Nashville, TN USA [email protected] A SNE R. B AUSKIN Centre for Immunology, St. Vincents Hospital and Department of Medicine University of New South Wales Sydney, NSW Australia [email protected] N ICOLE B EAUCHEMIN McGill Cancer Centre McGill University Montreal, Quebec Canada [email protected] J OHN F. B ECHBERGER Department of Cellular and Physiological Sciences The University of British Columbia Vancouver, BC Canada [email protected] G ERHILD B ECKER Department of Internal Medicine II (Gastroenterology, Hepatology, Endocrinology) University Hospital Freiburg Freiburg, Germany [email protected] H EINZ B ECKER Department of General and Visceral Surgery University Medical Center Göttingen Germany [email protected] M ARIE E. B ECKNER Department of Pathology University of Pittsburgh School of Medicine Pittsburgh, PA USA [email protected] C LAUS B ELKA Department of Radiation Oncology University Tübingen Tübingen Germany [email protected] M AURIZIO B ENDANDI University Clinic and Center for Applied Medical Research University of Navarra Pamplona Spain [email protected]

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YAACOV B EN -D AVID Division of Cancer Biology, Sunny brook Health Sciences University of Toronto Toronto, ONT Canada [email protected] S UZANNE M. B ENJES Cancer Genetics Research Group, University of Otago at Christchurch Christchurch, New Zealand [email protected] C ARMEN B ERASAIN Division of Hepatology and Gene Therapy, CIMA University of Navarra Pamplona Spain [email protected] A LAN B EREZOV Department of Pathology and Laboratory Medicine & Abramson Cancer Center University of Pennsylvania Philadelphia, PA USA [email protected] R OB J. W. B ERG University Medical Center Utrecht Utrecht The Netherlands [email protected]

S ANCHITA B HADRA Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology The University of Texas at Austin Austin, TX USA [email protected] K UMAR M. R. B HAT Department of Dermatology University of Wisconsin School of Medicine and Public Health Sciences Madison, WI USA [email protected] M ALAYA B HATTACHARYA -C HATTERJEE University of Cincinnati and The Barrett Cancer Center Cincinnatti, OH USA [email protected] C ATERINA B IANCO Mammary Biology and Tumorigenesis Laboratory National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected] J EAN -M ICHEL B IDART Institut de Cancérologie Gustave-Roussy Villejuif and Université Paris-Sud 11, Paris France [email protected]

C ORINNA B ERGELT Institute of Medical Psychology University Medical Center Hamburg-Eppendorf Hamburg Germany [email protected]

J ACLYN A. B IEGEL The Children’s Hospital of Philadelphia Philadelphia, PA USA [email protected]

R ENE B ERNARDS The Netherlands Cancer Institute Amsterdam The Netherlands [email protected]

M ARGHERITA B IGNAMI Istituto Superiore di Sanita’ Rome Italy [email protected]

Z WI B ERNEMAN Laboratory of Experimental Hematology Antwerp University Hospital Edegem Belgium [email protected]

I RENE V. B IJNSDORP Department of Medical Oncology VU University Medical Center Amsterdam The Netherlands [email protected]

J ÉRÔME B ERTHERAT Assistance Publique Hôpitaux de Paris, Hôpital Cochin Department of Endocrinology Reference Center for Rare Adrenal Diseases Paris France [email protected]

C HEN B ING Division of Cellular and Metabolic Medicine, School of Clinical Sciences University of Liverpool Liverpool UK [email protected]


M ARC B IRKHAHN Departments of Urology and Pathology University of Southern California Keck School of Medicine Los Angeles, CA USA [email protected] G IOVANNI B LANDINO Rome Oncogenomic Center, Department of Experimental Oncology Regina Elena Cancer Institute Rome Italy [email protected] D AVID E. B LASK Bassett Research Institute The Mary Imogene Bassett Hospital Cooperstown, NY USA [email protected] J ONATHAN B LAY Department of Pharmacology Dalhousie University Halifax, NS Canada [email protected] A DRIAN J. B LOOR The Christie NHS Trust Manchester UK [email protected] J ULIAN B LOW Wellcome Trust Biocentre University of Dundee Dundee, Scotland UK [email protected] P ETER B LUME -J ENSEN Serono Reproductive Biological Institute Randolph, MA USA

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PAOLO B OFFETTA Gene-Environment Epidemiology Group International Agency for Research on Cancer Lyon France [email protected] S TEFAN K. B OHLANDER Medizinische Klinik und Poliklinik III, Klinikum Großhadern Universität München München Germany [email protected] VALENTINA B OLLATI Department of Occupational and Environmental Health “Clinica del Lavoro L. Devoto” University of Milan Milan Italy [email protected] M ARIA G RAZIA B ORRELLO Department of Experimental Oncology IRCCS Istituto Nazionale Tumori Foundation Milan Italy [email protected] G IUSEPPE B ORZACCHIELLO Department of Pathology and Animal health University of Naples Federico II Naples Italy [email protected] VALERIE B OSCH Forschungsschwerpunkt Infektion und Krebs, F020 German Cancer Research Center (DKFZ) Heidelberg Germany [email protected] C HRIS B OSHOFF Cancer Research Campaign Viral Oncology Group Wolfson Institute for Biomedical Research University College London London UK [email protected]

S ARAH B OCCHINI Department of Experimental Medicine University of Rome “Sapienza” Rome Italy [email protected]

F RANCK B OURDEAUT Département de pédiatrie INSERM 830, Biologie et génétique des tumeurs Institut Curie Paris France [email protected]

A NN M. B ODE The Hormel Institute University of Minnesota Austin, MN USA [email protected]

J EAN -P IERRE B OURQUIN Pediatric Oncology University Children’s Hospital Zurich Zurich Switzerland [email protected]

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J UDITH V. M. G. B OVÉE Department of Pathology Leiden University Medical Center Leiden The Netherlands J.V.M.G. [email protected]

K ATJA B ROCKE -H EIDRICH Institute of Clinical Immunology and Transfusion medicine University Hospital of Leipzig Leipzig Germany [email protected]

N ORMAN B OYD Campbell Family Institute for Breast Cancer Research Ontario Cancer Institute Toronto, ONT Canada [email protected]

A NGELA B RODIE University of Maryland School of Medicine Baltimore, MD USA University of Maryland Greenebaum Cancer Center Baltimore, MD USA [email protected]

S VEN B RANDAU Department of Otorhinolaryngology University Duisburg-Essen Essen Germany [email protected] B URKHARD H. B RANDT Institute for Tumour Biology University Medical Centre Hamburg Germany [email protected] H ILTRUD B RAUCH Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University Tuebingen Stuttgart Germany [email protected] S AMUEL N. B REIT Centre for Immunology, St. Vincents Hospital and Department of Medicine University of New South Wales Sydney, NSW Australia [email protected] E DWIN B REMER Groningen University Institute for Drug Exploration (GUIDE), Department of Pathology & Laboratory Medicine Section Medical Biology, Laboratory for Tumor Immunology Groningen The Netherlands [email protected]

C HRISTOPHER L. B ROOKS Institute for Cancer Genetics, and Department of Pathology College of Physicians and Surgeons, Columbia University New York, NY USA M AI N. B ROOKS Surgical Oncology, School of Medicine University of California Los Angeles, CA USA [email protected] D AVID A. B ROWN Centre for Immunology, St. Vincents Hospital and Department of Medicine University of New South Wales Sydney, NSW Australia [email protected] K AREN B ROWN Cancer Biomarkers and Prevention Group Department of Cancer Studies, University of Leicester Leicester UK [email protected] K EVIN B ROWN University of Florida College of Medicine Gainesville, FL USA [email protected]

C ATHERINE B RENNER University of Versailles/SQY CNRS UMR 8159, Versailles France [email protected]

D IEDERIK DE B RUIJN Department of Human Genetics Radboud University Nijmegen Medical Centre Nijmegen The Netherlands [email protected]

D AVID J. B RENNER Department of Radiation Oncology Columbia University New York, NY USA [email protected]

T ILMAN B RUMMER Cancer Research Program Garvan Institute of Medical Research Sydney, NSW Australia [email protected]


A NTONIO B RUNETTI Department of Experimental and Clinical Medicine G. Salvatore University of Catanzaro Magna Græcia Catanzaro Italy [email protected] A NDREAS K. B UCK Department of Nuclear Medicine Technical University of Munich Munich Germany [email protected] L ASZLO B UDAY Department of Medical Chemistry Semmelweis University Medical School Budapest Hungary [email protected] M ARIE A NNICK B UENDIA Oncogenesis and Molecular Virology Unit Inserm U579 Institut Pasteur, Paris France [email protected] R ALF B UETTNER City of Hope National Medical Center and Beckman Research Institute Duarte, CA USA [email protected] N IGEL J. B UNDRED Department of Academic Surgery South Manchester University Hospital Manchester UK [email protected] I RINA B U ß Institute of Pharmacy University of Bonn Bonn Germany [email protected] A LEXANDER B ÜRKLE Department of Biology University of Konstanz Konstanz Germany [email protected] J ADISH B UTANY Laboratory Medicine and Pathobiology University Health Network/Toronto Toronto, ON Canada [email protected]

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N EVILLE J. B UTCHER School of Biomedical Sciences University of Queensland St Lucia, QLD Australia [email protected] T IMON P. H. B UYS Department of Cancer Genetics and Developmental Biology British Columbia Cancer Research Centre Vancouver, BC Canada [email protected] Y I C AI Department of Pathology Baylor College of Medicine Houston, TX USA Y IQIANG C AI Section of Nephrology Yale University School of Medicine New Haven, CT USA [email protected] B RUNO C ALABRETTA Thomas Jefferson University Kimmel Cancer Institute Philadelphia, PA USA [email protected] D ANIELE C ALISTRI Molecular Laboratory Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori Meldola (Forlì) Italy [email protected] J AVIER C AMACHO Pharmacology Division Centro de Investigación y de Estudios Avanzados del I.P.N. Mexico City, Mexico [email protected] W ILLIAM G. C ANCE Departments of Surgery University of Florida College of Medicine Gainesville, FL USA [email protected] A MPARO C ANO Departamento de Bioquímica Facultad de Medicina, UAM, Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-UAM Madrid Spain [email protected]

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S HARON C ANTOR Assistant Professor of Cancer Biology UMASS Medical School Worcester, MA USA [email protected]

E STEBAN C ELIS H. Lee Moffitt Cancer Center and Research Institute University of South Florida Tampa, FL USA [email protected]

A NTHONY J. C APOBIANCO The Wistar Institute Molecular and Cellular Oncogenesis Program Philadelphia, PA USA [email protected]

H O M AN C HAN Division of Biochemistry and Molecular Biology University of Glasgow Glasgow UK

S ALVATORE J. C ARADONNA University of Medicine and Dentistry of New Jersey Stratford, NJ USA [email protected] M ICHELE C ARBONE Cancer Research Center of Hawaii University of Hawaii Honolulu, HI USA [email protected] N EIL O C ARRAGHER Advanced Science and Technology Laboratory AstraZeneca R&D Charnwood Loughborough UK [email protected] M ICHELA C ASANOVA Pediatric Oncology Unit Fondazione IRCCS Istituto Nazionale Tumori Milano Italy [email protected] W OLFGANG H. C ASELMANN Medizinische Klinik und Poliklinik I Rheinische Friedrich-Wilhelms-Universität Bonn Germany [email protected] R OBERTO C ATTANEO Molecular Medicine Program and Virology and Gene Therapy Track Mayo Clinic College of Medicine Rochester, MN USA [email protected] W EBSTER K. C AVENEE Ludwig Institute for Cancer Research, UCSD La Jolla, CA USA [email protected]

D AWN S. C HANDLER Department of Pediatrics, Columbus Children’s Research Institute, Center for Childhood Cancer The Ohio State University School of Medicine and Public Health Columbus, OH USA [email protected] M AU -S UN C HANG Department of Medical Research Mackay Memorial Hospital Taipei County 251 Taiwan [email protected] M EI -C HI C HANG Biomedical Science Team Chang Gung Institute of Technology Taoyuan Taiwan [email protected] J ANE C.-J. C HAO School of Nutrition and Health Sciences Taipei Medical University Taipei Taiwan [email protected] C HRISTINE C HAPONNIER Department of Pathology and Immunology University of Geneva Geneva Switzerland [email protected] K ONSTANTINOS C HARALABOPOULOS Ioannina University Medical School Ioannina Greece [email protected] M ALAY C HATTERJEE Department of Pharmaceutical Technology Jadavpur University Calcutta, West Bengal India [email protected]


S UNIL K. C HATTERJEE University of Cincinnati and The Barrett Cancer Center Cincinnatti, OH USA [email protected] G AUTAM C HAUDHURI Department of Molecular and Medical Pharmacology and Department of Obstetrics and Gynecology David Geffen School of Medicine at UCLA Los Angeles, CA USA [email protected] M. A SIF C HAUDRY University Department of Surgery Royal Free and University College London Medical School London UK [email protected] D HARMINDER C HAUHAN Department of Medical Oncology The Jerome Lipper Multiple Myeloma Center Dana Farber Cancer Institute Harvard Medical School Boston, MA USA [email protected] J EREMY P. C HEADLE Institute of Medical Genetics Cardiff University Heath Park, Cardiff UK [email protected] S AI -J UAN C HEN State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital School of Medicine Shanghai Jiao Tong University Shanghai, People's Republic of China [email protected] TAOSHENG C HEN New Leads, Bristol-Myers Squibb Wallingford, CT USA [email protected] Z HU C HEN State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital School of Medicine Shanghai Jiao Tong University Shanghai, People's Republic of China [email protected] G EORGE Z. C HENG Mount Sinai School of Medicine New York, NY USA [email protected]

J IN Q. C HENG Molecular Oncology and Pathology, H. Lee Moffitt Cancer Center & Research Institute University of South Florida College of Medicine Tampa, FL USA [email protected] L IANG C HENG Department of Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, IN USA [email protected] YA -H UI C HI National Institute of Allergy and Infectious Disease, NIH Bethesda, MD USA [email protected] M ARTYN A. C HIDGEY Division of Medical Sciences University of Birmingham Clinical Research Block Queen Elizabeth Hospital Birmingham UK [email protected] Y OUNG -W ON C HIN College of Pharmacy The Ohio State University Columbus, OH USA [email protected] S UDHAKAR C HINTHARLAPALLI Department of Veterinary Physiology and Pharmacology Texas A&M University College Station, TX USA [email protected] A LEXANDRE C HLENSKI The Robert H. Lurie Comprehensive Cancer Center Northwestern University Feinberg School of Medicine Chicago, IL USA [email protected] W ILLIAM C HI -S HING C HO Department of Clinical Oncology Queen Elizabeth Hospital Kowloon Hong Kong [email protected] M ICHAEL C HOPP Neurology Research Henry Ford Health System Detroit, MI USA [email protected]

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P EI -L UN C HOU Division of Allergy-Immunology-Rheumatology, Department of Internal Medicine Lin Shin Hospital Taichung Taiwan

P IER PAOLO C LAUDIO Department of Surgery Marshall University Huntington, WV USA [email protected]

C LAUS C HRISTENSEN Department of Cancer Genetics Danish Cancer Society Copenhagen Denmark [email protected]

E LIZABETH B. C LAUS Department of Epidemiology and Public Health Yale University School of Medicine New Haven, CT USA [email protected]

R IKKE C HRISTENSEN Anatomy and Neurobiology, Institute of Medical Biology University of Southern Denmark Odense, Denmark [email protected]

PASCAL C LAYETTE SPI-BIO, Service de Neurovirologie, CEA, CRSSA, EPHE Fontenay aux Roses Cedex France [email protected]

G ERHARD C HRISTOFORI Department of Clinical-Biological Sciences, Centre of Biomedicine Institute of Biochemistry and Genetics, University of Basel Basel Switzerland [email protected]

S TEVEN C. C LIFFORD Northern Institute for Cancer Research Newcastle University Newcastle upon Tyne UK [email protected]

R ICHARD I. C HRISTOPHERSON School of Molecular and Microbial Biosciences University of Sydney Sydney, NSW Australia [email protected]

K EVIN A. C OCKELL Nutrition Research Division Health Canada Ottawa, ON Canada [email protected]

B ONG H YUN C HUNG BioNanotechnology Research Center Korea Research Institute of Bioscience and Biotechnology Yuseong, Daejeon Republic of Korea [email protected]

S USAN L. C OHN Clinical Sciences The Institute for Molecular Pediatric Sciences University of Chicago Chicago, IL USA [email protected]

F UNG -L UNG C HUNG Department of Oncology, Lombardi Comprehensive Cancer Center Georgetown University Medical Center Washington, DC USA [email protected]

G RAHAM A. C OLDITZ Washington University in St. Louis St. Louis, MO USA [email protected]

J ACKY K. H. C HUNG Department of Medical Genetics and Microbiology University of Toronto Toronto, Ontario Canada [email protected]

PAOLA C OLLINI Anatomic Pathology Department Fondazione IRCCS Istituto Nazionale Tumori Milano Italy [email protected]

S UE C LARK The Polyposis Registry St Mark’s Hospital Harrow UK [email protected]

A NDREW R. C OLLINS Department of Nutrition University of Oslo Oslo Norway [email protected]


J OAN W. C ONAWAY Stowers Institute for Medical Research Kansas, MO USA [email protected] R ONALD C. C ONAWAY Stowers Institute for Medical Research Kansas, MO USA [email protected] N ATHALIE C OOLS Laboratory of Experimental Hematology Antwerp University Hospital Edegem Belgium [email protected] H ELEN C. C OONEY UCD School of Biomolecular and Biomedical Science UCD Conway Institute University College Dublin Dublin Ireland [email protected] K UMARASEN C OOPER Department of Pathology University of Vermont Burlington, VT USA [email protected] L AURENCE J. N. C OOPER Division of Pediatrics, Department of Immunology M.D. Anderson Cancer Center Houston, TX USA [email protected] P ETER J. C OOPMAN CRBM/CNRS UMR 5237, Univ Montpellier II, Montpellier France [email protected] R ICHARD J. C OTE Departments of Urology and Pathology University of Southern California Keck School of Medicine Los Angeles, CA USA [email protected] M ARCUS V. C RONAUER Institute of General Zoology and Endocrinology University of Ulm Ulm Germany [email protected]

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S IDNEY C ROUL Department of Pathology, UHN University of Toronto ON Canada [email protected] V INCENT L. C RYNS Department of Medicine, Feinberg School of Medicine Northwesten University Chicago, IL USA [email protected] R ONALD G. C RYSTAL Department of Genetic Medicine Weill Medical College of Cornell University New York, NY USA [email protected] J IUWEI C UI Department of Medical Biophysics University of Toronto Toronto, ONT Canada [email protected] E DNA C UKIERMAN Basic Science/Tumor Cell Biology Fox Chase Cancer Center Philadelphia, PA USA [email protected] Z ORAN C ULIG Department of Urology Innsbruck Medical University Innsbruck Austria [email protected] D AVID T. C URIEL Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology and the Gene Therapy Center, Division of Human Gene Therapy University of Alabama at Birmingham Birmingham, AL USA [email protected] YATARO D AIGO Institute of Medical Science The University of Tokyo Tokyo Japan [email protected] A NGUS G. D ALGLEISH St. George’s Hospital Medical School London UK [email protected]

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A SHRAF D ALLOL Department of Medical and Molecular Genetics, Institute of Biomedical Research University of Birmingham Birmingham UK [email protected] TAMAS D ALMAY School of Biological Sciences University of East Anglia Norwich UK [email protected] I VAN D AMJANOV Department of Pathology University of Kansas School of Medicine Kansas City, KS USA [email protected] V INCENT D AMMAI Hollings Cancer Center and Department of Pathology and Laboratory Medicine Medical University of South Carolina Charleston, SC USA [email protected] C HENDIL D AMODARAN Department of Clinical Sciences University of Kentucky Lexington, Kentucky USA [email protected] J ANET E. D ANCEY Division of Cancer Treatment and Diagnosis National Cancer Institute Rockville, MD USA [email protected] C HI V. D ANG Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Johns Hopkins University School of Medicine Baltimore, MD USA [email protected] A LLA D ANILKOVITCH -M IAGKOVA National Cancer Institute-FCRDC Frederick, MD USA [email protected] M ASSIMINO D’A RMIENTO Department of Experimental Medicine University of Rome-Sapienza Rome Italy [email protected]

K AUSTUBH D ATTA Department of Urology Research, Department of Biochemistry and Molecular Biology Mayo Clinic College of Medicine Rochester, Minnesota USA [email protected] P RAN K. D ATTA Departments of Surgery and Cancer Biology Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine Nashville, TN USA [email protected] L EONOR D AVID IPATIMUP (Institute of Molecular Pathology and Immunology of the University of Porto) and Medical Faculty of the University of Porto Porto Portugal [email protected] H ELEN D AVIS Oxford University Medical School John Radcliffe Hospital Oxford UK [email protected] A LEXEY D AVYDOV Fox Chase Cancer Center Philadelphia, PA USA [email protected] R OBERT D AY Department of Pharmacology, Institut de Pharmacologie Faculté de Médecine Université de Sherbrooke Sherbrooke, QC Canada [email protected] J OCHEN D ECKER Bioscientia Institute Center for Human Genetics and University of Mainz Ingelheim Germany [email protected] A MIR R. D EHDASHTI Division of Neurosurgery University of Toronto Toronto, ON Canada [email protected]


M ARYSE D ELEHEDDE INSERM U774 Institut Pasteur de Lille Lille France [email protected] B ERNA D EMIRCAN University of Florida College of Medicine Gainesville, FL USA [email protected] D AVID A. D ENNING Department of Surgery Marshall University Huntington, WV USA [email protected] S YLVIANE D ENNLER The Netherlands Cancer Institute Amsterdam The Netherlands [email protected] C HANNING J. D ER University of North Carolina at Chapel Hill Chapel Hill, NC USA [email protected] B ARBARA D ESCHLER Medical Center, Department of Hematology-Oncology University of Freiburg Freiburg Germany [email protected] C HANTAL D ESDOUETS Institut Cochin Université Paris Descartes, CNRS Paris France [email protected] P ETER D EVILEE Leiden University Medical Center Leiden The Netherlands [email protected] M ARK W. D EWHIRST Department of Radiation Oncology Duke University Durham, NC USA [email protected]

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M ARC D IEDERICH Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC) Hôpital Kirchberg Steichen Luxembourg [email protected] J OSEPH D I F RANZA Department of Family Medicine and Community Health University of Massachusetts Medical Center Worcester, MA USA [email protected] M ARTIN D IGWEED Institute of Human Genetics Charité – Universitätsmedizin Berlin Berlin Germany [email protected] P ETER T EN D IJKE The Netherlands Cancer Institute Amsterdam The Netherlands [email protected] G ERARD D IJKSTRA University Medical Center Groningen University of Groningen Groningen The Netherlands [email protected] H ELEN D IMARAS Ontario Cancer Institute/Princess Margaret Hospital Toronto, ON Canada [email protected] J IAN D ING State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai P. R. China [email protected] J ÜRGEN D ITTMER Clinic for Gynecology University of Halle, Halle Germany [email protected] H ENRIK J. D ITZEL Medical Biotechnology Center Institute of Medical Biology University of Southern Denmark Odense C Denmark [email protected]

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C HOLPON S. D JUZENOVA Klinik für Strahlentherapie der Universität Würzburg Würzburg Germany [email protected]

B RIAN J. D RUKER Oregon Health & Science University Cancer Institute Portland, OR USA [email protected]

C HRISTIAN D OEHN Department of Urology University of Luebeck, Medical School Luebeck Germany [email protected]

R AYMOND N. D U B OIS Provost and Executive Vice President M.D. Anderson Cancer Center Houston, TX USA [email protected]

YASUFUMI D OI Department of Environmental Medicine Graduate School of Medical Sciences Kyushu University Fukuoka Japan [email protected]

D AN G. D UDA Steele Laboratory for Tumor Biology Department of Radiation Oncology Massachusetts General Hospital and Harvard Medical School Boston, MA USA [email protected]

Q IHAN D ONG Central Clinical School The University of Sydney, Department of Endocrinology and Sydney Cancer Centre, Royal Prince Alfred Hospital NSW Australia [email protected]

J AQUELIN P. D UDLEY Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology The University of Texas at Austin Austin, TX USA [email protected]

Z IGANG D ONG The Hormel Institute University of Minnesota Austin, MN USA [email protected]

R OY J. D UHÉ Department of Pharmacology and Toxicology University of Mississippi Medical Center Jackson, MS USA [email protected]

Q. P ING D OU The Prevention Program, Barbara Ann Karmanos Cancer Institute and Department of Pathology, School of Medicine Wayne State University Detroit, MI USA [email protected]

I GNACIO D URAN Department of Medical Oncology and Hematology Robert and Maggie Bras and Family New Drug Development Program Princess Margaret Hospital Toronto, ONT Canada [email protected]

T OMMASO A. D RAGANI Fondazione IRCCS Istituto Nazionale Tumori Milan Italy [email protected] K ENNETH D RAKE Department of Chemistry and Biochemistry University of Texas at Arlington Arlington, TX USA [email protected] M ARTIN D REYLING Department of Internal Medicine III University of Munich, Großhadern München Germany [email protected]

S TEPHEN T. D URANT University of Oxford Oxford UK [email protected] M EENAKSHI D WIVEDI Department of Life Science Gwangju Institute of Science and Technology Buk-Gu, Gwangju Korea [email protected] B EHFAR E HDAIE University of Virginia Charlottesville, VA USA [email protected]


J USTIS P. E HLERS Department of Ophthalmology and Visual Sciences Washington University School of Medicine St. Louis, MO USA [email protected] C. H. J. VAN E IJCK Department of Surgery Erasmus MC Rotterdam The Netherlands [email protected] G ERHARD E ISENBRAND Department of Chemistry, Division of Food Chemistry and Toxicology University of Kaiserslautern Kaiserslautern Germany [email protected]

A NDREAS E NGERT Hematology and Oncology University Hospital of Cologne, Department of Internal Medicine I Cologne Germany [email protected] C OCAV A. E NGMAN Department of Medicine Dartmouth Hitchcock Medical Center Lebanon, NH USA [email protected] M ARICA E OLI Unit of Clinical Neuro-Oncology Istituto Neurologico Besta Milano Italy

PATRICIA V. E LIZALDE Institute of Biology and Experimental Medicine (IBYME) CONICET Buenos Aires Argentina [email protected]

S ÜLEYMAN E RGÜN Institute of Anatomy University Hospital Essen Essen Germany [email protected]

B ASSEL F. E L -R AYES Karmanos Cancer Institute Wayne State University Detroit, MI USA [email protected]

PABLO V. E SCRIBÁ Department of Biology-IUNICS University of the Balearic Islands Palma de Mallorca Spain [email protected]

M ITSURU E MI Departments of Obstetrics and Gynecology Nippon Medical School Kawasaki and Tokyo Japan [email protected]

F RANCISCO J. E STEVA Breast Cancer Translational Research Laboratory The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected]

C AROLINE E ND Division of Molecular Genome Analysis, DKFZ Heidelberg Germany [email protected] M ANON VAN E NGELAND Department of Pathology Research Institute for Growth and Development (GROW) Maastricht University Hospital Maastricht The Netherlands [email protected] R AINER E NGERS Institute of Pathology University Hospital Düsseldorf Düsseldorf Germany [email protected]

M ARK F. E VANS Department of Pathology University of Vermont Burlington, VT USA [email protected] B. M ARK E VERS Department of Surgery, University of Texas Medical Branch Galveston, TX USA [email protected] C RISTINA M ARIA FAILLA Laboratory of Molecular and Cell Biology, IDI-IRCCS Rome Italy [email protected]

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M ARCO FALASCA Department of Medicine University College London London UK [email protected] FANG FAN Department of Pathology University of Kansas School of Medicine Kansas City, KS USA [email protected] S AIJUN FAN Long Island Jewish Medical Center Albert Einstein College of Medicine Bronx, NY USA B INGLIANG FANG Department of Thoracic and Cardiovascular Surgery The University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected] L EI FANG Dermatology Branch National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected] VALERIA R. FANTIN Merck Research Laboratories Boston, MA USA [email protected]

A NDREW P. F EINBERG Department of Medicine and Center for Epigenetics Institute for Basic Biomedical Sciences Johns Hopkins University School of Medicine Baltimore, MD USA [email protected] M ARK A. F EITELSON Department of Pathology, Anatomy and Cell Biology Thomas Jefferson University Philadelphia, PA USA [email protected] D AVID F ELDMAN Division of Endocrinology Department of Medicine, Stanford University School of Medicine Stanford, CA USA [email protected] F RANCESCO F EO Department of Biomedical Sciences Division of Experimental Pathology and Oncology Sassari Italy [email protected] M ARIE F ERNET INSERM U612, Institut Curie-Recherche Orsay France [email protected] A NDREA F ERRARI Pediatric Oncology Unit Fondazione IRCCS Istituto Nazionale Tumori Milano Italy [email protected]

O MID C. FAROKHZAD Laboratory of Nanomedicine and Biomaterials, Department of Anesthesiology Brigham and Women’s Hospital Boston, MA USA [email protected]

L ORENA L. F IGUEIREDO -P ONTES Medical School of Ribeirão Preto University of São Paulo Ribeirão Preto Brazil [email protected]

W ILLIAM L. FARRAR National Cancer Institute – Frederick Frederick, MD USA [email protected]

D ANIEL F INLEY Department of Cell Biology Harvard Medical School Boston, MA USA [email protected]

A LESSANDRO FATATIS Department of Pharmacology and Physiology Drexel University College of Medicine Philadelphia, PA USA [email protected]

G AETANO F INOCCHIARO Istituto Nazionale Neurologico Besta Unit of Experimental Neuro-Oncology Milano Italy [email protected]


P IER PAOLO D I F IORE IFOM, the FIRC Institute of Molecular Oncology Milan Italy [email protected]

R ICCARDO F ODDE Department of Pathology Josephine Nefkens Institute Erasmus MC, Rotterdam The Netherlands [email protected]

PAUL B. F ISHER Departments of Urology, Pathology and Neurosurgery Columbia University Medical Center College of Physicians and Surgeons New York, NY USA [email protected]

J UDAH F OLKMAN Children’s Hospital and Harvard Medical School Boston, MA USA [email protected]

J AMES F LANAGAN Wolfson Institute for Biomedical Research University College London London UK [email protected]

H AMIDREZA F ONOUNI Department of General Visceral and Transplantation Surgery University of Heidelberg Heidelberg Germany [email protected]

M ICHAEL F LEISCHHACKER Charité-Universitätsmedizin Berlin CCM, Berlin Germany [email protected]

K ENNETH A. F OON The Pittsburgh Cancer Institute Pittsburgh, PA USA [email protected]

E LIEZER F LESCHER Department of Human Microbiology, Sackler Faculty of Medicine Tel Aviv University Tel Aviv Israel [email protected] J ONATHAN A. F LETCHER Albany Medical College Albany NY [email protected] B ARBARA D. F LORENTINE Department of Pathology Henry Mayo Newhall Memorial Hospital Valencia, CA and Keck School of Medicine, University of Southern California, Los Angeles, CA USA [email protected] R OKA F LORIAN Department of Surgical Oncology The University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected] TAMARA F LOYD Cancer Vaccine Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected]

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A LESSANDRA F ORNI EPOCA Epidemiology Research Center, “Clinica del Lavoro L. Devoto” University of Milan Milan Italy [email protected] PAUL F OSTER Department of Endocrinology and Metabolic Medicine, Imperial College Faculty of Medicine St. Mary’s Hospital London UK [email protected] D AVID A. F RANK Dana-Farber Cancer Institute and Harvard Medical School Boston, MA USA [email protected] S TUART J. F RANK Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine University of Alabama at Birmingham Birmingham, AL USA [email protected] S TANLEY R. F RANKEL Merck Research Laboratories Upper Gwynedd, PA USA

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A LEKSANDRA F RANOVIC Department of Cellular and Molecular Medicine Faculty of Medicine, University of Ottawa Ottawa, ONT Canada [email protected]

ATSUKO F UJIHARA Department of Gene Therapy Science Osaka University, Graduate School of Medicine Suita, Osaka Japan [email protected]

M ICHAEL R. F REEMAN Urological Diseases Research Center Children’s Hospital Boston, Harvard Medical School Boston, MA USA [email protected]

J IRO F UJIMOTO Department of Obstetrics and Gynecology Gifu University School of Medicine Gifu City Japan [email protected]

E MIL F REI III Dana-Farber Cancer Institute Boston, MA USA [email protected] J EAN -N OËL F REUND INSERM, U682 Université Louis Pasteur Strasbourg France [email protected] E RROL C. F RIEDBERG University of Texas Southwestern Medical Center Dallas, TX USA [email protected] S TEVEN M. F RISCH Mary Babb Randolph Cancer Center and Department of Biochemistry West Virginia University Morgantown, WV USA [email protected] PAUL F RÉNEAUX Département de Pathologie Institut Curie Paris France [email protected] M ICHAEL C. F RÜHWALD Department of Pediatric Hematology and Oncology University Children’s Hospital Muenster Muenster Germany [email protected] A NDREW M. F RY University of Leicester Leicester UK [email protected]

J UN F UJITA Department of Clinical Molecular Biology, Graduate School of Medicine Kyoto University Kyoto Japan [email protected] K ENJI F UKASAWA Molecular Oncology Program H. Lee Mofitl Cancer Center & Research Institute Tampa, FL USA [email protected] S IMONE F ULDA University Children’s Hospital, University of Ulm Ulm Germany [email protected] K YLE F URGE Van Andel Research Institute Grand Rapids, MI USA [email protected] M UTSUO F URIHATA Department of Pathology Kochi Medical School Kochi Japan [email protected] R HOIKOS F URTWÄNGLER Division of Pediatric Hematology and Oncology Saarland University Hospital Homburg/Saar Germany [email protected] B ERNARD W. F UTSCHER Department of Pharmacology and Toxicology, Arizona Cancer Center and College of Pharmacy University of Arizona Tucson, AZ USA [email protected]


S HIRISH G ADGEEL Karmanos Cancer Institute Wayne State University Detroit, MI USA [email protected]

A NDREI L. G ARTEL Department of Medicine University of Illinois at Chicago Chicago, IL USA [email protected]

F EDERICO G AGO Departamento de Farmacología Universidad de Alcalá Alcalá de Henares, Madrid Spain [email protected]

R ONALD B. G ARTENHAUS The University of Maryland Marlene and Stewart Greenebaum Cancer Center Baltimore, MD USA [email protected]

W ILLIAM M. G ALLAGHER UCD School of Biomolecular and Biomedical Science UCD Conway Institute University College Dublin Dublin Ireland [email protected]

T HOMAS A. G ASIEWICZ University of Rochester Medical Center Rocheser, NY USA [email protected]

B ERNARD G ALLEZ Biomedical Magnetic Resonance Université Catholique de Louvain Brussels Belgium [email protected] B RENDA L. G ALLIE Ontario Cancer Institute/Princess Margaret Hospital Toronto, ON Canada [email protected]

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PATRIZIA G ASPARINI Molecular-Cytogenetic Unit, Department of Experimental Oncology Istituto Nazionale Tumori Milano Italy [email protected] G RÉGORY G ATOUILLAT Laboratory of Biochemistry IFR53, Faculty of Pharmacy Reims cedex, France [email protected]

P ING G AO Division of Hematology, Department of Medicine Johns Hopkins University School of Medicine Baltimore, MD USA [email protected]

A DI F. G AZDAR Hamon Center for therapeutic Oncology Research and Departments of Pathology, Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas USA [email protected]

R OY G ARCIA City of Hope National Medical Center and Beckman Research Institute Duarte, CA USA [email protected]

C HRISTIAN G EISLER The Leukemia and Lymphoma Marker Laboratory Department of Hematology, The Finsen Centre Rigshospitalet, Copenhagen Denmark [email protected]

L AWRENCE B. G ARDNER The NYU Cancer Institute New York University School of Medicine New York, NY USA [email protected]

S PYROS D. G EORGATOS Department of Basic Sciences The University of Crete, School of Medicine Heraklion, Crete Greece [email protected]

C ATHIE G ARNIS MIT Center for Cancer Research Cambridge, MA USA [email protected]

J ULIA M. G EORGE Department of Molecular and Integrative Physiology University of Illinois at Urbana-Champaign IL, USA [email protected]

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A RMIN G ERGER Department of Internal Medicine, Division of Oncology Medical University Graz Graz Austria [email protected]

M ICHAEL K. G IBSON University of Pittsburgh Cancer Institute Pittsburgh, PA USA [email protected]

U LRICH G ERMING Klinik für Hämatologie Onkologie und Klinische Immunologie Heinrich-Heine-Universität Düsseldorf Germany [email protected]

M ICHAEL Z. G ILCREASE Department of Pathology, Breast Section M. D. Anderson Cancer Center Houston, TX USA [email protected]

J EFFREY E. G ERSHENWALD Department of Surgical Oncology The University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected]

M.B. G ILLESPIE Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA

A NDREAS J. G ESCHER Cancer Biomarkers and Prevention Group Department of Cancer Studies, University of Leicester Leicester UK [email protected]

F RANÇOIS N OËL G ILLY Department of Digestive Oncologic Surgery Hospices Civils de Lyon–Université Lyon 1 Lyon France [email protected]

C HRISTIAN G ESPACH INSERM U. 673, Paris, France; Laboratory of Molecular and Clinical Oncology of Solid tumors, Faculté de Médecine Université Pierre et Marie Curie-Paris 6 Paris France [email protected]

T HOMAS D. G ILMORE Biology Department, Boston University Boston, MA USA [email protected]

B. M ICHAEL G HADIMI Department of General and Visceral Surgery University Medical Center Göttingen Germany [email protected]

O LIVER G IMM Department of Surgery University Hospital Linköping Sweden [email protected]

R ICCARDO G HIDONI Laboratory of Biochemistry and Molecular Biology San Paolo Medical School University of Milan Milan Italy [email protected] R ONALD A. G HOSSEIN Department of Pathology Memorial Sloan-Kettering Cancer Center New York, NY USA [email protected] L ORENZO G IANNI Department of Oncology Infermi Hospital Rimini Italy [email protected]

L UTZ G ISSMANN DKFZ, Heidelberg Germany [email protected] M ORTEN F. G JERSTORFF Department of Oncology Odense University Hospital Odense C Denmark [email protected] S HANNON S. G LASER Departments of Medicine and Research and Education Scott & White Hospital and Texas A&M University System Health Science Center Temple, TX USA [email protected]


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H ANSRUEDI G LATT German Institute of Human Nutrition (DIfE) Potsdam-Rehbruecke Nuthetal Germany [email protected]

S USANNE M. G OLLIN Department of Human Genetics University of Pittsburgh Graduate School of Public Health Pittsburgh, PA USA [email protected]

O LIVIER G LEHEN Department of Digestive Oncologic Surgery Hospices Civils de Lyon–Université Lyon 1 Lyon France [email protected]

R OY M. G OLSTEYN Senior Scientist, Cancer Research Division Institut de Recherches Servier Croissy-Sur-Seine France [email protected]

A LEKSANDRA G LOGOWSKA Department of Human Anatomy and Cell Science University of Manitoba Winnipeg, MB Canada

E LLEN L. G OODE Mayo Clinic College of Medicine Rochester, MN USA [email protected]

T HOMAS W. G LOVER Department of Human Genetics University of Michigan Ann Arbor, MI USA [email protected]

G REGORY J. G ORES Miles and Shirley Fiterman Center for Digestive Diseases Division of Gastroenterology and Hepatology Mayo Clinic College of Medicine Rochester, MN USA [email protected]

U LRICH G ÖBEL Clinic of Pediatric Oncology Hematology and Immunology Heinrich-Heine-University Düsseldorf Düsseldorf Germany [email protected] J. G ODDARD Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA [email protected] A NDREW K. G ODWIN Department of Medical Oncology Fox Chase Cancer Center Philadelphia, PA USA [email protected] G ARY S. G OLDBERG Molecular Biology University of Medicine and Dentistry of New Jersey Stratford, NJ USA [email protected] I TZHAK D. G OLDBERG Long Island Jewish Medical Center Albert Einstein College of Medicine Bronx, NY USA

T OBIAS G ÖRGE Department of Dermatology University of Münster Münster Germany [email protected] N ORIKO G OTOH Institute of Medical Sciences The University of Tokyo Minatoku, Tokyo Japan [email protected] S TEPHANIE G OUT Le Centre de recherche en cancérologie de l’Université Laval Québec, QC Canada [email protected] A MMI G RAHN Department of Clinical Chemistry and Transfusion Medicin Institute of Biomedicine Sahlgrenska academy at Göteborg University Göteborg Sweden [email protected] P ETER G REAVES Department of Cancer Studies and Molecular Medicine University of Leicester Leicester UK [email protected]

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M ARK I. G REENE Department of Pathology and Laboratory Medicine & Abramson Cancer Center University of Pennsylvania Philadelphia, PA USA [email protected] W. M. U. VAN G REVENSTEIN Department of Surgery Erasmus MC Rotterdam The Netherlands [email protected]

VALENTINA G UARNERI Department of Oncology and Hematology University of Modena and Reggio Emilia, Policlinico via del Pozzo, Modena Italy [email protected] W EI G U Institute for Cancer Genetics, and Department of Pathology College of Physicians and Surgeons, Columbia University New York, NY USA [email protected]

A RJAN W. G RIFFIOEN Angiogenesis Laboratory, Department of Pathology Maastricht University Maastricht The Netherlands [email protected]

L ILIANA G UEDEZ Extracellular Matrix Pathology Section, Cell and Cancer Biology Branch National Cancer Institute Bethesda, MD USA [email protected]

D IRK G RIMM University of Heidelberg, Cluster of Excellence Cell Networks BIOQUANT Heidelberg, Germany [email protected]

F. P ETER G UENGERICH Department of Biochemistry and Center in Molecular Toxicology Vanderbilt University School of Medicin Nashville, TN USA [email protected]

M ATTHEW J. G RIMSHAW Breast Cancer Biology Group, King’s College London School of Medicine, Guy’s Hospital London UK [email protected] S TEPHEN R. G ROBMYER Department of Surgery, Division of Surgical Oncology University of Florida Gainesville, FL USA [email protected]

A BHIJIT G UHA Division of Neurosurgery University of Toronto ON Canada [email protected] K ATHERINE A. G UINDON Department of Pharmacology and Toxicology Queen’s University Kingston, ONT Canada [email protected]

B ERND G ROSCHE Department of Radiation Protection and Health Bundesamt für Strahlenschutz (Federal Office for Radiation Protection) Oberschleissheim Germany [email protected]

E RICH G ULBINS Department of Molecular Biology University of Duisburg-Essen Essen Germany [email protected]

H ANS H. G RUNICKE Biocenter, Division of Medical Biochemistry Medical University of Innsbruck Innsbruck Austria [email protected]

B ILL G ULLICK Department of Biosciences University of Kent at Canterbury Canterbury, Kent UK [email protected]

J ULIANA G UARIZE Department of Thoracic Surgery European Institute of Oncology Milan Italy [email protected]

U RSULA G ÜNTHERT Department of Clinical and Biological Sciences Institute for Medical Microbiology, University of Basel Basel Switzerland [email protected]


J AMES F. G USELLA Molecular Neurogenetics Unit Massachusetts General Hospital Charlestown, MA USA [email protected] G RAEME R. G UY Signal Transduction Laboratory Institute of Molecular and Cell Biology Singapore [email protected] M ANUEL G UZMÁN Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid Spain [email protected] G EUM -Y OUN G WAK Department of Medicine Samsung Medical Center Sungkyunkwan University School of Medicine Gangnam-gu, Seoul South Korea [email protected] C LAUDIA H AFERLACH MLL Munich Leukemia Laboratory Munich Germany [email protected] T ORSTEN H AFERLACH MLL Munich Leukemia Laboratory Munich Germany [email protected] S TEPHAN A. H AHN University of Bochum Bochum Germany [email protected] J ÖRG H AIER Department of General Surgery University Hospital Münster Münster Germany [email protected] N UMSEN H AIL Department of Pharmaceutical Sciences The University of Colorado at Denver and Health Sciences Center Denver, CO USA [email protected]

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P IERRE H AINAUT Group of Molecular Carcinogenesis and Biomarkers International Agency for Research on Cancer World Health Organization Lyon cedex 08 France [email protected] B RETT M. H ALL Department of Pediatrics Columbus Children’s Research Institute The Ohio State University Columbus, OH USA [email protected] J ANET H ALL INSERM U612, Institut Curie-Recherche Orsay France [email protected] J OYCE L. H AMLIN Department of Biochemistry and Molecular Genetics University of Virginia School of Medicine Charlottesville, VA USA [email protected] R ASHA S. H AMOUDA National Institutes of Health Bethesda, MD USA, [email protected] J. W ILLIAM H ARBOUR Department of Ophthalmology and Visual Sciences Washington University School of Medicine St. Louis, MO USA [email protected] M ARK H ARLAND Section of Epidemiology and Biostatistics Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St. James’s University Hospital Leeds UK [email protected] A DRIAN L. H ARRIS University of Oxford, Cancer Research UK Weatherall Institute of Molecular Medicine, John Radcliffe Hospital Headington, Oxford UK [email protected] U ZMA H ASAN Infections and Cancer Biology Group International Agency for Research on Cancer Lyon France [email protected]

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M IA H ASHIBE Gene-Environment Epidemiology Group International Agency for Research on Cancer Lyon France [email protected]

O LAF H EIDENREICH Northern Institute for Cancer Research Newcastle University Newcastle upon Tyne UK [email protected]

M ASAHARU H ATA Department of Radiology Yokohama City University, Graduate School of Medicine Yokohama, Kanagawa Japan [email protected]

D AVID G. H EIDT Department of Surgery University of Michigan Medical Center Ann Arbor, MI USA

J OHN . D. H AYES Biomedical Research Centre, Ninewells Hospital and Medical School University of Dundee Dundee, Scotland UK [email protected] L ILI H E Molecular Oncology and Pathology H. Lee Moffitt Cancer Center & Research Institute Tampa, FL USA [email protected] L I -Z HEN H E Memorial Sloan-Kettering Cancer Center Weill Cornell Graduate School of Medical Sciences NY USA [email protected]

W ERNER H ELD Ludwig Institute for Cancer Research Lausanne Branch, and University of Lausanne Epalinges Switzerland [email protected] C ARL -H ENRIK H ELDIN Ludwig Institute for Cancer Research Uppsala Sweden [email protected] W IJNAND H ELFRICH Groningen University Institute for Drug Exploration (GUIDE), Department of Pathology & Laboratory Medicine, Section Medical Biology, Laboratory for Tumor Immunology Groningen The Netherlands [email protected]

R UTH H E Georgetown University Lombardi Comprehensive Cancer Center Washington, DC USA [email protected]

D EBBY H ELLEBREKERS Department of Pathology Research Institute for Growth and Development (GROW) Maastricht University Hospital Maastricht The Netherlands [email protected]

S TEPHEN S. H ECHT The Cancer Center University of Minnesota Minneapolis, MN USA [email protected]

PAUL W. S. H ENG Department of Pharmacy National University of Singapore Singapore Singapore [email protected]

A HMED E. H EGAB Department of Geriatric and Respiratory Medicine Tohoku University Hospital Sendai Japan [email protected]

K AI -O LIVER H ENRICH DKFZ, German Cancer Research Center Heidelberg Germany [email protected]

A XEL H EIDENREICH Division of Oncological Urology, Department of Urology University of Köln Köln Germany [email protected]

M ARIE H ENRIKSSON Department of Microbiology Tumor and Cell Biology (MTC) Karolinska Institutet Stockholm Sweden [email protected]


E LIZABETH P ETRI H ENSKE Fox Chase Cancer Center Philadelphia, PA USA [email protected] D ONALD E. H ENSON The George Washington University Cancer Institute Washington, DC USA [email protected] M EENHARD H ERLYN The Wistar Institute Philadelphia, PA USA [email protected] B LANCA H ERNANDEZ -L EDESMA Dept of Nutritional Sciences and Toxicology University of California Berkeley, CA USA [email protected] W OLFGANG H ERR Department of Medicine III, Hematology and Oncology Johannes Gutenberg-University of Mainz Mainz Germany [email protected] H ELEN E. H ESLOP Center for Cell and Gene Therapy, Baylor College of Medicine Texas Children’s Hospital, and The Methodist Hospital Houston, TX USA [email protected] J OCHEN H ESS Deutsches Krebsforschungszentrum Division of Signal Transduction and Growth Control Heidelberg Germany [email protected] M ARRY M. VAN DEN H EUVEL -E IBRINK Department of Pediatric Oncology/Hematology Erasmus MC-Sophia Children’s Hospital Rotterdam The Netherlands [email protected] D OMINIQUE H EYMANN Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives University of Nantes Nantes France [email protected]

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M ARTHA H ICKEY Department of Gynaecology, School of Women’s and Infants’ Health WIRF and University of Western Australia Crawley, WA Australia [email protected] J AMES H ICKS Cold Spring Harbor Laboratory Cold Spring Harbor NY USA [email protected] K EVIN O. H ICKS Auckland Cancer Society Research Centre The University of Auckland Auckland New Zealand [email protected] I AN D. H ICKSON ICRF, Institute of Molecular Medicine University of Oxford John Radcliffe Hospital, Oxford UK [email protected] C OLIN K. H ILL Department of Radiation Oncology USC Keck School of Medicine Los Angeles, CA USA B OAZ H IRSHBERG Cardiovascular and Metabolic Diseases, Pfizer Inc Groton, CT USA [email protected] A RI H IRVONEN Finnish Institute of Occupational Health Helsinki Finland [email protected] YASUYUKI H ITOSHI Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center Dartmouth Medical School Hanover, NH USA [email protected] R ICARDO H ITT Medical Oncology Service University Hospital 12 de Octubre Madrid Spain [email protected]

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E ISO H IYAMA Natural Science Center for Basic Research and Development Department of Pediatric Surgery, Hiroshima University Hospital Hiroshima University Hiroshima Japan [email protected] FALK H LUBEK Department of Pathology Ludwig-Maximilians-University of Munich Munich Germany [email protected] S TEVEN N. H OCHWALD Departments of Surgery Departments of Molecular Genetics and Microbiology University of Florida College of Medicine Gainesville, FL USA [email protected] M ICHAEL H ODSDON Department of Laboratory Medicine Yale University School of Medicine New Haven, CT USA [email protected] K ASPER H OEBE Department of Immunology The Scripps Research Institute San Diego, CA USA [email protected] L ORNE J. H OFSETH Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy University of South Carolina Columbia, SC USA [email protected] PANCRAS C. W. H OGENDOORN Department of Pathology Leiden University Medical Center Leiden The Netherlands [email protected] S TEFAN H OLDENRIEDER Institute of Clinical Chemistry University Hospital of Munich Ludwig-Maximilians-University Munich Germany [email protected]

P ETRA D EN H OLLANDER Molecular and Cellular Oncology The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected] C AROLINE L. H OLLOWAY BC Cancer Agency Centre for the Southern Interior Kelowna, BC Canada [email protected] A RNE H OLMGREN Department of Medical Biochemistry and Biophysics Karolinska Institutet Stockholm Sweden [email protected] JUN HYUK HONG The Cancer Institute of NJ, Robert Wood Johnson Medical School Division of Urologic Oncology New Brunswick, NJ USA C HRISTINE H ORAK Women’s Cancers Section, Laboratory of Molecular Pharmacology, Center for Cancer Research National Cancer Institute Bethesda, MD USA [email protected] A DÍLIA H ORMIGO Department of Neurology Memorial Sloan-Kettering Cancer Center New York, NY USA [email protected] J. H ORNIG Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA M ICHAEL R. H ORSMAN Department of Experimental Clinical Oncology Aarhus University Hospital Aarhus Denmark [email protected] A NDREA K RISTINA H ORST Institute of Clinical Chemistry University Medical Center Hamburg Eppendorf Hamburg Germany [email protected]


D AVID W. H OSKIN Departments of Pathology, and Microbiology and Immunology Dalhousie University Halifax, NS Canada [email protected]

M AUREEN B. H UHMANN Department of Primary Care, School of Health Related Professions, University of Medicine and Dentistry of New Jersey The Cancer Institute of New Jersey New Brunswick, NJ USA [email protected]

A NDREAS F. H OTTINGER Department of Neurology Memorial Sloan-Kettering Cancer Center New York, NY USA [email protected]

W EN -C HUN H UNG Institute of Biomedical Sciences National Sun Yat-Sen University Kaohsiung, Taiwan Republic of China [email protected]

P ETER J. H OUGHTON Department of Molecular Pharmacology St. Jude Children’s Research Hospital Memphis, TN USA [email protected]

K ENT H UNTER Laboratory of Population Genetics, CCR/NCI/NIH Bethesda, MD USA [email protected]

A NTHONY H OWELL CRUK Department of Medical Oncology University of Manchester, Christie Hospital NHS Trust Manchester UK [email protected] S HIE -L IANG H SIEH Department of Microbiology and Immunology National Yang-Ming University Immunology Research Center, Taipei Veterans General Hospital Genomics Research Center Academia Sinica, Taipei Taiwan [email protected] C HENG -L ONG H UANG Department of Second Surgery Kagawa University Kagawa Japan [email protected] K AY H UEBNER Department of Molecular Virology Immunology and Medical Genetics, Ohio State University Comprehensive Cancer Center Columbus, OH USA [email protected] P ERE H UGUET Department of Pathology Vall d’Hebron University Hospital Barcelona, Spain [email protected]

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T ONY H UNTER Salk Institute Molecular and Cell Biology Laboratory La Jolla, CA USA [email protected] T EH -I A H UO Institute of Pharmacology, School of Medicine National Yang-Ming University Taipei, TAIWAN and Department of Medicine Taipei Veterans General Hospital Taipei, TAIWAN People’s Rebublic of China [email protected] J ACQUES H UOT Le Centre de recherche en cancérologie de l’Université Laval Québec, QC Canada [email protected] D OUGLAS R. H URST Department of Pathology and Comprehensive Cancer Center University of Alabama at Birmingham Birmingham, AL USA [email protected] K AREN L. H UYCK Department of Pathology Brigham and Women’s Hospital Boston, MA USA [email protected] S AM T. H WANG Dermatology Branch National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected]

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B RANDY D. H YNDMAN Department of Pathology and Molecular Medicine Queen’s University Cancer Research Institute Queen’s University Kingston, ON Canada [email protected] TAKAFUMI I CHIDA Department of Hepatology and Gastroenterology Juntendo University School of Medicine, Shizuoka Hospital Shizuoka Japan [email protected] Y OSHITO I HARA Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute Nagasaki University Graduate School of Biomedical Sciences Nagasaki Japan [email protected] H ITOSHI I KEDA Department of Pediatric Surgery Dokkyo Medical University Koshigaya Hospital Koshigaya, Saitama Japan [email protected] K AZUHIKO I NO Department of Obstetrics and Gynecology Nagoya University Graduate School of Medicine Nagoya Japan [email protected] J UAN I OVANNA INSERM, Stress Cellulaire, Parc Scientifique et Technologique de Luminy Marseille Cedex France [email protected] I RMGARD I RMINGER -F INGER Molecular Gynecology and Obstetrics Laboratory, Department of Gynecology and Obstetrics Geneva University Hospitals Geneva, Switzerland [email protected]

T OSHIHISA I SHIKAWA Department of Biomolecular Engineering Graduate School of Bioscience and Biotechnology Tokyo Institute of Technology Meguro-ku Tokyo [email protected] M ARK A. I SRAEL Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center Dartmouth Medical School Hanover, NH USA [email protected] A NTOINE I TALIANO Laboratory of Solid Tumors Genetics Nice University Hospital and CNRS UMR 6543, Faculty of Medicine Nice France [email protected] N ORIMASA I TO University of Pittsburgh, Departments of Surgery and Bioengineering Pittsburgh, PA USA [email protected] M ICHAEL I TTMANN Department of Pathology Baylor College of Medicine Houston, TX USA [email protected] R ICHARD I VELL Head of School of Molecular and Biomedical Sciences The University of Adelaide SA Australia [email protected]

M EREDITH S. I RWIN Cell Biology Program and Division of Hematology-Oncology Hospital for Sick Children University of Toronto Toronto, ON Canada [email protected]

N OBU I WAKUMA Department of Surgery, Division of Surgical Oncology University of Florida Gainesville, FL USA [email protected]

P LO I SABELLE INSERM U790, Hématopoièse et cellules souches Institut Gustave Roussy–PR1 Villejuif France [email protected]

S HAI I ZRAELI Pediatric Hemato-Oncology Sheba Medical Center and Tel Aviv University Ramat Gan Israel [email protected]


PAOLA I ZZO Dipartimento di Biochimica e Biotechnologie Medicine a Chirurgia, Facoltà di Medicina Università di Napoli Federico II Napoli Italy [email protected] M ARK J ACKMAN Wellcome/CRC Institute Cambridge UK [email protected]

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G ORDON C. J AYSON Cancer Research UK Department of Medical Oncology Christie Hospital Manchester UK [email protected] K UAN -T EH J EANG National Institute of Allergy and Infectious Disease, NIH Bethesda, MD USA [email protected]

A LAN J ACKSON Imaging Science University of Manchester Manchester UK [email protected]

J IIANG -H UEI J ENG Laboratory of Pharmacology and Toxicology, School of Dentistry National Taiwan University Hospital and National Taiwan University Medical College Taipei Taiwan [email protected]

D EBORAH J ACKSON -B ERNITSAS Department of Systems Biology, The University of Texas M.D. Anderson Cancer Center Houston, TX USA [email protected]

E LWOOD V. J ENSEN National Institute of Health Bethesda, MD USA [email protected]

U LRICH J AEHDE Institute of Pharmacy University of Bonn Bonn Germany [email protected]

L IN J I Department of Thoracic & Cardiovascular Surgery The University of Texas M.D. Anderson Cancer Center Houston, TX USA [email protected]

D AVID J AMIESON School of Clinical and Laboratory Sciences Newcastle University Newcastle upon Tyne UK [email protected]

Y UFEI J IANG Cancer Vaccine Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected]

P IDDER J ANSEN -D ÜRR Institute for Biomedical Ageing Research Austrian Academy of Sciences Innsbruck Austria [email protected]

C HARLOTTE J IN Departments of Clinical Genetics University Hospital Lund, Sweden [email protected]

S IEGFRIED J ANZ Department of Pathology University of Iowa, Carver College of Medicine Iowa City, IA USA [email protected] D ANIEL G. J AY Tufts University School of Medicine Boston, Ma USA [email protected]

A NDREW K. J OE Department of Medicine Herbert Irving Comprehensive Cancer Center New York, NY USA [email protected] A LAN L. J OHNSON Walther Cancer Research Center University of Notre Dame Notre Dame, IN USA [email protected]

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S ARA M. J OHNSON Department of Surgery University of Texas Medical Branch Galveston, TX USA [email protected] F LORIS A ART DE J ONG Department of Medical Oncology Erasmus University Medical Center Rotterdam Rotterdam The Netherlands [email protected] W ON -A J OO The Wistar Institute Philadelphia, PA USA [email protected] V. C RAIG J ORDAN Fox Chase Cancer Center Philadelphia, PA USA [email protected] S ERENE J OSIAH Shire Pharmaceuticals Cambridge, MA USA [email protected] R ICHARD J OVE City of Hope National Medical Center and Beckman Research Institute Duarte, CA USA [email protected] J AROSLAW J OZWIAK Department of Histology and Embryology Medical University of Warsaw Warsaw, Poland [email protected] J ESPER J URLANDER Department of Hematology The Finsen Centre Rigshospitalet, Copenhagen Denmark [email protected]

TADAO K AKIZOE National Cancer Center Tokyo Japan [email protected] T UULA K ALLUNKI Apoptosis Department Institute of Biological Cancer Research Danish Cancer Society Copenhagen Denmark [email protected] TAKEHIKO K AMIJO Division of Biochemistry Chiba Cancer Center Research Institute Chuoh-ku, Chiba Japan [email protected] YASUFUMI K ANEDA Department of Gene Therapy Science Osaka University Graduate School of Medicine Suita, Osaka Japan [email protected] K AZUHIRO K ANEKO Second Department of Internal Medicine Showa University School of Medicine Tokyo Japan [email protected] I NKYUNG K ANG Department of Surgery University of California San Francisco, CA USA [email protected] D AVID E. K APLAN Division of Gastroenterology University of Pennsylvania Philadelphia, PA USA [email protected]

C HAIM K AHANA Department of Molecular Genetics Weizmann Institute of Science Rehovot Israel [email protected]

M ICHALIS V. K ARAMOUZIS Department of Biological Chemistry Medical School University of Athens Goudi, Athens Greece [email protected]

B ERND K AINA Department of Toxicology University of Mainz Mainz Germany [email protected]

A DAM R. K ARPF Department of Pharmacology and Therapeutics Roswell Park Cancer Institute Buffalo, NY USA [email protected]


N ILESH D. K ASHIKAR Departments of Surgery and Cancer Biology Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine Nashville, TN USA [email protected] J UHAYNA K ASSEM Division of Pulmonary and Critical Care Medicine Weill Medical College of Cornell University New York, NY USA [email protected]

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S TANLEY B. K AYE Section of Medicine Institute of Cancer Research, The Royal Marsden Hospital London UK [email protected] R ICHARD K EFFORD Westmead Institute for Cancer Research and Sydney Melanoma Unit, University of Sydney Westmead, NSW Australia [email protected]

M ATILDA K ATAN CRC Centre for Cell and Molecular Biology Institute of Cancer Research London UK [email protected]

E VAN T. K ELLER Departments of Urology and Pathology University of Michigan Ann Arbor, MI USA [email protected]

S ASSER A. K ATE Department of Pediatrics Columbus Children’s Research Institute, The Ohio State University Columbus, OH USA [email protected]

S TEPHEN K ENNEDY Nuffield Department of Obstetrics and Gynaecology Oxford University John Radcliffe Hospital, Oxford UK [email protected]

W ILLIAM K. K AUFMANN Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill Chapel Hill, NC USA [email protected]

D ANIEL K EPPLER Department of Cell Biology & Anatomy and Feist-Weiller Cancer Center LSUHSC-School of Medicine Shreveport, LA USA [email protected]

M ANJINDER K AUR Department of Pharmaceutical Sciences School of Pharmacy University of Colorado Health Sciences Center Denver, CO USA [email protected]

W OLFGANG K ERN MLL Munich Leukemia Laboratory Munich Germany [email protected]

I NGO K AUSCH Department of Urology University of Luebeck, Medical School Luebeck Germany [email protected] K OJI K AWAKAMI Graduate School of Medicine and Public Health Kyoto University Kyoto Japan [email protected] F REDERIC J. K AYE National Cancer Institute NIH and National Naval Medical Center Bethesda, MD USA [email protected]

J ORMA K ESKI -O JA Departments of Pathology and of Virology Haartman Institute, University of Helsinki Helsinki, Finland [email protected] A D G EURTS VAN K ESSEL Department of Human Genetics Radboud University Nijmegen Medical Centre Nijmegen The Netherlands [email protected] K HANDAN K EYOMARSI Department of Experimental Radiation Oncology Department of Surgical Oncology Unit 66, University of Texas, MD Anderson Cancer Center Houston, TX USA [email protected]

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C HAND K HANNA National Cancer Institute, Center for Cancer Research Comparative Oncology Program Bethesda, MD USA [email protected]

S EONG J IN K IM Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute Bethesda, MD USA [email protected]

S AMIR K HLEIF Cancer Vaccine Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected]

S U Y OUNG K IM Pediatric Oncology Branch, Center for Cancer Research National Cancer Institute Bethesda, MD USA [email protected],gov

R OYA K HOSRAVI -FAR Department of Pathology Harvard Medical School, Beth Israel Deaconess Medical Center Boston, MA USA [email protected] T OBIAS K IESSLICH Department of Molecular Biology University of Salzburg Hellbrunnerstrasse 34, 5020 Salzburg Austria [email protected] F UMITAKA K IKKAWA Department of Obstetrics and Gynecology Nagoya University Graduate School of Medicine Nagoya Japan [email protected] N ERBIL K ILIC Department of Hematology and Oncology University Hospital Hamburg-Eppendorf Germany [email protected] I SAAC Y I K IM The Cancer Institute of NJ, Robert Wood Johnson Medical School Division of Urologic Oncology New Brunswick, NJ USA [email protected] J UNG - WHAN K IM Division of Hematology, Department of Medicine Johns Hopkins University School of Medicine Baltimore, MD USA [email protected] M OONIL K IM BioNanotechnology Research Center Korea Research Institute of Bioscience and Biotechnology Yuseong, Daejeon Republic of Korea [email protected]

A DI K IMCHI Department of Molecular Genetics Weizmann Institute of Science Rehovot Israel [email protected] A. D OUGLAS K INGHORN College of Pharmacy, The Ohio State University Columbus, OH USA [email protected] D AVID K IRN Jennerex Biotherapeutics Inc. San Francisco, CA USA [email protected] Y OULIA M. K IROVA Department of Radiation Oncology Institut Curie Paris France [email protected] S HINICHI K ITADA Burnham Institute for Medical Research La Jolla, CA USA [email protected] K AREL K ITHIER Department of Pathology Wayne State University School of Medicine Detroit, MI USA [email protected] C ELINA G. K LEER Department of Pathology and Comprehensive Cancer Center University of Michigan Medical School MI USA [email protected] G EORGE K LEIN Microbiology, Tumor and Cell Biology Karolinska Institute Stockholm Sweden [email protected]


M ICHAEL J. K LEIN Center for Metabolic Bone Disease The University of Alabama at Birmingham Birmingham, AL USA [email protected]

L IN K ONG Department of Radiation Oncology Cancer Hospital of Fudan University Shanghai China [email protected]

E LENA K LENOVA Department of Biological Sciences University of Essex Colchester Essex, UK [email protected]

R OLAND E. K ONTERMANN Institute of Cell Biology and Immunology University of Stuttgart Germany [email protected]

T HOMAS K LONISCH Department of Human Anatomy and Cell Science University of Manitoba Winnipeg, MB Canada [email protected] E LIZABETH K NOBLER Department of Dermatology Columbia College of Physicians and Surgeons New York, NY USA [email protected] R OBERT K NOBLER Department of Dermatology Medical University of Vienna Vienna Austria [email protected] B EATRICE K NUDSEN Divisions of Public Health Sciences, Human Biology and Clinical Sciences Fred Hutchinson Cancer Research Center Seattle, WA USA [email protected] S TEFAN K OCHANEK Division of Gene Therapy University of Ulm Cologne Germany [email protected]

J ANKO K OS Department of Pharmaceutical Biology University of Ljubljana Ljubljana Slovenia [email protected] M ARTA K OSTROUCHOVA Laboratory of Molecular Biology and Genetics Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University Prague Czech Republica [email protected] H EINRICH K OVAR Children’s Cancer Research Institute St. Anna Kinderkrebsforschung, Vienna Austria [email protected] C RAIG K OVITZ Department of Medical Oncology University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected] B ARNETT S. K RAMER Office of Disease Prevention National Institutes of Health Bethesda, MD USA [email protected]

C HRISTIAN K OLLMANNSBERGER Division of Medical Oncology, British Columbia Cancer Agency, Vancouver Cancer Centre University of British Columbia Vancouver, BC Canada [email protected]

B ARBARA K RAMMER Department of Molecular Biology University of Salzburg Salzburg Austria [email protected]

Y UTAKA K ONDO Division of Molecular Oncology Aichi Cancer Center Research Institute Nagoya Japan [email protected]

H ENK J. VAN K RANEN National Institute of Public Health and Environment Bilthoven The Netherlands [email protected]

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T HOMAS K RAUSZ Department of Pathology University of Chicago Chicago, IL USA [email protected]

D EEPAK K UMAR Department of Biological and Environmental Sciences University of the District of Columbia Washington, DC USA [email protected]

J ÜRGEN K RAUTER Department of Haematology Haemostasis and Oncology Hannover Medical School Hannover Germany [email protected]

H IROKI K UNIYASU Department of Molecular Pathology Nara Medical University School of Medicine Kashihara, Nara Japan [email protected]

B ERNHARD K REMENS Department of Pediatric Hematology Oncology and Respiratory Medicine, University Hospitals of Essen Essen Germany [email protected] A RUNA K RISHNAN Division of Endocrinology Department of Medicine, Stanford University School of Medicine Stanford, CA USA [email protected] B IN B. R. K ROON Department of Surgery The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Amsterdam The Netherlands [email protected] YASUSEI K UDO Department of Oral and Maxillofacial Pathobiology Division of Frontier Medical Science Graduate School of Biomedical Sciences Hiroshima University Hiroshima Japan [email protected] R AKESH K UMAR Molecular and Cellular Oncology The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected] PARVESH K UMAR Department of Radiation Oncology USC Keck School of Medicine Los Angeles, CA USA [email protected]

S IAVASH K. K URDISTANI Department of Biological Chemistry David Geffen School of Medicine at UCLA Los Angeles, CA USA [email protected] R ALF K ÜPPERS Institute for Cell Biology (Tumor Research) University of Duisburg-Essen, Medical School Essen Germany [email protected] E LENA K URENOVA Departments of Surgery University of Florida College of Medicine Gainesville, FL USA [email protected] K EISUKE K UROSE Departments of Obstetrics and Gynecology Nippon Medical School Kawasaki and Tokyo Japan PAULA M. K UZONTKOSKI Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center Dartmouth Medical School Hanover, NH USA [email protected] R OBERT M. K YPTA Cell Biology and Stem Cells Unit CIC bioGUNE, Derio, Bilbao, Spain; Imperial College London London UK [email protected] J UAN C ARLOS L ACAL Instituto de Investigaciones Biomedicas CSIC, Madrid Spain [email protected]


J AMES C. L ACEFIELD Departments of Electrical and Computer Engineering and Medical Biophysics University of Western Ontario London, ONT Canada [email protected] S TEPHAN L ADISCH Children’s Research Institute, Children’s National Medical Center, The George Washington University School of Medicine Washington, DC USA [email protected] H ERMANN L AGE Charité Campus Mitte Institute of Pathology Berlin Germany [email protected]

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J OSEPH R. L ANDOLPH Departments of Molecular Microbiology and Immunology, and Pathology; USC/Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Health Sciences Campus University of Southern California Los Angeles, CA USA [email protected] R OBERT L ANGER Department of Chemical Engineering and Center for Cancer Research Massachusetts Institute of Technology Cambridge, MA USA [email protected] PAOLA L ARGHI Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected]

C HARLES P. K. L AI Department of Cellular and Physiological Sciences The University of British Columbia Vancouver, BC Canada [email protected]

J AMES M. L ARNER Department of Therapeutic Radiology and Oncology University of Virginia School of Medicine Charlottesville, VA USA [email protected]

D ALE W. L AIRD Department of Anatomy and Cell Biology University of Western Ontario London, ON Canada [email protected]

G ÖRAN L ARSON Department of Clinical Chemistry and Transfusion Medicine Institute of Biomedicine Sahlgrenska Academy at Göteborg University Göteborg Sweden [email protected]

J ANICE B. B. L AM Department of Medicine and Genome Research Center University of Hong Kong Hong Kong, China [email protected]

S USANNA C. L ARSSON Division of Nutritional Epidemiology, Institute of Environmental Medicine Karolinska Institutet Stockholm Sweden [email protected]

WAN L. L AM Department of Cancer Genetics and Developmental Biology British Columbia Cancer Research Centre Vancouver, BC Canada [email protected] H UI Y. L AN Department of Medicine The University of Hong Kong Li Ka Shing Faculty of Medicine Hong Kong China [email protected]

P HILIPPE L ASSALLE INSERM U774 Institut Pasteur de Lille Lille France [email protected] FARIDA L ATIF Department of Medical and Molecular Genetics, Institute of Biomedical Research University of Birmingham Birmingham UK [email protected]

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V IRPI L AUNONEN Department of Medical Genetics, Biomedicum Helsinki University of Helsinki Helsinki Finland [email protected]

W ILLIAM P. J. L EENDERS Department of Pathology Radboud University Nijmegen Medical Center Nijmegen The Netherlands [email protected]

V INCENZO DE L AURENZI Department of Experimental Medicine and Biochemical Sciences University of Tor Vergata Rome Italy [email protected]

A NDREAS L EIBBRANDT Institute of Molecular Biotechnology of the Austrian Academy of Sciences Vienna Austria [email protected]

G WENDAL L AZENNEC INSERM Montpellier France [email protected]

M ANUEL C. L EMOS Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Centre for Diabetes Endocrinology and Metabolism (OCDEM), Churchill Hospital Headington, Oxford UK [email protected]

G AIL S. L EBOVIC Director of Women’s Services The Cooper Clinic Dallas, TX USA [email protected] D AVID P. L E B RUN Department of Pathology and Molecular Medicine Queen’s University Cancer Research Institute, Queen’s University Kingston, ON Canada [email protected] C HEONG J. L EE Department of Surgery University of Michigan Medical Center Ann Arbor, MI USA J ONG -H EUN L EE Women’s Cancers Section, Laboratory of Molecular Pharmacology, Center for Cancer Research National Cancer Institute Bethesda, MD USA [email protected]

E RIC J. L ENTSCH Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA [email protected] Y UN -C HUNG L EUNG Lo Ka Chung Centre for Natural Anti-cancer Drug Development and Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong China [email protected] F RANCIS L ÉVI INSERM, Rythmes Biologiques et Cancers Hospital Paul Brousse, Villejuif Cedex France Université Paris Sud XI Orsay France [email protected]

S EAN B ONG L EE Genetics of Development and Disease Branch, National Institute of Diabetes & Digestive & Kidney Diseases National Institutes of Health Bethesda, MD USA [email protected]

J AY A. L EVY University of California, School of Medicine San Francisco, CA USA [email protected]

S TEPHEN L EE Department of Cellular and Molecular Medicine Faculty of Medicine, University of Ottawa Ottawa, ONT Canada [email protected]

K AIYI L I Department of Surgery Baylor College of Medicine Houston, TX USA [email protected]


D AIQING L IAO Department of Anatomy and Cell Biology, Shands Cancer Center University of Florida College of Medicine Gainesville, FL USA [email protected] E MMANUELLE L IAUDET-C OOPMAN INSERM U826 CRLC Val d’Aurelle Parc Euromédecine, Montpellier Cedex 5 France [email protected] R OSSELLA L IBÈ INSERM U567, Institut Cochin Endocrinology, Metabolism and Cancer Department Paris France K E L IN Department of Haematology Royal Liverpool University Hospital Liverpool UK [email protected] S HENG -C AI L IN Department of Biomedical Sciences, School of Life Sciences Xiamen University, Xiamen Fujian China [email protected] S HIAW-Y IH L IN Department of Systems Biology, The University of Texas M.D. Anderson Cancer Center Houston, TX USA [email protected] WAN -WAN L IN Department of Pharmacology College of Medicine National Taiwan University Taipei Taiwan [email protected] J ANET C. L INDSEY Northern Institute for Cancer Research Newcastle University Newcastle upon Tyne UK [email protected] C HRISTOPHER A. L IPINSKI Scientific Advisor, Melior Discovery Waterford USA [email protected]

J OSEPH L IPSICK Stanford University Stanford, CA USA [email protected] F EI -F EI L IU Department of Medical Biophysics, Princess Margaret Hospital/Ontario Cancer Institute University Health Network University of Toronto Toronto, ON Canada [email protected] X IANGGUO L IU Winship Cancer Institute Emory University Atlanta, GA USA [email protected] T ING L ING L O Signal Transduction Laboratory Institute of Molecular and Cell Biology Singapore [email protected] V ICTOR L OBANENKOV Section of Molecular Pathology, NIAID National Institutes of Health Bethesda, MD USA [email protected] H OLGER N. L ODE Charité University Medicine Berlin Pediatrics, Berlin Germany [email protected] L AWRENCE A. L OEB University of Washington Seattle, WA USA [email protected] R OBERT L OEWE Department of Dermatology, Division of General Dermatology Medical University of Vienna Vienna Austria [email protected] S TEFFEN L OFT Institute of Public Health Department of Occupational and Environmental Health University of Copenhagen, København K Denmark [email protected]

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D IETMAR L OHMANN Institut für Humangenetik Universitätsklinikum Essen Essen Germany [email protected] M ATTHIAS L ÖHR Molecular Gastroenterology Unit, German Cancer Research Center (dkfz E180) Heidelberg and Department of Medicine II, Mannheim Medical Faculty University of Heidelberg Heidelberg Germany [email protected] V INATA B. L OKESHWAR Department of Urology, Cell Biology and Anatomy Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine Miami, FL USA [email protected] A LEXANDRE L OKTIONOV Colonix Medical Limited Babraham Research Campus Cambridge UK [email protected] E LIAS L OLIS Department of Pharmacology Yale University School of Medicine New Haven, CT USA [email protected] P IER -L UIGI L OLLINI Section of Cancer Research Department of Experimental Pathology University of Bologna Bologna Italy [email protected] M IGUEL L OPEZ -L AZARO Department of Pharmacology Faculty of Pharmacy, University of Seville, Seville Spain [email protected] C HARLES L. L OPRINZI Division of Oncology Department of Internal Medicine Mayo Clinic College of Medicine Rochester, MN USA [email protected]

J OCHEN L ORCH Dana Farlur Cancer Institute Head and Neck Oncology Program Boston, MA USA [email protected] E DITH M. L ORD Department of Microbiology & Immunology University of Rochester School of Medicine and Dentistry Rochester, NY USA [email protected] G IUSEPPE D I L ORENZO Cattedra di Oncologia Medica, Dipartimento di Endocrinologia e Oncologia molecolare e clinica Università degli Studi “Federico II” Napoli Italy [email protected] R EUBEN L OTAN Department of Thoracic Head and Neck Medical Oncology The University of Texas, MD Anderson Cancer Center Houston, TX USA [email protected] R AGNHILD A. L OTHE Department of Cancer Prevention Rikshospitalet-Radiumhospitalet Medical Centre Oslo Norway [email protected] M ICHAEL T. L OTZE University of Pittsburgh, Departments of Surgery and Bioengineering Pittsburgh, PA USA [email protected] C HRYSTAL U. L OUIS Center for Cell and Gene Therapy, Baylor College of Medicine Texas Children’s Hospital, and The Methodist Hospital Houston, TX USA [email protected] D MITRI L OUKINOV Section of Molecular Pathology NIAID NIH WT-I bldg Rockville, MD USA [email protected] D AVID B. L OVEJOY Department of Pathology University of Sydney NSW Australia [email protected]


J IADE J. L U Department of Radiation Oncology Cancer Hospital of Fudan University Shanghai China [email protected]

J IAN -H UA L UO Department of Pathology University of Pittsburgh Pittsburgh, PA USA [email protected]

T ZONG -S HI L U Division of Experimental Medicine Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine Boston, MA USA [email protected]

G ARY H. LYMAN Cancer Center University of Rochester Medical Center Rochester, NY USA [email protected]

Y UANAN L U Department of Public Health Science University of Hawaii Honolulu, HI USA [email protected]

H ENRY LYNCH Hereditary Cancer Institute Creighton University Omaha, NE USA [email protected]

I RINA A. L UBENSKY National Cancer Institute National Institutes of Health Bethesda, MD USA [email protected]

E LSEBETH LYNGE Institute of Public Health University of Copenhagen Denmark, Copenhagen [email protected]

D ARIO D I L UCA Department of Experimental and Diagnostic Medicine University of Ferrara Ferrara, Italy [email protected]

S COTT K. LYONS Molecular Imaging Group CRUK Cambridge Research Institute, Li Ka Shing Centre Cambridge UK [email protected]

J ARED L UCAS Divisions of Public Health Sciences, Human Biology and Clinical Sciences Fred Hutchinson Cancer Research Center Seattle, WA USA [email protected]

M ICHAEL M AC M ANUS Department of Radiation Oncology Peter MacCallum Cancer Institute East Melbourne, VIC Australia [email protected]

A NDREAS L UCH Federal Institute for Risk Assessment Berlin Germany [email protected]

B RITTA M ÄDGE DKFZ Heidelberg Germany

B EN O. DE L UMEN Dept of Nutritional Sciences and Toxicology University of California Berkeley, CA USA [email protected]

C LAUDIE M ADOULET Laboratory of Biochemistry IFR53, Faculty of Pharmacy Reims Cedex France [email protected]

M ARIA L I L UNG Department of Biology and Center for Cancer Research Hong Kong University of Science and Technology Clearwater Bay Kowloon, Hong Kong (SAR) People's Republic of China [email protected]

F ÉLIX F ERNÁNDEZ M ADRID Department of Internal Medicine Karmanos Cancer Institute and Center for Molecular Medicine and Genetics, Wayne State University Detroit, MI USA [email protected]

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R OLANDO F. D EL M AESTRO Brain Tumour Research Centre, Montreal Neurological Institute, McGill University Montreal, QC Canada [email protected] B RINDA M AHADEVAN Department of Environmental and Molecular Toxicology Oregon State University Corvallis, OR USA [email protected] C SABA M AHOTKA Institute of Pathology Heinrich Heine Universität Düsseldorf Germany [email protected] S OURINDRA N. M AITI Division of Pediatrics, Department of Immunology M.D. Anderson Cancer Center Houston, TX USA [email protected] C ÉDRIC M ALICET INSERM, Stress Cellulaire, Parc Scientifique et Technologique de Luminy Marseille Cedex France [email protected] A LESSANDRA M ANCINO Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected] E VELYNE M ANET INSERM U758, Ecole Normale Supérieure de Lyon Lyon France [email protected] S RIDHAR M ANI Department of Medicine, Oncology and Molecular Genetics Albert Einstein College of Medicine NY USA [email protected] M ARCEL M ANNENS Academic Medical Centre, University of Amsterdam Amsterdam The Netherlands [email protected]

A LBERTO M ANTOVANI Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected] A SHLEY A. M ANZOOR Department of Radiation Oncology Duke University Durham, NC USA [email protected] L UCIA M ARCOCCI Department of Biochemical Sciences “A. Rossi Fanelli” Sapienza University of Rome Rome Italy [email protected] D IETER M ARMÉ Tumor Biology Center Institute of Molecular Oncology Freiburg Germany [email protected] D EBORAH J. M ARSH Kolling Institute of Medical Research University of Sydney NSW Australia [email protected] J OHN L. M ARSHALL Georgetown University Lombardi Comprehensive Cancer Center Washington, DC USA [email protected] R ENÉE M. M ARSHALL The Wistar Institute Molecular and Cellular Oncogenesis Program Philadelphia, PA USA [email protected] A NGELA M ÄRTEN National Centre for Tumour Diseases, Department of Surgery University Hospital Heidelberg Heidelberg Germany [email protected] E DMUND M ASER Institute of Toxicology and Pharmacology for Natural Scientists University Medical School Kiel Germany [email protected]


T HOMAS E. M ASSEY Department of Pharmacology and Toxicology Queen’s University Kingston, ONT Canada [email protected]

J OSEPH H. M C C ARTY University of Texas, M.D. Anderson Cancer Center Houston, TX USA [email protected]

N ORIYUKI M ASUDA Department of Respiratory Medicine Kitasato University School of Medicine Sagamihara, Kanagawa Japan [email protected]

K ATHERINE A. M C G LYNN Division of Cancer Epidemiology and Genetics National Cancer Institute National Institutes of Health Bethesda, MD USA [email protected]

ATSUKO M ASUMI National Institute of Infectious Diseases, Musashimurayama-shi Tokyo Japan [email protected]

W. G LENN M C G REGOR James Graham Brown Cancer Center University of Louisville School of Medicine Louisville, KY USA [email protected]

YASUNOBU M ATSUDA Department of Hepatology and Gastroenterology Juntendo University School of Medicine, Shizuoka Hospital Shizuoka Japan

I AIN H. M C K ILLOP Department of Biology The University of North Carolina at Charlotte Charlotte, NC USA [email protected]

S ACHIKO M ATSUHASHI Department of Internal Medicine Saga Medical School Saga University Saga Japan [email protected]

R OGER E. M C L ENDON Department of Pathology Duke University Medical Center Durham, NC USA [email protected]

TAKAYA M ATSUZUKA Department of Anatomy and Physiology Kansas State University Manhattan, KS USA [email protected]

D ONALD C. M C M ILLAN University Department of Surgery Royal Infirmary Glasgow UK [email protected]

M ALGORZATA M ATUSIEWICZ Department of Medical Biochemistry Wroclaw Medical University Wroclaw Poland [email protected]

A RIANEB M EHRABI Department of General Visceral and Transplantation Surgery University of Heidelberg Heidelberg Germany [email protected]

WARREN L. M AY Department of Preventive Medicine University of Mississippi Medical Center Jackson, MS USA [email protected] M ATTHEW A. M C B RIAN Department of Biological Chemistry David Geffen School of Medicine at UCLA Los Angeles, CA USA [email protected]

A NIL M EHTA Maternal and Child Health Sciences University of Dundee Dundee UK [email protected] K APIL M EHTA The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected]

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R EKHA M EHTA Toxicology Research Division, Bureau of Chemical Safety Food Directorate, HPFB, Health Canada Ottawa, ONT Canada [email protected] E LVIRA DE M EJIA Department of Food Science and Human Nutrition University of Illinois Urbana-Champaign, IL USA [email protected] B AR -E LI M ENASHE Department of Cancer Biology The University of Texas, M.D. Anderson Cancer Center Houston, TX USA [email protected] H EATHER M ERNITZ Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University Boston, MA USA [email protected] J ENNIFER A. M ERTZ Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology The University of Texas at Austin Austin, TX USA [email protected] K ARL -H EINZ M ERZ Department of Chemistry, Division of Food Chemistry and Toxicology University of Kaiserslautern Kaiserslautern Germany [email protected] J UN M I Department of Therapeutic Radiology and Oncology University of Virginia School of Medicine Charlottesville, VA USA [email protected] D ENNIS F. M ICHIEL Biopharmaceutical Development Program SAIC-Frederick, Inc. National Cancer Institute-Frederick Frederick, MD USA [email protected]

S TEPHAN M IELKE Hematology Branch, National Heart, Lung and Blood Institute (NHLBI) National Institutes of Health (NIH) Bethesda, MD USA [email protected] TAKEO M INAGUCHI Department of Obstetrics and Gynecology Toranomon Hospital Tokyo Japan [email protected] N AGAHIRO M INATO Department of Immunology and Cell Biology Graduate School of Medicine, Kyoto University Kyoto Japan [email protected] R ODNEY F. M INCHIN School of Biomedical Sciences University of Queensland St Lucia, QLD Australia [email protected] J OHN D. M INNA Hamon Center for Therapeutic Oncology Research and Departments of Pathology, Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, TX USA [email protected] C LAUDIA M ITCHELL Institut Cochin Université Paris Descartes, CNRS Paris France [email protected] K AZUO M IYASHITA Department of Bioresources Chemistry, Faculty of Fisheries Sciences Hokkaido University Hakodate Japan [email protected] E IJI M IYOSHI Department of Biochemistry Osaka University Graduate School of Medicine Suita Japan [email protected]


J UN M IYOSHI Department of Molecular Biology Osaka Medical Center for Cancer and Cardiovascular Diseases Osaka Japan [email protected] T OSHIHIKO M IZUTA Department of Internal Medicine Saga Medical School Saga Japan [email protected] K. T HOMAS M OESTA Klinik für Chirurgie und Chirurgische Onkologie Robert Rössle-Klinik, Charité Berlin-Buch, Berlin Germany [email protected] S ONIA M OHINTA Department of Medical Microbiology, Immunology and Cell Biology Southern Illinois University, School of Medicine Springfield, IL USA [email protected] J AN M OLLENHAUER Division of Molecular Genome Analysis, DKFZ Heidelberg Germany [email protected] M ICHAEL B. M ØLLER Department of Pathology, Division of Hematopathology Odense University Hospital Odense Denmark [email protected] B RUNO M ONDOVÌ Department of Biochemical Sciences “A. Rossi Fanelli” Sapienza University of Rome Rome Italy [email protected] A LESSANDRA M ONTECUCCO Istituto di Genetica Molecolare CNR via Abbiategrasso, Pavia Italy [email protected] R UGGERO M ONTESANO Courmayeur Italy [email protected]

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W OLTER J. M OOI Department of Pathology VU Medical Center Amsterdam The Netherlands [email protected] A MY C. M OORE Vanderbilt University Medical Center Nashville, TN USA [email protected] M ALCOLM A. S. M OORE Department of Cell Biology Memorial-Sloan-Kettering Cancer Center New York, NY USA [email protected] C ESAR A. M ORAN Department of Pathology M D Anderson Cancer Center Houston, TX USA [email protected] J AQUELINE M ORENO Division of Endocrinology Department of Medicine, Stanford University School of Medicine Stanford, CA USA [email protected] S ERGIO M ORENO Instituto de Biología Molecular y Celular del Cáncer CSIC/Universidad de Salamanca Campus Miguel de Unamuno, Salamanca Spain [email protected] FABIOLA M ORETTI Institute of Neurobiology and Molecular Medicine, National Council of Research/Molecular Oncogenesis Laboratory “Regina Elena” Cancer Institute Rome Italy [email protected] E IICHIRO M ORI Department of Biology Nara Medical University School of Medicine Kashihara, Nara Japan [email protected] A KIRA M ORIMOTO Department of Pediatrics Kyoto Prefectural University of Medicine Kyoto Japan [email protected]

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PAT J. M ORIN Laboratory of Cellular and Molecular Biology National Institute on Aging, NIH Baltimore, MD USA [email protected]

H. K. M ÜLLER -H ERMELINK Institute of Pathology University of Würzburg Würzburg Germany [email protected]

C HRISTINE M. M ORRIS Cancer Genetics Research Group University of Otago at Christchurch Christchurch New Zealand [email protected]

L OIS M. M ULLIGAN Department of Pathology & Molecular Medicine Queen’s University Kingston, ON Canada [email protected]

G ABRIELA M ÖSLEIN Chefärztin für Allgemein- und Viszeralchirurgie Bochum Germany [email protected] C YNTHIA C. M ORTON Department of Pathology Brigham and Women’s Hospital Boston, MA USA [email protected] J USTIN L. M OTT Miles and Shirley Fiterman Center for Digestive Diseases Division of Gastroenterology and Hepatology Mayo Clinic College of Medicine Rochester, MN USA [email protected] S PYRO M OUSSES Cancer Genetics Branch National Human Genome Research Institute, NIH Bethesda, MD USA [email protected] S EBASTIAN M UELLER Department of Medicine Salem Medical Center, Heidelberg, Center of Alcohol Research Liver Disease and Nutrition and University of Heidelberg Heidelberg, Germany [email protected] S USETTE C. M UELLER Lombardi Comprehensive Cancer Center Georgetown University Medical Center Washington, DC USA [email protected] R OLF M ÜLLER Institute of Molecular Biology and Tumor Research (IMT) Philipps-University Marburg Marburg Germany [email protected]

R AMACHANDRAN M URALI Department of Pathology and Laboratory Medicine & Abramson Cancer Center University of Pennsylvania Philadelphia, PA USA [email protected] K ENJI M URO Department of Neurological Surgery, Northwestern University Feinberg School of Medicine Robert H. Lurie Comprehensive Cancer Center Chicago, IL USA [email protected] E DWARD L. M URPHY University of California, School of Medicine San Francisco, CA USA [email protected] PAUL G. M URRAY CRUK Institute for Cancer Studies, Molecular Pharmacology, Medical School University of Birmingham Birmingham UK [email protected] M ARKUS M ÜSCHEN Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center University of Southern California Los Angeles, CA USA [email protected] R UTH J. M USCHEL Radiation Oncology and Biology University of Oxford Oxford, UK [email protected] A KIRA N AGANUMA Laboratory of Molecular and Biochemical Toxicology Graduate School of Pharmaceutical Sciences Tohoku University Sendai Japan [email protected]


S HIGEKAZU N AGATA Osaka University Medical School Osaka Japan [email protected] R ITA N AHTA Department of Breast Medical Oncology The University of Texas MD Anderson Cancer Center Houston, TX USA Breast Cancer Translational Research Laboratory The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected] A KIRA N AKAGAWARA Chiba Cancer Center Research Institute Chiba Japan [email protected] T ETSUYA N AKATSURA Section for Frontier Medicine, Investigative Treatment Division, Research Center for Innovative Oncology National Cancer Center Hospital East Kashiwa City, Chiba Prefecture Japan [email protected] PATRIZIA N ANNI Section of Cancer Research Department of Experimental Pathology University of Bologna Bologna Italy [email protected] Z VI N AOR Department of Biochemistry, The George S. Wise Faculty of Life Sciences Tel Aviv University Ramat Aviv Israel [email protected] K EVIN T. N ASH Department of Pathology and Comprehensive Cancer Center University of Alabama-Birmingham Birmingham, AL USA [email protected] C HRISTIAN C. N AUS Department of Cellular and Physiological Sciences The University of British Columbia Vancouver, BC Canada [email protected]

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T IM S. N AWROT Division of Lung Toxicology Department of Occupational and Environmental Medicine (T.S.N.) and the Studies Coordinating Centre (J.A.S.) Division of Hypertension and Cardiovascular Rehabilitation Department of Cardiovascular Diseases University of Leuven Leuven Belgium [email protected] D AVID F. N ELLIS Biopharmaceutical Development Program SAIC-Frederick, Inc. National Cancer Institute-Frederick Frederick, MD USA [email protected] P ETER N ELSON Divisions of Public Health Sciences, Human Biology and Clinical Sciences Fred Hutchinson Cancer Research Center Seattle, WA USA [email protected] K ENNETH P. N EPHEW Medical Sciences Indiana University School of Medicine Bloomington, IN USA [email protected] K ORNELIA N EVELING Department of Human Genetics University of Wurzburg Wurzburg Germany [email protected] B RAD W N EVILLE Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA [email protected] K LAUS N EUHAUS Department of Operative, Preventive and Paediatric Dentistry School of Dental Medicine, University of Bern Bern Switzerland [email protected] I RENE O. L. N G Department of Pathology The University of Hong Kong Hong Kong [email protected]

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D UC N GUYEN Associate Research Scientist Yale University School of Medicine New Haven, CT USA [email protected]

L ARRY N ORTON Breast Cancer Medicine Service Department of Medicine Memorial Sloan-Kettering Cancer Center New York, NY USA [email protected]

C AROLE N ICCO Faculté de Médecine Paris – Descartes UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul Paris France [email protected]

F RANCISCO J. N OVO Department of Genetics University of Navarra Pamplona, Spain [email protected]

S ANTO V. N ICOSIA Molecular Oncology Program & Research Institute, H. Lee Moffitt Cancer Center University of South Florida College of Medicine Tampa, FL USA [email protected]

R USLAN N OVOSYADLYY Division of Endocrinology, Diabetes and Bone Diseases Departmant of Medicine Mount Sinai School of Medicine New York, NY USA [email protected]

A NNE T. N IES Division of Tumor Biochemistry German Cancer Research Center Heidelberg Germany [email protected] M. A NGELA N IETO Instituto de Neurociencias de Alicante CSIC-UMH Sant Joan d’Alacant Spain [email protected] O MGO E. N IEWEG Department of Surgery The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Amsterdam The Netherlands [email protected] J ONAS N ILSSON Department of Clinical Chemistry and Transfusion Medicin Institute of Biomedicine Sahlgrenska academy at Göteborg University Göteborg Sweden [email protected] E WA N INIO INSERM UMRS Université Pierre et Marie Curie-Paris Paris France [email protected] D OUGLAS N OONAN University of Insubria Varese Italy [email protected]

N OA N OY Department of Pharmacology Case-Western Reserve University School of Medicine Cleveland, OH USA [email protected] H ALA H. N SOULI Department of Epidemiology and Biostatistics The George Washington University School of Public Health and Health Services Washington, DC USA [email protected] L AUREN M. N UNEZ Department of Cell Biology & Anatomy and Feist-Weiller Cancer Center LSUHSC-School of Medicine Shreveport, LA USA [email protected] A NDRÉ O BERTHÜR Department of Pediatric Oncology and Hematology Children’s Hospital University of Cologne Cologne Germany [email protected] TAKAHIRO O CHIYA Section for Studies on Metastasis National Cancer Center Research Institute Chuo-ku, Tokyo Japan [email protected]


J AMES P. B. O’C ONNOR Imaging Science University of Manchester Manchester UK james.o'[email protected] S ARAH T. O’D WYER Department of Surgery University of Manchester Christie Hospital NHS Foundation Trust Manchester UK [email protected] S TEFAN O FFERMANNS Institute of Pharmacology, University of Heidelberg Heidelberg Germany [email protected] A NAT O HALI Cancer Vaccine Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected] TAKEO O HNISHI Department of Biology Nara Medical University School of Medicine Kashihara, Nara Japan [email protected] H ITOSHI O HNO Department of Internal Medicine, Faculty of Medicine Kyoto University Kyoto Japan [email protected] K EVIN R. O LDENBURG MatriCal, Inc. Spokane, WA USA [email protected] M AGALI O LIVIER Group of Molecular Carcinogenesis and Biomarkers International Agency for Research on Cancer World Health Organization Lyon Cedex 08 France [email protected] M ILITSAKH O N Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA [email protected]

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R UTH M. O’R EGAN Winship Cancer Institute Emory University Atlanta, GA USA [email protected] G ERTRAUD O REND Departement Klinisch-Biologische Wissenschaften (DKBW) Center for Biomedicine, Institute of Biochemistry and Genetics University of Basel Basel Switzerland [email protected] M AKOTO O SANAI Department of Pathology Sapporo Medical University School of Medicine Sapporo Japan [email protected] E DUARDO O SINAGA Departamento de Inmunobiología, Facultad de Medicina Universidad de la República Montevideo Uruguay [email protected] F RANCISO R UIZ -C ABELLO O SUNA Hospital Universitario Virgen de las Nieves Granada Spain [email protected] G. O TT Institute of Pathology Robert-Bosch Krankenhaus Stuttgart Germany [email protected] C HRISTIAN O TTENSMEIER CRC Wessex Oncology Unit, Southampton General Hospital and Tenovous Laboratory Southampton University Hospital Trust Southampton UK [email protected] I WATA O ZAKI Health Administration Center Saga Medical School Saga University Saga Japan [email protected]

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S HUJI O ZAKI Department of Medicine and Bioregulatory Sciences The University of Tokushima Graduate School of Health Biosciences Tokushima Japan [email protected] H ELEN PACE Department of Molecular Virology Immunology and Medical Genetics, Ohio State University Comprehensive Cancer Center Columbus, OH USA [email protected] S IMON C. PACEY Cancer Research UK Center for Cancer Therapeutics The Institute of Cancer Research Sutton, Surrey UK [email protected] P IER PAOLO PANDOLFI Memorial Sloan-Kettering Cancer Center Weill Cornell Graduate School of Medical Sciences NY USA [email protected] K LAUS PANTEL Universitäts-Krankenhaus Eppendorf Hamburg Germany [email protected] M ELISSA C. PAOLONI National Cancer Institute, Center for Cancer Research Comparative Oncology Program Bethesda, MD USA [email protected],gov E VANGELIA PAPADIMITRIOU Department of Pharmacy University of Patras Patras Greece [email protected] ATHANASIOS G. PAPAVASSILIOU Department of Biological Chemistry, Medical School University of Athens Goudi, Athens Greece [email protected] S ABITHA PAPINENI Department of Veterinary Physiology and Pharmacology Texas A&M University College Station, TX USA [email protected]

B EN H O PARK The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University Baltimore, MD USA [email protected] G EOFF J. M. PARKER Imaging Science University of Manchester Manchester UK [email protected] S ARAH J. PARSONS University of Virginia Charlotteville, VA USA [email protected] O NEEL PATEL Department of Surgery University of Melbourne, Austin Health Melbourne, VIC Australia [email protected] PATRIZIA PATERLINI -B RÉCHOT INSERM Unit 807, Faculté de Médecine Necker Enfants Malades, Paris V, Paris France [email protected] Y VONNE PATERSON Professor of Microbiology University of Pennsylvania Philadelphia, PA USA [email protected] K ONAN P ECK Institute of Biomedical Sciences Academia Sinica Taipei Taiwan Republic of China [email protected] F LORENCE P EDEUTOUR Laboratory of Solid Tumors Genetics Nice University Hospital and CNRS UMR 6543 Faculty of Medicine Nice France [email protected] D AN P EER Departments of Anesthesia and Immunology, CBR Institute for Biomedical Research Harvard Medical School Boston, MA USA [email protected]


M IGUEL A. P EINADO Institue of Predictive and Personalized Medicine of Cancer (IMPPC) Badalona Barcelona, Spain [email protected]

G ODEFRIDUS J. P ETERS Department of Medical Oncology VU University Medical Center Amsterdam The Netherlands [email protected]

A NGEL P ELLICER Department of Pathology New York University School of Medicine New York, NY USA [email protected]

M ARLEEN M. R. P ETIT Department of Human Genetics University of Leuven Leuven Belgium [email protected]

J UHA P ELTONEN Department of Anatomy, Institute of Biomedicine University of Turku Turku Finland [email protected]

P ETER P ETZELBAUER Department of Dermatology, Division of General Dermatology Medical University of Vienna Vienna Austria [email protected]

S IRKKU P ELTONEN Department of Dermatology University of Turku Turku Finland [email protected]

C LAUDIA P FÖHLER Department of Dermatology Saarland University Medical School Homburg/Saar, Germany [email protected]

J OSEF M. P ENNINGER Institute of Molecular Biotechnology of the Austrian Academy of Sciences Vienna Austria [email protected]

M ICHAEL P FREUNDSCHUH Klinik für Innere Medizin I Universität des Saarlandes Homburg Germany [email protected]

M AIKEL P. P EPPELENBOSCH University Medical Center Groningen University of Groningen Groningen The Netherlands [email protected]

P HILIP A. P HILIP Karmanos Cancer Institute Wayne State University Detroit, MI USA [email protected]

M ÓNICA P ÉREZ -R ÍOS Department of Preventive Medicine and Public Health University of Santiago de Compostela C/San Francisco Spain [email protected]

M ARCO A. P IEROTTI Scientific Director IRCCS Istituto Nazionale Tumori Foundation Milan Italy [email protected]

F RANCISCO G. P ERNAS National Institute on Deafness and Other Communication Disorders and National Cancer Institute NIH Bethesda, MD USA

PAOLA P IETRANGELI Department of Biochemical Sciences-A. Rossi Fanelli Sapienza University of Rome Rome Italy [email protected]

S ILVERIO P ERROTTA Department of Pediatrics Second University of Naples Naples Italy [email protected]

T ORSTEN P IETSCH Institut für Neuropathologie Universitätskliniken Bonn, Bonn Germany Kinderchirurgie [email protected]

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M ICHAEL P ISHVAIAN Georgetown University Lombardi Comprehensive Cancer Center Washington, DC USA [email protected] E LLEN S. P IZER Laboratory of Cellular and Molecular Biology National Institute on Aging, NIH Baltimore, MD USA C HRISTOPH P LASS German cancer Research cener (DKFZ) Heidelberg Germany [email protected] L EONIDAS C. P LATANIAS Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Department of Medicine Northwestern University Medical School Chicago, IL USA [email protected] J EFFREY L. P LATT Transplantation Biology and the Departments of Surgery Immunology and Pediatrics Mayo Clinic Rochester, MN USA [email protected] M ARK R. P LAYER Johnson & Johnson Pharmaceutical Research & Development Exton, PA USA [email protected] I RIS M. C. VAN DER P LOEG Department of Surgery The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Amsterdam The Netherlands [email protected] K LAUS P ODAR Department of Medical Oncology, Jerome Lipper Multiple Myeloma Center Dana-Farber Cancer Institute Boston, MA USA [email protected] M IRIAM C. P OIRIER National Cancer Institute, NIH Bethesda, MD USA [email protected]

J EFFREY W. P OLLARD Department of Developmental and Molecular Biology, Center for the Study of Reproductive Biology and Women’s Health Albert Einstein College of Medicine Bronx, NY USA [email protected] S IMONA P OLO University of Milan, Medical School Milan Italy [email protected] B RUCE A. J. P ONDER Department of Oncology University of Cambridge Cambridge UK [email protected] M IRCO P ONZONI Differentiation Therapy Unit, Laboratory of Oncology G. Gaslini Children’s Hospital Genoa Italy [email protected] B EATRICE L. P OOL -Z OBEL Nutritional Toxicology Friedrich-Schiller-University of Jena Jena Germany [email protected] C HRISTOPHER S. P OTTEN Epistem Ltd and School of Biological Sciences University of Manchester Manchester UK [email protected] A NNEMARIE P OUSTKA Division of Molecular Genome Analysis, DKFZ Heidelberg Germany [email protected] M ARISSA V. P OWERS Cancer Research UK Centre for Cancer Therapeutics The Institute of Cancer Research Sutton, Surrey UK [email protected] G ARTH P OWIS Department of Experimental Therapeutics M.D. Anderson Cancer Center Houston, TX USA [email protected]


G RAZIELLA P RATESI Fondazione IRCCS Istituto Nazionale dei Tumori Milan Italy [email protected] G EORGE C. P RENDERGAST Department of Pathology, Anatomy and Cell Biology Jefferson Medical School Lankenau Institute for Medical Research Wynnewood, PA USA [email protected] V ICTOR G. P RIETO Department of Pathology The University of Texas M.D. Anderson Cancer Center Houston, TX USA [email protected] K EVIN M. P RISE Centre for Cancer Research and Cell Biology Queen’s University Belfast Belfast UK [email protected] K ATHY P RITCHARD -J ONES Institute of Cancer Research/Royal Marsden Hospital Sutton, Surrey UK [email protected] A MANDA H. P ROWSE Nuffield Department of Obstetrics and Gynaecology Oxford University John Radcliffe Hospital Oxford UK [email protected] C HING -H ON P UI St. Jude Children’s Research Hospital Memphis, TN USA [email protected] K AREN P ULFORD Department of Clinical Laboratory Sciences University of Oxford, John Radcliffe Hospital Oxford UK [email protected] T ERESA G ÓMEZ D EL P ULGAR Instituto de Investigaciones Biomedicas CSIC, Madrid Spain

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C HAO -N AN Q IAN Laboratory of Cancer Genetics Van Andel Research Institute Grand Rapids, MI USA [email protected] J IAHUA Q IAN Cancer Vaccine Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected] H ARTMUT M. R ABES Institute of Pathology University of Munich Munich Germany [email protected] D IRK R ADES Department of Radiation Oncology University Hospital Schleswig-Holstein Campus Luebeck Germany [email protected] J ERALD P. R ADICH Clinical Research Division Fred Hutchinson Cancer Research Center Seattle, WA USA [email protected] N ORMAN S. R ADIN Department of Psychiatry University of Michigan Ann Arbor, MI USA [email protected] F ULVIO D ELLA R AGIONE Department of Biochemistry and Biophysics Second University of Naples Naples Italy [email protected] RYAN L. R AGLAND Department of Human Genetics University of Michigan Ann Arbor, MI USA [email protected] AYYAPPAN K. R AJASEKARAN Department of Pathology and Laboratory Medicine University of California Los Angeles, CA USA [email protected]

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S IGRID A. R AJASEKARAN Department of Pathology and Laboratory Medicine University of California Los Angeles, CA USA [email protected] J AYADEV R AJU Toxicology Research Division, Bureau of Chemical Safety Food Directorate, HPFB, Health Canada Ottawa, ONT Canada [email protected] S. R AMAKRISHNAN Department of Pharmacology University of Minnesota Minneapolis, MN USA [email protected] K OTA V. R AMANA Dept of Biochemistry and Molecular Biology University Of Texas Medical Branch Galveston, TX USA [email protected] S ANTIAGO R AMÓN Y C AJAL Department of Pathology, Vall d’ Hebron University Hospital Barcelona Spain [email protected] G IORGIA R ANDI Department of Epidemiology Institute for Farmacological Research Mario Negri Milan Italy [email protected] M ARIUSZ Z. R ATAJCZAK Stem Cell Institute at James Graham Brown Cancer Center University of Louisville Louisville, KY USA [email protected] A NKE R ATTENHOLL Department of Dermatology University of Münster Münster Germany [email protected] A LBERTO R AVAIOLI Department of Oncology Infermi Hospital Rimini Italy [email protected]

M IRA R. R AY Department of Urologic Sciences The Prostate Centre at Vancouver General Hospital Vancouver, BC Canada [email protected] R OGER R EDDEL Children’s Medical Research Institute Westmead, NSW Australia [email protected] M AY R EED Department of Medicine, Division of Geriatric Medicine University of Washington Seattle, WA USA [email protected] E DUARDO M. R EGO Medical School of Ribeirão Preto University of São Paulo Ribeirão Preto Brazil [email protected] R EUVEN R EICH Department of Pharmacology, School of Pharmacy, Faculty of Medicine The Hebrew University of Jerusalem Jerusalem Israel [email protected] J EAN -M ARIE R EIMUND Service d’Hépato-Gastro-Entérologie et Nutrition, Centre Hospitalier Universitaire de Caen Université de Caen-Basse Normandie Caen France [email protected] C ELSO A. R EIS Institute of Molecular Pathology and Immunology of the University of Porto Medical Faculty of Porto Porto Portugal [email protected] L ING R EN National Cancer Institute, Center for Cancer Research Pediatric Oncology Branch Bethesda, MD USA [email protected] ANDREW G. RENEHAN Department of Surgery University of Manchester Christie Hospital NHS Foundation Trust Manchester UK [email protected]


M ARCUS R ENNER Division of Molecular Genome Analysis, DKFZ Heidelberg Germany [email protected] PAUL S. R ENNIE The Richard Ivey School of Business University of Western Ontario London, ONT Canada [email protected] D OMENICO R IBATTI Department of Human Anatomy and Histology University of Bari Medical School Bari Italy [email protected] R AUL C. R IBEIRO Department of Oncology St. Jude Children’s Research Hospital Memphis, TN USA [email protected] D ES R. R ICHARDSON Department of Pathology University of Sydney NSW, Australia [email protected] A NN R ICHMOND Department of Cancer Biology Vanderbilt University Medical School Nashville, TN USA [email protected] V ICTORIA M. R ICHON Merck Research Laboratories Boston, MA USA [email protected]

C ARRIE R INKER -S CHAFFER Department of Surgery, Section of Urology The University of Chicago Chicago, IL USA [email protected] TADEUSZ R OBAK Department of Hematology Medical University of Lodz Lodz Poland [email protected] F REDIKA M. R OBERTSON The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected] C RISTINA R ODRÍGUEZ National Cancer Institute of Research (CNIO) Madrid Spain J OSE L UIS R ODRÍGUEZ -F ERNÁNDEZ Departamento de Fisiología Celular y Molecular Centro de Investigaciones Biológicas Madrid Spain [email protected] C ARLOS RODRIGUEZ-GALINDO Department of Oncology St. Jude Children’s Research Hospital Memphis, TN USA [email protected]

J USTIN L. R ICKER Merck Research Laboratories Upper Gwynedd, PA USA

D EREK L E R OITH Division of Endocrinology, Diabetes and Bone Diseases Departmant of Medicine Mount Sinai School of Medicine New York, NY USA [email protected]

T HOMAS R IED Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH Bethesda, MD USA [email protected]

W OLF C. R OLAND Biomedical Research Centre University of Dundee Dundee UK [email protected]

M AARTJE C. VAN R IJK Department of Surgery The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Amsterdam The Netherlands [email protected]

Z E ’ EV R ONAI Signal Transduction Program Burnham Institute for Medical Research La Jolla, CA USA [email protected]

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L UCA R ONCUCCI Department of Medicine University of Modena and Reggio Emilia Modena Italy [email protected]

L UCA R UBINO Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected]

I GOR B. R ONINSON Department of Molecular Genetics University of Illinois at Chicago Chicago, IL USA [email protected]

M ARCO R UGGIERO Department of Experimental Pathology and Oncology University of Firenze Firenze Italy [email protected]

E LIOT M. R OSEN Long Island Jewish Medical Center Albert Einstein College of Medicine Bronx, NY USA [email protected] C AROL L. R OSENBERG Boston Medical Center and Boston University School of Medicine Boston, MA USA [email protected] S TEVEN A. R OSENZWEIG Department of Cell and Molecular Pharmacology and Experimental Therapeutics Medical University of South Carolina Charleston, SC USA [email protected] A NGELO R OSOLEN Department of Pediatrics, Hemato-oncology Unit University of Padua Padova Italy [email protected] J EFFREY S. R OSS Albany Medical College Albany, NY USA [email protected]

Z ORAN R UMBOLDT Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA T HOMAS M. R ÜNGER Department of Dermatology Boston University School of Medicine Boston, MA USA [email protected] E RKKI R UOSLAHTI Vascular Mapping Center, Burnham Institute for Medical Research at University of California Santa Barbara, CA USA [email protected] D ARIO R USCIANO Friedrich Miescher Institute Basel Switzerland [email protected] G IANDOMENICO R USSO Istituto Demopatico dell’Immacolata Instituto di Ricovero e Cura a Carattere Scientifico Roma, Italy [email protected]

T HEODORA S. R OSS Department of Internal Medicine University of Michigan Ann Arbor, MI USA [email protected]

I RMA H. R USSO Breast Cancer Research Laboratory Fox Chase Cancer Center Philadelphia, PA USA [email protected]

A LBERTO R UANO -R AVINA Department of Preventive Medicine and Public Health University of Santiago de Compostela C/San Francisco Spain [email protected]

J OSE R USSO Breast Cancer Research Laboratory Fox Chase Cancer Center Philadelphia, PA USA [email protected]


J. RYAN Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA

T OSHIYUKI S AKAI Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science Kyoto Prefectural University of Medicine Kyoto Japan [email protected]

J AMES T. R UTKA The Arthur and Sonia Labatt Brain Tumour Research Centre The Hospital for Sick Children The University of Toronto Toronto, ONT USA [email protected]

B ODOUR S ALHIA Cancer and Cell Biology Division The Translational Genomics Research Institute Phoenix, AZ USA [email protected]

A NNE T HOUSTRUP S ABER National Research Centre for the Working Environment Copenhagen Denmark [email protected]

H ELMUT R AINER S ALIH Department of Internal Medicine II University Hospital of Tuebingen Eberhard-Karls-University Tuebingen Germany [email protected]

G AURI S ABNIS University of Maryland School of Medicine Baltimore, MD USA [email protected] M OHAMAD S EYED S ADR Brain Tumour Research Centre, Montreal Neurological Institute, McGill University Montreal, QC Canada [email protected] S TEPHEN S AFE Department of Veterinary Physiology and Pharmacology Texas A&M University College Station, TX USA [email protected]

B ETH A. S ALMON Department of Pharmacology and Therapeutics University of Florida Gainesville, FL USA [email protected] H OWARD W. S ALMON Department of Radiation Oncology North Florida Radiation Oncology Gainesville, FL USA [email protected] R AED S AMARA Cancer Vaccine Branch, National Cancer Institute National Institutes of Health Bethesda, MD USA [email protected]

X AVIER S AGAERT Department of Pathology University Hospitals of K.U. Leuven Leuven Belgium [email protected]

J ULIAN R. S AMPSON Institute of Medical Genetics Cardiff University Heath Park, Cardiff UK [email protected]

A SIM S AHA University of Cincinnati and The Barrett Cancer Center Cincinnatti, OH USA [email protected]

M ANORANJAN S ANTRA Neurology Research Henry Ford Health System Detroit, MI USA [email protected]

K UNAL S AIGAL National Institute on Deafness and Other Communication Disorders and National Cancer Institute NIH Bethesda, MD USA [email protected]

F RANK S ARAN Department of Radiotherapy and Paediatric Oncology Royal Marsden Hospital NHS Foundation Trust Sutton, Surrey UK [email protected]

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D EVANAND S ARKAR Departments of Urology, Pathology and Neurosurgery Columbia University Medical Center College of Physicians and Surgeons New York, NY USA [email protected] FAZLUL H. S ARKAR Karmanos Cancer Institute Wayne State University Detroit, MI USA [email protected] D EBASHIS S ARKER Cancer Research UK Center for Cancer Therapeutics The Institute of Cancer Research Sutton, Surrey UK [email protected] S TEPHANIE S ASSE Hematology and Oncology University Hospital of Cologne, Department of Internal Medicine I Cologne Germany [email protected] L EONARD A. S AUER Bassett Research Institute Cooperstown, NY USA [email protected] C HRISTOBEL S AUNDERS School of Surgery and Pathology, QEII Medical Centre University of Western Australia Crawley, WA Australia [email protected] C ONSTANCE L. L. S AW Department of Pharmacy National University of Singapore Singapore [email protected]

R ON H. N. VAN S CHAIK Department of Clinical Chemistry Erasmus University Medical Center Rotterdam The Netherlands [email protected] M ANFRED S CHARTL Biozentrum, Universität Würzburg Würzburg Germany [email protected] H UUB S CHELLEKENS Department of Innovation Studies Central Laboratory Animal Institute Utrecht University, TD Utrecht The Netherlands [email protected] D ETLEV S CHINDLER Department of Human Genetics University of Wurzburg Wurzburg, Germany [email protected] P ETER M. S CHLAG Klinik für Chirurgie und Chirurgische Onkologie Robert Rössle-Klinik, Charité Berlin-Buch Germany [email protected] M ARTIN S CHLUMBERGER Institut de Cancérologie Gustave-Roussy, Villejuif and Université Paris-Sud 11, Paris France [email protected] P ETER S CHMEZER Division of Toxicology and Cancer Risk Factors German Cancer Research Center (DKFZ) Heidelberg Germany [email protected]

A NURAG S AXENA Department of Pathology and Laboratory Medicine Royal University Hospital, Saskatoon Health Region/ University of Saskatchewan Saskatoon, Saskatchewan Canada [email protected]

A NNETTE S CHMITT-G RAEFF Institute of Pathology University Hospital Freiburg Freiburg Germany [email protected]

R EINHOLD S CHÄFER Molecular Tumor Pathology Institute of Pathology Berlin Germany [email protected]

D OMINIK T. S CHNEIDER Clinic of Pediatrics Klinikum Dortmund Dortmund Germany [email protected]


S TEFAN W. S CHNEIDER Department of Dermatology University of Münster Münster Germany [email protected]

M ARKUS S CHWAIGER Department of Nuclear Medicine Technical University of Munich Munich Germany [email protected]

S USANNE S CHNITTGER MLL Munich Leukemia Laboratory Munich Germany [email protected]

E DWARD L. S CHWARTZ Department of Oncology Albert Einstein College of Medicine Bronx, NY USA [email protected]

N ATHALIE S CHOLLER Center for Research on Early Detection and Cure of Ovarian Cancer, School of Medicine University of Pennsylvania Biomedical Research Building (BRB) II/III Philadelphia, PA USA [email protected] A XEL H. S CHÖNTHAL University of Southern California Keck School of Medicine Los Angeles, CA USA [email protected] Y VONNE M. S CHRAGE Department of Pathology Leiden University Medical Center Leiden The Netherlands [email protected] H. W. B ART S CHREUDER Department of Orthopaedics Radboud University Medical Centre Nijmegen The Netherlands [email protected] L AURA W. S CHRUM Department of Biology The University of North Carolina at Charlotte Charlotte, NC USA [email protected] W OLFGANG A. S CHULZ Department of Urology Heinrich-Heine University Düsseldorf Germany [email protected] M ANFRED S CHWAB Tumour Genetics German Cancer Research Center, DKFZ Heidelberg Germany [email protected]

D IETRICH VON S CHWEINITZ Universitäts-Kinderspital beider Basel (UKBB) Basel Switzerland B ÉATRICE S ECRETAN IARC/WHO, Group Carcinogen Identification and Evaluation Lyon Cedex France [email protected] R ONY S EGER Department of Biological Regulation Weizmann Institute of Science Rehovot Israel [email protected] G AIL M. S EIGEL Department of Ophthalmology University at Buffalo Buffalo, NY USA [email protected] H IROYUKI S EIMIYA Division of Molecular Biotherapy, Cancer Chemotherapy Center Japanese Foundation for Cancer Research Koto-ku, Tokyo Japan [email protected] PAULE S EITE Pôle Biologie Santé University of Poitiers Poitiers cedex France [email protected] H ELMUT K. S EITZ Department of Medicine Salem Medical Center, Heidelberg, Center of Alcohol Research Liver Disease and Nutrition and University of Heidelberg Heidelberg, Germany [email protected]

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W ILLIAM R. S ELLERS Harvard Medical School Dana-Farber Cancer Institute Boston, MA USA [email protected] P ERIASAMY S ELVARAJ Department of Pathology Emory University School of Medicine Atlanta, GA USA [email protected] M ARIE G. S ELZER Department of Urology, Cell Biology and Anatomy Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine Miami, FL USA [email protected] S UBRATA S EN Department of Molecular Pathology (Unit 951) The University of Texas, M.D. Anderson Cancer Center Houston, TX USA [email protected] V ITALYI S ENYUK Department of Medicine (M/C 737), College of Medicine Research Building University of Illinois at Chicago Chicago, IL USA [email protected] N EDIME S ERAKINCI Anatomy and Neurobiology Institute of Medical Biology University of Southern Denmark Odense Denmark [email protected] C HRISTINE S ERS Institute of Pathology University Medicine Charité Berlin Germany [email protected] V IJAYASARADHI S ETALURI Department of Dermatology University of Wisconsin School of Medicine and Public Health Sciences Madison, WI USA [email protected]

J OHN F. S EYMOUR Peter MacCallum Cancer Center and the University of Melbourne Melbourne, VIC Australia [email protected] R ABIA K S HAHID Saskatoon Cancer Center University of Saskatchewan Saskatoon, SK Canada [email protected] G IRISH V. S HAH Department of Pharmacology University of Louisiana College of Pharmacy Monroe, LA USA [email protected] A. S HARMA Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA R ICKY A. S HARMA Radiation Oncology and Biology University of Oxford Churchill Hospital Oxford UK [email protected] J ERRY W. S HAY University of Texas Southwestern Medical Center Dallas, TX USA [email protected] S HIJIE S HENG Department of Pathology, Wayne State University School of Medicine Karmanos Cancer Institute Detroit, MI USA [email protected] D ONNA S HEWACH Department of Pharmacology University of Michigan Medical School Ann Arbor, MI USA [email protected] I E -M ING S HIH Department of Pathology Johns Hopkins University School of Medicine Baltimore, MD USA [email protected]


K ENTARO S HIKATA Department of Environmental Medicine Graduate School of Medical Sciences Kyushu University Fukuoka Japan [email protected] Y OSEF S HILOH Sackler School of Medicine, Tel Aviv University Tel Aviv, Israel [email protected] H YUNSUK S HIM Department of Hematology/Oncology Winship Cancer Institute, Emory University Atlanta, GA USA [email protected] Y UTAKA S HIMADA Department of Surgery Graduate School of Medicine Kyoto University Kyoto Japan [email protected] Y ONG -B EOM S HIN BioNanotechnology Research Center Korea Research Institute of Bioscience and Biotechnology Yuseong, Daejeon Republic of Korea [email protected]

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A NTONIO S ICA Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected] G ENE P. S IEGAL Departments of Pathology, Cell Biology, and Surgery and the Gene Therapy Center University of Alabama at Birmingham Birmingham, AL USA [email protected] D IETMAR W. S IEMANN Department of Radiation Oncology University of Florida Gainesville, FL USA [email protected] C HRISTINE L. E. S IEZEN National Institute of Public Health and Environment Bilthoven The Netherlands [email protected] A LEXANDRA C. S ILVEIRA Department of Pathology and Comprehensive Cancer Center University of Alabama at Birmingham Birmingham, AL USA [email protected]

T OSHI S HIODA Massachusetts General Hospital Center for Cancer Research Charlestown, MA USA [email protected]

D IANE M. S IMEONE Department of Surgery University of Michigan Medical Center Ann Arbor, MI USA [email protected]

J ANET S HIPLEY The Institute of Cancer Research, Sutton Surrey UK [email protected]

H ANS -U WE S IMON Department of Pharmacology University of Bern Bern, Switzerland [email protected]

G IRJA S. S HUKLA Department of Surgery, Vermont Comprehensive Cancer Center, College of Medicine University of Vermont Burlington, VT USA [email protected]

A MRIK J. S INGH Department of Pathology Harvard Medical School, Beth Israel Deaconess Medical Center Boston, MA USA [email protected]

A RTHUR S HULKES Department of Surgery University of Melbourne, Austin Health Melbourne, VIC Australia [email protected]

S HREE R AM S INGH Mouse Cancer Genetics Program National Cancer Institute at Frederick Frederick, MD USA [email protected]

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V INEETA S INGH School of Surgery and Pathology Sir Charles Gairdner Hospital, QEII Medical Centre Nedlands, WA Australia [email protected]

J OSEF S MOLLE Department of Dermatology Medical University Graz Graz Austria [email protected]

L ILLIAN L. S IU Department of Medical Oncology and Hematology Robert and Maggie Bras and Family New Drug Development Program Princess Margaret Hospital Toronto, ONT Canada [email protected]

LYNN S NIDERHAN Department of Microbiology and Immunology University of Rochester Rochester, NY USA [email protected]

A NITA S JÖLANDER Cell and Experimental Pathology, Department of Laboratory Medicine Lund University, Malmo University Hospital Malmo Sweden [email protected]

R OBERT W. S OBOL Department of Pharmacology University of Pittsburgh School of Medicine, Hillman Cancer Center, University of Pittsburgh Cancer Institute Pittsburgh, PA USA [email protected]

J. S KONER Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA K EITH S KUBITZ Division of Hematology, Oncology and Transplantation University of Minnesota Medical School Minneapolis, MN USA [email protected] C HRISTOPHER S LAPE Genetics Branch, Center for Cancer Research National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected] K EIRAN S. M. S MALLEY The Wistar Institute Philadelphia, PA USA [email protected]

A LEXANDER S. S OBOLEV Department of Molecular Genetics of Intracellular Transport Institute of Gene Biology Russian Academy of Sciences Moscow Russia [email protected] G RAZIELLA S OLINAS Department of Immunology Fondazione Humanitas per la Ricerca Rozzano, Milan Italy [email protected] T OSHIYA S OMA Department of Surgery Graduate School of Medicine Kyoto University Kyoto Japan [email protected]

L UBOMIR B. S MILENOV Department of Radiation Oncology Columbia University New York, NY USA [email protected]

PAVEL S OUCEK Group for Biotransformations, Center of Occupational Medicine National Institute of Public Health Prague Czech Republic [email protected]

B RUCE F. S MITH Scott-Ritchey Research Center College of Veterinary Medicine Auburn University Auburn, AL USA [email protected]

T HOMAS D. S OUTHGATE Paterson Institute for Cancer Research University of Manchester Manchester UK [email protected]


L ORENZO S PAGGIARI Department of Thoracic Surgery European Institute of Oncology Milan Italy [email protected] D AVID W. S PEICHER The Wistar Institute Philadelphia, PA USA [email protected] VALERIE S PEIRS Leeds Institute of Molecular Medicine University of Leeds Leeds UK [email protected] D IETMAR S PENGLER Max-Panck-Institut für Psychiatrie München Germany [email protected] P HILLIPPE E. S PIESS Department of Urologic Oncology University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected] C HRISTOPH S PRINGFELD Department of Internal Medicine IV University of Heidelberg Hospital Heidelberg Germany [email protected]

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M. S HARON S TACK Northwestern University Medical School Chicago, IL USA [email protected] J AN A. S TAESSEN Division of Lung Toxicology Department of Occupational and Environmental Medicine (T.S.N.) and the Studies Coordinating Centre (J.A.S.) Division of Hypertension and Cardiovascular Rehabilitation Department of Cardiovascular Diseases University of Leuven Leuven Belgium [email protected] L. J OE S TAFFORD Department of Pathology and Comprehensive Cancer Center University of Alabama-Birmingham Birmingham, AL USA [email protected] E RIC S TANBRIDGE Department of Microbiology and Molecular Genetics University of California Irvine, CA USA [email protected] B ARRY S TAYMATES Department of Pathology Henry Mayo Newhall Memorial Hospital Valencia, CA USA [email protected] S TACEY S TEIN Center for Advanced Biotechnology and Medicine UMDMJ – Robert Wood Johnson Medical School Piscataway, NJ USA

L AKSHMAIAH S REERAMA Department of Chemistry St. Cloud State University St. Cloud, MN USA [email protected]

M ARTIN S TEINHOFF Department of Dermatology University of Münster Münster Germany [email protected]

S ATISH K. S RIVASTAVA Dept of Biochemistry and Molecular Biology University Of Texas Medical Branch Galveston, TX USA [email protected]

A LEXANDER S TEINLE Department of Immunology Institute for Cell Biology Eberhard-Karls-University Tuebingen Germany [email protected]

S UDHIR S RIVASTAVA Division of Cancer Prevention National Cancer Institute Rockville, MD USA [email protected]

C ARSTEN S TEPHAN Department of Urology, Charité Universitätsmedizin Campus Charité Mitte Berlin Germany [email protected]

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P ETER L. S TERN Cancer Research UK Immunology Group Paterson Institute for Cancer Research Manchester, UK [email protected] W ILLIAM G. S TETLER -S TEVENSON Extracellular Matrix Pathology Section, Cell and Cancer Biology Branch National Cancer Institute Bethesda, MD USA [email protected] R ICHARD G. S TEVENS University of Connecticut Health Center Farmington, CT USA [email protected] F REDA S TEVENSON CRC Wessex Oncology Unit, Southampton General Hospital and Tenovous Laboratory Southampton University Hospital Trust Southampton UK W ILLIAM P. S TEWARD Cancer Biomarkers and Prevention Group Department of Cancer Studies, University of Leicester Leicester UK [email protected] C ONSTANTINE A. S TRATAKIS Department of Pediatric Endocrinology NICHD, NIH Bethesda, MD USA [email protected] A LEX Y. S TRONGIN Burnham Institute for Medical Research La Jolla, CA USA [email protected] G ARNET S UCK Health Sciences Authority Centre for Transfusion Medicine Singapore [email protected] PAUL H. S UGARBAKER Washington Cancer Institute, Washington Hospital Center Washington, DC USA [email protected] M AXWELL S UMMERHAYES Roche Products Limited Welwyn Garden City UK [email protected]

B AOCUN S UN Department of Pathology Tianjin Cancer Hospital and Tianjin Cancer Institute Tianjin P.R of China [email protected] D UXIN S UN Division of Pharmaceutics, College of Pharmacy The Ohio State University Columbus, OH USA [email protected] S HI -Y ONG S UN Winship Cancer Institute Emory University Atlanta, GA USA [email protected] Z HIFU S UN Department of Health Sciences Research Mayo Clinic College of Medicine Rochester, MN USA [email protected] S AUL S USTER The Ohio State University Columbus, OH USA [email protected] S RILATHA S WAMI Division of Endocrinology Department of Medicine, Stanford University School of Medicine Stanford, CA USA [email protected] R USSELL S ZMULEWITZ Department of Medicine, Section of Hematology/Oncology The University of Chicago Chicago, IL USA [email protected] D AY TA Head and Neck Tumor Program Hollings Cancer Center Medical University of South Carolina Charleston, SC USA [email protected] D IRK TAEGER Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin (BGFA) Institute of Ruhr University Bochum Bochum Germany [email protected]


M ASATOSHI TAGAWA Division of Pathology Chiba Cancer Center Research Institute Chiba, Chuo-ku Japan [email protected]

M ASAAKI TAMURA Department of Anatomy and Physiology Kansas State University Manhattan, KS USA [email protected]

Y OSHIKAZU TAKADA UC Davis School of Medicine Sacramento, CA USA [email protected]

TAKUJI TANAKA Deaprtment of Oncologic Pathology Kanazawa Medical University Kanazawa Japan [email protected]

A KIHISA TAKAHASHI Department of Biology Nara Medical University School of Medicine Kashihara, Nara Japan [email protected] T SUTOMU TAKAHASHI Laboratory of Molecular and Biochemical Toxicology Graduate School of Pharmaceutical Sciences Tohoku University Sendai Japan [email protected] Y OSHIMI TAKAI Osaka University Graduate School of Medicine/Faculty of Medicine Suita Japan [email protected] TAKASHI TAKATA Department of Oral and Maxillofacial Pathobiology, Division of Frontier Medical Science, Graduate School of Biomedical Sciences Hiroshima University Hiroshima Japan [email protected] TAMOTSU TAKEUCHI Department of Pathology Kochi Medical School Kochi Japan [email protected] C ONSTANTINE S. TAM The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected] H ARALD TAMMEN Digilab BioVisioN GmbH Hannover Germany [email protected]

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D AVID S. P. TAN Section of Medicine Institute of Cancer Research, The Royal Marsden Hospital London UK [email protected] D EAN G. TANG Department of Carcinogenesis, The University of Texas M.D Anderson Cancer Center Science Park-Research Division Smithville, TX USA [email protected] N IZAR M. TANNIR Department of Genitourinary Medical Oncology University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected] W EIKANG TAO Department of Cancer Research Merck Research Laboratories West Point, PA USA [email protected] C HI TARN Department of Medical Oncology Fox Chase Cancer Center Philadelphia, PA USA [email protected] C LIVE R. TAYLOR Departments of Urology and Pathology University of Southern California Keck School of Medicine Los Angeles, CA USA [email protected] J ENNIFER TAYLOR Committee on Cancer Biology The University of Chicago Chicago, IL USA [email protected]

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A NDREW R. T EE Institute of Medical Genetics Cardiff University Heath Park, Cardiff UK [email protected] AYALEW T EFFERI Division of Hematology Mayo Clinic College of Medicine Rochester, MN USA [email protected] B IN T EAN T EH Laboratory of Cancer Genetics Van Andel Research Institute Grand Rapids, MI USA [email protected] V IGGO VAN T ENDELOO Laboratory of Experimental Hematology Antwerp University Hospital Edegem Belgium [email protected] J OSEPH R. T ESTA Fox Chase Cancer Center Philadelphia, PA USA [email protected] J OHN T HACKER Medical Research Council Radiation and Genome Stability Unit Harwell, Oxfordshire UK [email protected] R AJESH V. T HAKKER Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Centre for Diabetes Endocrinology and Metabolism (OCDEM), Churchill Hospital, Headington Oxford UK [email protected] N ICHOLAS B. L A T HANGUE Division of Biochemistry and Molecular Biology Davidson Building University of Glasgow Glasgow UK [email protected] D AN T HEODORESCU Department of Urology University of Virginia Charlottesville, VA USA [email protected]

PANAYIOTIS A. T HEODOROPOULOS Department of Basic Sciences The University of Crete, School of Medicine Heraklion, Crete Greece K ARL -H EINZ T HIERAUCH Therapeutic Research Group Oncology Bayer Schering Pharma AG Berlin Germany [email protected] M EGAN N. T HOBE University of Cincinnati College of Medicine Cincinnati, OH USA [email protected] G ARETH J. T HOMAS Institute of Cancer, Bart’s and the London School of Medicine and Dentistry Queen Mary University of London London UK [email protected] N ATALIE T HOMAS Department of Biochemistry and Molecular Biology Monash University Melbourne, VIC Australia [email protected] P ETER T HOMAS Departments of Surgery and Biomedical Sciences Creighton University Omaha, NE USA [email protected] S VEN T HOMS Department of Paediatrics and Paediatric Neurology University of Göttingen Göttingen Germany [email protected] A NDREW T HORBURN University of Colorado at Denver and Health Sciences Center Aurora, CO USA [email protected] M AGNUS T HÖRN Department of Surgery (MT) Karolinska Institutet Stockholm Sweden [email protected]


D ERYA T ILKI Department of Urology University Hospital Groβhadern Munich Germany [email protected]

M ASSIMO T OMMASINO Infections and Cancer Biology Group International Agency for Research on Cancer Lyon France [email protected]

D ONALD J. T INDALL Department of Urology Research, Department of Biochemistry and Molecular Biology Mayo Clinic College of Medicine Rochester, Minnesota USA [email protected]

A NTONIO T ONINELLO Department of Biochemical Sciences “A. Rossi Fanelli” Sapienza University of Rome Rome Italy [email protected]

U MBERTO T IRELLI National Cancer Institute Aviano Italy [email protected] M ARTIN T OBI Section of Gastroenterology Detroit VAMC, Detroit MI USA [email protected] P EIKERT T OBIAS Division of Pulmonary and Critical Care Medicine Department of Internal Medicine Mayo Clinic College of Medicine Rochester USA [email protected] P HILIP J. T OFILON Department of Interdisciplinary Oncology, Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute University of South Florida Tampa, FL USA [email protected] H ENRIK T OFT S ØRENSEN Department of Clinical Epidemiology Aarhus University Hospital Aarhus C Denmark M ASAKAZU T OI Department of Surgery (Breast Surgery) Graduate School of Medicine, Kyoto University Bunkyo-ku, Kyoto Japan [email protected] A MANDA E WART T OLAND Division of Human Cancer Genetics The Ohio State University Columbus, OH USA [email protected]

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J EFFREY A. T ORETSKY Departments of Pediatric Hematology and Oncology Lombardi Comprehensive Cancer Center, Georgetown University Washington DC, NW USA [email protected] J ORGE R. T ORO National Institutes of Health Bethesda, MD USA [email protected] T IFFANY A. T RAINA Breast Cancer Medicine Service Department of Medicine Memorial Sloan-Kettering Cancer Center New York, NY USA [email protected] L UBA T RAKHTENBROT Head of Molecular Cytogenetics Laboratory Institute of Hematology, The Chaim Sheba Medical Center Tel Hashomer, Israel [email protected] P IERRE -L UC T REMBLAY Le Centre de recherche en cancérologie de l’Université Laval Québec, QC Canada [email protected] G REGORY J. T SAY Department of Medicine, Institute of Immunology Chung Shan Medical University Taichung Taiwan [email protected] A POSTOLIA -M ARIA T SIMBERIDOU Department of Investigational Cancer Therapeutics, Division of Cancer Medicine The University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected]

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K UNIHIRO T SUCHIDA Division for Therapies against Intractable Diseases Institute for Comprehensive Medical Science (ICMS) Fujita Health University Toyoake Japan [email protected]

S PECKS U LRICH Division of Pulmonary and Critical Care Medicine Department of Internal Medicine Mayo Clinic College of Medicine Rochester USA [email protected]

N OBUO T SUCHIDA Department of Molecular Cellular Oncology and Microbiology Tokyo Medical and Dental University Bunkyo-ku Tokyo Japan [email protected]

N ICK U NDERHILL -D AY School of Biosciences University of Birmingham Birmingham UK [email protected]

M EHMET K EMAL T UR Department of Experimental Medicine and Immunotherapy Helmholtz Institute for Biomedical Engineering University Hospital RWTH Aachen Aachen Germany [email protected] G REG T URENCHALK Senior Bioinformatics Developer 454 Life Sciences Branford, CT USA [email protected] A NDREW S. T URNELL Cancer Research UK Institute for Cancer Studies The Medical School The University of Birmingham Birmingham UK [email protected] M ICHELLE C. T URNER McLaughlin Centre for Population Health Risk Assessment Institute of Population Health University of Ottawa Ottawa, ON Canada [email protected]

R OSEMARIE A. U NGARELLI Boston Medical Center and Boston University School of Medicine Boston, MA USA [email protected] A NTTI VAHERI Haartman Institute University of Helsinki Helsinki Finland [email protected] K EDAR S. VAIDYA Department of Pathology and Comprehensive Cancer Center University of Alabama at Birmingham Birmingham, AL USA [email protected] I LAN VAKNIN The Lautenberg Center for General and Tumor Immunology The Hebrew University-Hadassah Medical School Jerusalem Israel [email protected] A NNE M. VAN B USKIRK Department of Surgery The Ohio State University Colombus, OH USA [email protected]

T HOMAS T ÜTING Laboratory for Experimental Dermatology Department of Dermatology University of Bonn Bonn Germany [email protected]

M ICHAEL W. VAN D YKE Department of Molecular and Cellular Oncology The University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected]

S ALVATORE U LISSE Department of Experimental Medicine University of Rome-Sapienza Rome Italy [email protected]

C ARTER VAN WAES National Institute on Deafness and Other Communication Disorders and National Cancer Institute NIH Bethesda, MD USA [email protected]


G EORGE F. VANDE W OUDE Van Andel Research Institute Grand Rapids, MI USA [email protected] W IM VANDEN B ERGHE Lab of Eukaryotic Gene Expression LEGEST-University Gent Gent Belgium [email protected] S AKARI VANHARANTA Department of Medical Genetics Biomedicum Helsinki University of Helsinki Helsinki Finland [email protected] R OBERTA VANNI Department of Biomedical Science & Technology University of Cagliari Monserrato (CA), Italy [email protected] J UDITH A. VARNER Moores UCSD Cancer Center University of California San Diego La Jolla, CA USA [email protected] A IKATERINI T. VASILAKI University Department of Surgery Royal Infirmary Glasgow UK [email protected] P ETER VAUPEL Institute of Physiology and Pathophysiology University of Mainz Mainz, Germany [email protected]

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M ARCEL V ERHEIJ Department of Radiotherapy The Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital Amsterdam The Netherlands [email protected] M UKESH V ERMA Analytical Epidemiology Research Branch, Epidemiology and Genetics Research Program, Division of Cancer Control and Population Sciences National Cancer Institute, Bethesda MD USA [email protected] R AKESH V ERMA Antisoma Research Laboratories, St. George’s Hospital Medical School London, UK [email protected] S RDAN V ERSTOVSEK MD Anderson Cancer Center University of Texas Houston, TX USA [email protected] R ENÉ P. H. V ETH Department of Orthopaedics Radboud University Medical Centre Nijmegen The Netherlands [email protected] GJ V ILLARES Department of Cancer Biology The University of Texas, M.D. Anderson Cancer Center Houston, TX USA A KILA N. V ISWANATHAN Brigham and Women’s/Dana-Farber Cancer Center Boston, MA USA [email protected]

G UILLERMO V ELASCO Department of Biochemistry and Molecular Biology I School of Biology Complutense University Madrid Spain [email protected]

K RIS V LEMINCKX Department of Molecular Biology Ghent University – VIB Ghent Belgium [email protected]

A SHOK R. V ENKITARAMAN Hutchison/MRC Cancer Research Centre Cambridge UK [email protected]

I SRAEL V LODAVSKY Department of Oncology Hadassah Hospital, Jerusalem Israel [email protected]

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M ARTINA V OCKERODT Department of Pediatrics I, Children’s Hospital Georg-August University of Goettingen Goettingen Germany [email protected]

D ONGMEI WANG Georgetown University Lombardi Comprehensive Cancer Center Washington, DC USA [email protected]

C HARLES L. V OGEL Sylvester Cancer Center, School of Medicine University of Miami Plantation, FL USA [email protected]

G ANG WANG Department of Neurology and Neuroscience Weill Medical College of Cornell University New York, NY USA [email protected]

T ILMAN V OGEL Department of Surgery Krankenhaus Maria Hilf Mönchengladbach Germany [email protected]

J IANGHUA WANG Department of Pathology Baylor College of Medicine Houston, TX USA

U LLA V OGEL National Research Centre for the Working Environment København Ø Denmark [email protected] D ANIEL D. VON H OFF Arizona Cancer Center Tucson, AZ USA [email protected] A LIREZA V OSOUGH Department of Radiotherapy Royal Marsden Hospital NHS Foundation Trust Sutton, Surrey UK [email protected] C HRISTOPH WAGENER Institute of Clinical Chemistry University Medical Center Hamburg Eppendorf Hamburg Germany [email protected] S ABINE WAGNER Department of Pediatrics Klinik St. Hedwig, Krankenhaus der Barmherzigen Brüder Regensburg Germany [email protected] H ÅKAN WALLIN National Research Centre for the Working Environment København Ø Denmark [email protected] S USAN E. WALTZ Shriner’s Hospital for Children Cincinnati, OH USA [email protected]

R ONG -F U WANG Departments of Pathology and Immunology Center for Cell and Gene Therapy Baylor College of Medicine Houston, TX USA [email protected] X IANG -D ONG WANG Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University Boston, MA USA [email protected] X IANGHONG WANG Department of Anatomy The University of Hong Kong Hong Kong China [email protected] Y U WANG Department of Medicine and Genome Research Center University of Hong Kong Hong Kong, China [email protected] PATRICK WARNAT Department of Theoretical Bioinformatics German Cancer Research Center Heidelberg Germany [email protected] K OUNOSUKE WATABE Department of Medical Microbiology, Immunology and Cell Biology Southern Illinois University, School of Medicine Springfield, IL USA [email protected]


VALERIE M. W EAVER Department of Surgery University of California San Francisco, CA USA [email protected] S COTT A. W EED Department of Neurobiology and Anatomy, Mary Babb Randolph Cancer Center West Virginia University Morgantown, WV USA [email protected] O LIVER W EIGERT Department of Internal Medicine III University of Munich, Großhadern München Germany [email protected] E UGENE D. W EINBERG Biology and Medical Sciences Indiana University Bloomington, IN USA [email protected] I. B ERNARD W EINSTEIN Columbia University New York, NY USA [email protected] E LLEN W EISBERG Department of Medical Oncology Dana Farber Cancer Institute Boston, MA USA [email protected] L AWRENCE M. W EISS Division of Pathology City of Hope National Medical Center Duarte, CA USA [email protected] D ANNY R. W ELCH Department of Pathology and Comprehensive Cancer Center University of Alabama at Birmingham Birmingham, AL USA [email protected] S ARAH J. W ELSH Harris Manchester College University of Oxford Oxford UK [email protected]

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TANIA M. W ELZEL Division of Cancer Epidemiology and Genetics National Cancer Institute National Institutes of Health Bethesda, MD USA [email protected] C LAUDIA W ENDEL Department of General Surgery University Hospital Münster Münster Germany [email protected] TAMRA E. W ERBOWETSKI -O GILVIE McMaster Stem Cell and Cancer Research Institute Hamilton, ON Canada [email protected] F RANK W ESTERMANN German Cancer Research Center Department of Tumour Genetics Heidelberg Germany [email protected] A INSLEY W ESTON National Institute for Occupational Safety and health, CDC Morgantown, WV USA [email protected] L INDA C. W HELAN UCD School of Biomolecular and Biomedical Science UCD Conway Institute University College Dublin Dublin Ireland [email protected] R OBERT P. W HITEHEAD Department on Medicine, Division of Hematology/Oncology University of Texas Medical Branch Galveston, TX USA [email protected] T HERESA L. W HITESIDE University of Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine Pittsburgh, PA USA [email protected] A NDREAS W ICKI Department of Clinical-Biological Sciences, Centre of Biomedicine Institute of Biochemistry and Genetics, University of Basel Basel Switzerland [email protected]

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C AROL W ICKING Institute for Molecular Bioscience The University of Queensland St Lucia, QLD Australia [email protected]

C HRISTIANE D E W OLF -P EETERS Department of Pathology University Hospitals of K.U. Leuven Leuven Belgium [email protected]

E DWIN VAN W IJNGAARDEN Department of Community and Preventive Medicine University of Rochester School of Medicine and Dentistry Rochester, NY USA [email protected]

C. M. W ONG Department of Pathology The University of Hong Kong Hong Kong [email protected]

E LIZABETH D. W ILLIAMS Cancer Metastasis Laboratory Monash Institute of Medical Research, Monash University Clayton, VIC Australia [email protected] J AMES P. W ILMOT Cancer Center University of Rochester Medical Center Rochester, NY USA O LA W INQVIST Department of Medicine (OW) Karolinska Institutet Stockholm Sweden [email protected] J OHN P IERCE W ISE Department of Applied Medical Sciences, Maine Center for Toxicology and Environmental Health University of Southern Maine Portland, ME USA [email protected] O LAF W ITT CCU Pediatric Oncology German Cancer Research Center Heidelberg Germany [email protected]

D ORI C. W OODS Walther Cancer Research Center University of Notre Dame Notre Dame, IN USA [email protected] PAUL W ORKMAN Cancer Research UK Centre for Cancer Therapeutics The Institute of Cancer Research Sutton, Surrey UK [email protected] T HOMAS W ORZFELD Institute of Pharmacology, University of Heidelberg Heidelberg Germany [email protected] X IFENG W U Department of Epidemiology The University of Texas MD Anderson Cancer Center Houston, TX USA [email protected] G UANG -H UI X IAO Fox Chase Cancer Center Philadelphia, PA USA [email protected]

I SAAC P. W ITZ Department of Cell Research and Immunology Tel Aviv University Tel Aviv, Israel [email protected]

J IANMING X U Department of Molecular and Cellular Biology Baylor College of Medicine Houston, TX USA [email protected]

I DO W OLF Institute of Oncology and the Cancer Research Center, Chaim Sheba Medical Center and the Sackler Faculty of Medicine Tel Aviv University Tel Aviv Israel [email protected]

T IAN X U Professor and Vice Chair of Genetics Howard Hughes Medical Institute Yale University School of Medicine New Haven, CT USA [email protected]


J UDY W. P. YAM Department of Pathology The University of Hong Kong Hong Kong [email protected]

H ELEN L. Y IN Department of Physiology University of Texas Southwestern Medical Center Dallas, TX USA [email protected]

S HO - ICHI YAMAGISHI Division of Cardiovascular Medicine, Department of Medicine Kurume University School of Medicine Kurume Japan [email protected]

X IAO -M ING Y IN Department of Pathology University of Pittsburgh School of Medicine Pittsburgh, PA USA [email protected]

M ICHIKO YAMAMOTO Department of Respiratory Medicine Kitasato University School of Medicine Sagamihara, Kanagawa Japan [email protected]

Z HIMIN Y IN College of Life Sicence Nanjing Normal University Nanjing People’s Republic of China [email protected]

H AINING YANG Cancer Research Center of Hawaii University of Hawaii Honolulu, HI USA

K ENNETH W. Y IP Burnham Institute for Medical Research La Jolla CA USA [email protected]

H ONG YANG Cancer Vaccine Branch, National Cancer Institute, National Institutes of Health Bethesda, MD USA [email protected]

H ARRY H. Y OON Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Baltimore, MD USA [email protected]

P ING YANG Department of Health Sciences Research Mayo Clinic College of Medicine Rochester, MN USA [email protected]

J UNG -H WAN Y OON Department of Internal Medicine Seoul National University College of Medicine Chongno-gu Seoul South Korea [email protected]

M ASAKAZU YASHIRO Department of Surgical Oncology Osaka City University Graduate School of Medicine Osaka Japan [email protected]

K AZUHIRO Y OSHIDA Department of Surgical Oncology Gifu University School of Medicine Gifu Japan [email protected]

W. A NDREW Y EUDALL Philips Institute of Oral and Craniofacial Molecular Biology Virginia Commonwealth University Richmond, VA USA [email protected]

TATSUSHI Y OSHIDA Department of Molecular-Targeting Cancer Prevention Graduate School of Medical Science Kyoto Prefectural University of Medicine Kyoto Japan

M AKSYM V. Y EZHELYEV Winship Cancer Institute Emory University Atlanta, GA USA [email protected]

A NAS Y OUNES Department of Lymphoma and Myeloma M. D. Anderson Cancer Center Houston, TX USA [email protected]

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G RAEME P. Y OUNG Flinders Cancer Control Alliance Flinders University Adelaide, SA Australia [email protected]

L AURA P. Z ANELLO Department of Biochemistry University of California-Riverside Riverside, CA USA [email protected]

X IAO Y UAN Research and Development Center, Wuhan Botanical Garden Chinese Academy of Science Wuhan, Hubei People’s Republic of China [email protected]

U WE Z ANGEMEISTER -W ITTKE Department of Pharmacology University of Bern Bern, Switzerland [email protected]

J IAN Y U Departments of Pathology and Pharmacology University of Pittsburgh Cancer Institute University of Pittsburgh School of Medicine Pittsburgh, PA USA [email protected]

YAN P ING Y U Department of Pathology University of Pittsburgh Pittsburgh, PA USA [email protected]

YU YU Department of Pathology University of Sydney NSW Australia [email protected]

A NTHONY P O -W ING Y UEN Division of Otorhinolaryngology Department of Surgery, The University of Hong Kong Hong Kong, SAR China [email protected]

L EO R. Z ACHARSKI VA Hospital White River Junction VT USA [email protected]

G ERARD P. Z AMBETTI Department of Biochemistry St. Jude Children’s Research Hospital Memphis, TN USA [email protected]

A NDREW C. W. Z ANNETTINO IMVS Main Laboratory Adelaide, SA Australia [email protected] B ERTON Z BAR Laboratory of Immunobiology NIH – Frederick Frederick, MD USA [email protected] H ERBERT J. Z EH III University of Pittsburgh, Departments of Surgery and Bioengineering, Pittsburgh, PA USA [email protected] J ASON A. Z ELL Cancer Prevention Program Division of Hematology/Oncology and Epidemiology Department of Medicine School of Medicine, Chao Family Comprehensive Cancer Center, University of California Irvine, CA USA [email protected] D ANFANG Z HANG Department of Pathology Tianjin Cancer Hospital and Tianjin Cancer Institute Tianjin P.R of China [email protected] H AO Z HANG The University of Texas M. D. Anderson Cancer Center Houston, TX USA [email protected] H ONG Z HANG Biogen Idec San Diego, CA USA [email protected]


J I -H U Z HANG Lead Discovery Center Novartis Institute for Biomedical Research Cambridge, MA USA [email protected] J INPING Z HANG Departments of Pathology and Immunology, Center for Cell and Gene Therapy Baylor College of Medicine Houston, TX USA L IN Z HANG Biogen Idec San Diego, CA USA [email protected] L IN Z HANG Departments of Pathology and Pharmacology University of Pittsburgh Cancer Institute University of Pittsburgh School of Medicine Pittsburgh, PA USA [email protected] R UIWEN Z HANG University of Alabama at Birmingham Birmingham, AL USA [email protected]

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Y UESHENG Z HANG Roswell Park Cancer Institute Buffalo, NY USA [email protected] W EILING Z HAO Department of Radiation Oncology and Brain Tumor Center of Excellence Wake Forest University School of Medicine Winston-Salem, NC USA [email protected] G UANG -B IAO Z HOU State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital School of Medicine Shanghai Jiao Tong University Shanghai, People's Republic of China [email protected] ZENG B. ZHU Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology and the Gene Therapy Center, Division of Human Gene Therapy University of Alabama at Birmingham Birmingham, AL USA [email protected] M. Z IGLER Department of Cancer Biology The University of Texas, M.D. Anderson Cancer Center Houston, TX USA

S HIWU Z HANG Department of Pathology Tianjin Cancer Hospital and Tianjin Cancer Institute Tianjin P.R of China [email protected]

M ARGOT Z OELLER DKFZ Heidelberg Germany [email protected]

X IN A. Z HANG Vascular Biology Center, Cancer Institute, and Departments of Medicine and Molecular Sciences University of Tennessee Health Science Center Memphis, TN USA [email protected]

M ASSIMO Z OLLO Department of Genetics Faculty of Biotechnological Science Federico II, Naples CEINGE, Biotecnologie Avanzate Naples Italy [email protected]

X UEFENG Z HANG Beth Israel Deaconess Medical Center and Harvard Medical School Boston, MA USA [email protected]

E NRIQUE Z UDAIRE NCI Angiogenesis Core Facility, National Cancer Institute National Institutes of Health Advanced Technology Center Gaithersburg, MD USA [email protected]

Y U -W EN Z HANG Laboratory of Molecular Oncology Van Andel Research Institute Grand Rapids, MI USA [email protected]

C ARSTEN Z WICK Klinik für Innere Medizin I Universität des Saarlandes Homburg Germany [email protected]

A

2ar ▶Osteopontin

AAA+ Definition Superfamily of proteins characterized by a segment of ~220 aminoacids (the AAA domain) containing several conserved motifs including those necessary for ATP binding and hydrolysis.

17-1A

▶APAF-1 Signaling

▶EpCAM

AAMP M ARIE E. B ECKNER

A Disintegrin and Metalloprotease ▶ADAM Molecules

A-Scan Ultrasonography Definition Ophthalmologic ultrasound that provides singledimension information on the ultrasonic echogenicity of the ocular tissues providing information regarding axial length, size of intraocular structures (such as tumor height), and homogeneity of individual tissues within the eye. ▶Uveal Melanoma

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Definition AAMP stands for angio-associated migratory cell protein and its gene is found on chromosome 2q35. ▶HUGO nomenclature symbol is AAMP. AAMP has been conserved in evolution, is distributed intracellularly in many cells and also extracellularly on vascular cells, shares an ▶epitope with motility-related proteins (▶alpha-actinin and a fast twitch skeletal muscle protein) and contains potential heparin binding and thrombin cleavage sites. Antibody and antisense studies have indicated compartment (intracellular or extracellular) specific roles for AAMP in angiogenesis, cell-cell and cell-matrix interactions, and cell migration.

Characteristics The cDNA derived from mRNA encoding AAMP was originally cloned from a human melanoma cell library (A2058) in a search for migration-related proteins. AAMP has been found in the cytoplasm of many

2

AAMP

nucleated cells, in an extracellular mesh-like network on monolayers of endothelial and vascular-associated smooth muscle cells, and on the apical membranes of endometrial glandular cells. AAMP expression when normalized for tissue source has shown the highest levels of distribution in the esophagus (7.17% of tissue clones) (http://smd.stanford.edu/cgi-bin/source/sourceImage? File = Hs.83347). Local homologies discovered initially to human immunodeficiency viral proteins led to identification of two immunoglobulin-like ▶domains in AAMP. In addition to melanoma, expression of AAMP has been observed in a variety of malignant cells, including poorly differentiated colon adenocarcinoma within lymphatics, gastric adenocarcinoma, Jurkat lymphoma, gastrointestinal stromal tumors with mutated ▶c-kit, breast cancer cell lines and ductal adenocarcinoma in situ with necrosis, and brain tumor cells. Co-culture of astrocytes with endothelial cells (without physical contact) led to increased amounts of extracellular AAMP associated with the endothelial cells. Stimulation of T lymphocytes and monocytes by a ▶phorbol ester led to greatly increased AAMP expression, 1.6 kb message and 52 kDa protein. Hypoxia increased expression of the AAMP gene in a breast carcinoma cell line. AAMP has demonstrated compartment-specific effects on endothelial cell migration. Affinity-purified antibodies, that interacted with the extracellular form of AAMP on non-permeabilized endothelial cells, inhibited cell migration and endothelial tube formation. However, anti-sense oligonucleotides, that decreased total AAMP expression, paradoxically increased cell migration, presumably via loss of intracellular AAMP. The structure of AAMP was initially characterized as having two immunoglobulin-like domains and six ▶WD repeats. Now eight WD repeats have been identified in AAMP, UniProt KB/Swiss-Prot Q13685. AAMP has been conserved in evolution. Comparisons of reference sequences for human AAMP (433 aa) with related forms in mouse (434 aa), rat (471 aa), chicken (419 aa), frog (438 aa), and zebrafish (408 aa) have shown 99.5, 98.9, 86.7, 76.5, and 69.0% identity, respectively (UniGene, NCBI, NIH). An acid box (short contiguous run of glutamic or aspartic acid residues) has been identified in the amino terminal regions of several AAMP homologs. They are comprised of seven glutamic acids in human, eight glutamic acids in mouse and rat, and six aspartic acid residues in the zebrafish forms of AAMP. AAMP contains a strongly immunoreactive ESESES epitope at its ▶amino terminal end that has been used to generate an anti-peptide antibody. Under normal reducing conditions, the epitope is immunoreactive for AAMP only in lysates of human brain and activated T lymphocytes. AAMP (52 kDa) shares this epitope with non-skeletal alpha-actinin (100 kDa) and an

unidentified fast twitch skeletal muscle fiber protein (23 kDa), as demonstrated with anti-RRLRRMESESES (anti-P189) and related anti-peptide antibodies. The ESESES epitope is linear in AAMP but is discontinuous or conformational (formed by ▶secondary structure) in alpha-actinin. The fast twitch skeletal muscle fiber protein with immunoreactivity for anti-P189 was found in the periodic bands (Z discs). An alternatively spliced, slightly longer form of AAMP (452 aa) includes coding sequence upstream from MESESES. The immediate upstream sequence, RRLRR, potentially functions as a heparin binding site. In addition to an alternative initiating methionine, the upstream human coding sequence differs by only two of seventeen codons when compared to an even longer form of AAMP in rat. The coding sequence of AAMP in rat includes the sequence GRFRRMESESES that corresponds to RRLRRMESESES in the alternative form of human AAMP. In peptide studies, the bipolar RRLRRMESESES sequence was strongly self-aggregating, sensitive to thrombin digestion, and displayed binding to heparin and cells as either an immobilized, single peptide or as an aggregated peptide, without affecting cell viability or adhesion to collagen. Peptide sequencing verified the presence of RLRR in recombinant AAMP translated in Escherichia coli following thrombin digestion that cleaved the first R. Although anti-P189 (RRLRRMESESES) did not demonstrate reactivity with the RRLRR epitope in tissue that displayed reactivity with ESESES, the lack of reactivity for RRLRR could have been due to interference by strongly adherent ▶glycosaminoglycans. Thus initial studies of AAMP’s distribution and structure are supportive of a role for this protein in cell migration and angiogenesis.

References 1. Beckner ME, Krutzsch HC, Stracke ML et al. (1995) Identification of a new immunoglobulin superfamily protein expressed in blood vessels with a heparin-binding consensus sequence. Cancer Res 55:2140–2149 2. Allander SV, Nupponen NN, Ringner M et al. (2001) Gastrointestinal stromal tumors with KIT mutations exhibit a remarkably homogeneous gene expression profile. Cancer Res 61:8624–8628 3. Adeyinka A, Emberley E, Niu Y et al. (2002) Analysis of gene expression in ductal carcinoma in situ of the breast. Clin Cancer Res 8:3788–3795 4. Beckner ME, Jagannathan S, Peterson VA (2002) Extracellular angio-associated migratory cell protein plays a positive role in angiogenesis and is regulated by astrocytes in coculture. Microvasc Res 63:259–269 5. Beckner ME, Krutzsch HC, Klipstein S et al. (1996) AAMP, a newly identified protein, shares a common epitope with alpha-actinin and a fast skeletal muscle fiber protein. Exp Cell Res 225:306–314

AAV

3

A

AAPC ▶APC Gene in Familial Adenomatous Polyposis

AAV D IRK G RIMM University of Heidelberg, Cluster of Excellence Cell Networks, BIOQUANT, Heidelberg

Definition Adeno-associated viruses (AAV) are small DNAcontaining viruses that belong to the family of Parvoviridae. Thus far, 11 ▶serotypes of adeno-associated viruses (AAV-1 to AAV-11) have been cloned from humans and primates, and multiple further isolates were identified in various other species, including birds, bovines, mice, rats and goats. According to current knowledge, none of these naturally occurring viruses are pathogenic in humans. AAV type 2 (AAV-2) has been studied for over 40 years and is the best characterized AAV isolate, hence its frequent referral as the AAV prototype. All AAV serotypes are currently being developed and evaluated as gene transfer ▶vectors for the human ▶gene therapy of various inherited or acquired diseases, including different types of cancer.

Characteristics

As typical members of the ▶Parvovirus family, AAV are characterized by non-enveloped, icosahedral capsids of about 18–24 nm in diameter. These capsids carry linear single-stranded DNA genomes of ~4.6–4.8 kb. The genomes of all known AAV serotypes have been cloned and sequenced. With the exception of AAV-4 and -5, which are distinct (>30%) from the other serotypes at both the nucleotide and amino acid level, all human and primate AAV genomes are related and highly homologous (>80%). Accordingly, their genomic structure and organization are also very similar. AAV Genome Structure As an example, the organization of the 4,681 nucleotide AAV-2 prototype genome is described (Fig. 1). The AAV-2 genome consists of two large ▶open reading frames (orf), one at the left end encoding the nonstructural proteins (replication, rep orf), and one at the right end encoding the structural proteins (capsid, cap orf). In addition, a single intron sequence is found in the

AAV. Figure 1 Structure of the AAV-2 genome. The 4,681 nucleotide single-stranded genome is depicted as a solid line; by convention, AAV genomes are drawn in 3′-5′ orientation. Shown are the locations of the rep and cap orfs and the single intron (caret), as well as the position of the three promoters (p5, p19, p40) and the polyA signal, which is used for polyadenylation of all AAV-2 transcripts. Further depicted at the ends of the genome are the palindromic inverted terminal repeat (ITR) sequences in their hairpin configuration.

center of the genome, where the rep and cap orfs overlap. The AAV-2 rep gene encodes four closely related proteins (Rep proteins) with partially shared amino acid sequences. On the basis of their molecular weights, these proteins were designated Rep78, Rep68, Rep52 and Rep40. Unspliced and spliced transcripts originating from a ▶promoter located at map unit 5 (p5) are translated into the two large Rep proteins, Rep78 and Rep68. Rep52 and Rep40 are expressed from similarly spliced mRNAs that initiate from a second promoter, p19. The third AAV-2 promoter, p40, controls transcription of the cap gene. Translation of differentially spliced cap mRNAs results in expression of the three proteins that form the AAV-2 capsid, VP1, VP2 and VP3 (in a 1:1:10 ratio). The two viral genes are flanked by short (AAV-2: 145 nucleotides) inverted terminal repeats (ITR), palindromic sequences that are able to fold into T-shaped stem loop structures. The ITRs are necessary and sufficient for replication and encapsidation of the viral genome during a productive infection of cells. Moreover, they are important for integration and rescue of the AAV DNA into, or from, the genome of latently infected cells, respectively. Thereby, the ITRs serve as minimal cis-acting sequences during the two different AAV life cycles (see also below). AAV Life Cycles AAV serotypes belong to the Parvovirus genus Dependovirus, indicative of their dependence on an unrelated helpervirus to undergo a productive infection of cells. In fact, AAV genomes can only express their genes, replicate and become encapsidated if the cell is simultaneously co-infected by one of these helperviruses. The typical helpervirus for AAV-2 is human ▶Adenovirus type 2 or 5, but many other human viruses can also provide full or partial helper functions, including Herpes simplex virus, Vaccinia virus and Cytomegalovirus.

4

AAV

In the case of Adenovirus, one of the major helper functions is to stimulate AAV gene expression, by trans-activating the AAV-2 promoters. Additional help for the AAV life cycle is mediated at the posttranscriptional level, where adenoviral proteins and RNAs help to facilitate the cytoplasmic transport of AAV-2 mRNAs. Concurrently, adenoviral functions help to stabilize replicated AAV-2 genomic DNA later in the AAV infection. Notably, once expressed in the infected cell, AAV-2 Rep proteins subsequently further regulate and coordinate gene expression from the AAV promoters. They also play important roles for AAV DNA replication, as well as for packaging of viral genomes into empty new capsids (assembled from AAV-2 VP proteins). To mediate these diverse functions, Rep proteins bind to the AAV-2 ITRs and to sequences located in the AAV-2 promoters. They also interact with various cellular proteins, e.g. the TATAbox binding protein ▶(TBP), as well as with each other and the AAV-2 VP proteins. The final step in a productive AAV-2 infection is the helpervirus-mediated lysis of the infected cell. This results in cell death and release of both new AAV-2 and helpervirus particles. In contrast to this productive (or lytic) phase, AAV2 can establish latency in the absence of any helpervirus. Rather than replicating, the AAV-2 DNA then integrates into the target cell genome, where it stably persists as a so-called provirus. Important to point out, wildtype AAV-2 integration is not random, as is the case for retroviruses (▶Retroviral Insertional Mutagenesis) and other integrating viruses. Instead, it is targeted to a specific region on the long arm of human chromosome 19 (19q13.3-ter). The large Rep proteins (albeit only weakly expressed in the absence of a helpervirus) mediate this site-specific integration through binding to the AAV-2 ITRs, as well as to homologous sequences (AAVS1) located in chromosome 19. However, if a latently AAV-infected cell is later super-infected with a helpervirus, AAV-2 gene expression is induced and the AAV-2 genome is rescued from its integrated state. From this point on, a typical productive AAV-2 infection will occur. Thus, the helpervirus can act as an efficient switch between the two different phases that characterize the AAV-2 life cycle, lytic and latent. Clinical Relevance In theory, due to its inherent anti-tumor properties (see below), wildtype AAV-2 (and probably other serotypes alike) could be used as a therapeutic agent for the treatment of human cancers. However, more widely studied and applied are ▶recombinant vectors derived from wildtype AAVs. Typically, these vectors are generated by replacing the two viral genes (rep and cap) with a foreign ▶gene expression cassette, encoding RNAs or proteins that mediate an anti-tumor effect (if used for cancer therapy). The general clinical relevance of

wildtype and recombinant AAVs is briefly discussed below; for more depth, the reader is referred to recent excellent reviews on the use of AAV for the treatment of human disease (see References below). Are Wildtype AAVs Pathogenic in Humans? According to the bulk data available, wildtype AAV serotypes are believed to be non-pathogenic in humans. In fact, despite estimates that up to 80% of adults are ▶seropositive for AAV-2, no human disease has ever been causally linked to infection with the wildtype virus. This is even more remarkable considering that AAV-2 can infect a large variety of cells from diverse organs and tissues. Yet, although without gross pathological consequences for the cell, a latent AAV-2 infection can induce subtle changes in the cell ▶phenotype. Examples are an increased ability to respond to stress factors, or a perturbation of the cell cycle, resulting in retarded cell growth. Most probably, these various effects are mediated by the large AAV-2 Rep proteins, even at the low expression levels typical for the latent stage. Is There a Natural Connection Between AAV Infection and Cancer? One frequently reported observation is that AAV-2infected cells exhibit an increased resistance to ▶oncogene- or tumorvirus-induced transformation. It is moreover known that AAV-2 infection can inhibit the proliferation of cultured cells derived from human cancers, e.g. ▶melanomas. Cumulatively, these data strongly suggest that wildtype AAV-2 is not only nonpathogenic, but in fact has oncosuppressive properties. Moreover, certain human cancer cell lines become more sensitive to gamma irradiation (▶Ionizing Radiation Therapy) and chemotherapeutic drugs (▶Chemotherapy of Cancer, Progress and Perspectives) upon experimental infection with wildtype AAV-2, as compared to noninfected controls. From a clinical point of view, these findings are of particular interest, since a major limitation of cancer chemotherapies is increasing resistance of transformed cells towards the drugs used. The observations of AAV-2-mediated cell sensitization therefore suggest that wildtype AAV might help to improve cancer chemotherapy, when applied in combination with conventional drugs. What are Recombinant AAV Vectors? Recombinant AAV (rAAV) vectors are derivatives of wildtype AAV which lack the rep and cap genes, and instead carry a foreign gene expression cassette inserted between the two viral ITRs. By definition, AAV vectors are thus “gutless” or “gutted” (i.e., devoid of any viral genes). The generation of rAAV vectors is technically feasible and simple, due to the wide availability of molecular clones of the various wildtype viruses. These clones are easily modified using standard molecular

AAV

laboratory techniques. Particularly beneficial is that wildtype and recombinant AAV are very small as compared to all other viruses developed as vectors, which aids in their experimental manipulation. Except for the replacement of the wildtype genes with a recombinant DNA, AAV vectors are identical in structure and organization to wildtype viruses and thus also function alike. In fact, AAV vectors will infect the target cell via the same molecular and cellular pathways as the wildtype virus. Ultimately, this will lead to expression of the encapsidated recombinant gene in the cell and thus to the intended therapeutic effect. As gene transfer vehicles, AAV vectors hold enormous promise for therapeutic intervention for a multitude of human acquired or innate genetic diseases, including cancer. Is AAV Unique as a Human Gene Therapy Vector? AAV vectors possess a multitude of advantages over all other virus-derived gene transfer vectors currently in (pre-)clinical development. One asset already mentioned is the lack of pathogenicity of the wildtype virus, which is in stark contrast e.g. to Adenovirus, another commonly used virus for gene therapy. Consequently, the production and handling of AAV vectors requires the lowest biosafety levels (S1, i.e., causing minimal risks for humans and the environment). The safety of AAV vectors is further increased by their “gutted” nature, precluding the expression of viral gene products which could cause cellular immune responses in the treated patient (a frequent adverse reaction to adenoviral vectors). A third unique asset, and a further difference to other viral vectors, is the availability of a wide spectrum of human, mammalian and nonmammalian natural serotypes. These isolates typically differ in their ▶tropism, i.e., the range of cells and tissues they can infect. Fortunately, it is technically very simple to generate recombinant AAV vectors which carry the same expression cassette, but differ in the viral capsid. This process is called “pseudotyping” and allows for the targeted delivery of a given recombinant DNA to virtually any desired cell or tissue, provided it can be infected by a known wildtype AAV (or a mutant thereof, see below). A plethora of reports have already demonstrated the power of this approach, to use AAV vectors for therapeutic and specific gene transfer to all clinically relevant target organs, including liver, muscle, lung, eye and brain. Last but not least, AAV vectors also differ from all other viral vectors by their capability to mediate persistent and long-term gene expression, both in actively dividing and in quiescent (i.e., non-dividing) cells, and most importantly, without integrating into the host chromosome. Instead, the vector forms stable but extra-chromosomal DNA molecules, which are not capable of perturbing chromosome structures and thus do not pose a mutational risk. This is clinically most pertinent, as many gene therapy

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applications will require stable gene expression, ideally for the life-span of the patient. The only other viral vectors able to mediate long-term gene expression (and in non-dividing cells) are derived from retroviruses or lentiviruses (HIV). However, these vectors are associated with drastically higher concerns about biosafety, due to the inherent pathogenic nature of the parental wildtype virus, as well as due to their propensity for integration into the human genome. The latter can readily result in insertional mutagenesis, i.e., activation of endogenous oncogenes, or vice versa, inactivation of ▶tumor suppressor genes. In both cases, the result is malignant transformation of the infected cell. This potentially serious adverse event from the use of retroviral vectors has indeed been observed in a recent clinical study, where multiple children developed leukemias, and some even died. Likewise, adenoviral vectors and the associated immune response have been blamed for the death of a patient in an early gene therapy trial in 1999. In striking contrast, thus far, none of the over 30 clinical trials using AAV vectors has yielded any evidence for a tumorigenic or lethal potential of this particular vector system. What are Recent Advances in AAV Vector Technology? In the early years, AAV vectors have been criticized for their small size (preventing packaging and therapeutic transfer of recombinant DNA >5kb in length), their relatively slow transduction kinetics (resulting from the single-stranded DNA genome and its need for conversion into a transcriptionally active DNA duplex), and their restricted cell and tissue tropism (based on the sole availability of the AAV-2 capsid in the early phase of AAV vector development). Nonetheless, even with those presumed limitations, AAV-2 vectors have been tested successfully in various large animal models and in human patients, addressing diverse diseases such as cystic fibrosis or hemophilia B. Most importantly, all three initial limitations of the AAV vector system have now been overcome, leading to the rapid expansion of AAV-based human gene therapy, especially for cancer treatment. First of all, the issue of limited packaging capacity has been solved with the creation of “split” AAV vectors which exploit the virus’ natural propensity for ▶concatamerization. In an infected cell, rAAV genomes frequently recombine with each other, resulting in large “head-to-tail” concatamers (i.e., multiple copies of an rAAV genome in the same orientation). This can be exploited experimentally, by splitting a large recombinant DNA (e.g., a gene and its promoter) into two halves, each of which is then delivered by a separate rAAV vector. This strategy effectively doubles the packaging limit of AAV vectors to up to 10 kb, which is sufficient even for large DNAs such as the factor VIII gene (encoding a blood clotting factor missing or defect in hemophilia A patients). Secondly, the inherently slow transduction kinetics of AAV

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AAV

have been overcome with the development of selfcomplementary or double-stranded vectors. In these, two copies of a foreign gene expression cassette are cloned and packaged in an inverted format, only separated by a minimal version of an AAV ITR. In the transduced cell, these two inverted copies then rapidly anneal with each other without the need for conversion into a duplex AAV DNA molecule. This results in an extremely rapid onset as well as maximum efficacy of gene expression, both far superior to what is obtained with conventional single-stranded AAV vectors, or most other viral vector systems. Thirdly, the limited host range of AAV-2 was readily overcome with the engineering of the over 100 alternative naturally occurring AAV serotypes as vectors. This approach has not only substantially broadened the range of cells and tissues that can now be infected with AAV vectors, but it has also alleviated concerns over the prevalence of neutralizing antibodies against the AAV-2 prototype in the human population. In fact, a wealth of studies have shown that AAV vectors derived from non-type2 serotypes are functional in many tissues that are refractory to AAV-2 infection, and most importantly, transduction readily occurred in the (experimentally induced) presence of anti-AAV-2 antibodies, mimicking the situation in most humans. Moreover, very recent work demonstrated the feasibility to create synthetic AAV capsids which are further unique from the AAV2 prototype, as well as from any of the naturally occurring isolates. Multiple strategies are currently being pursued, including the random mutagenesis of the AAV(-2) cap gene, the insertion of peptide pools into exposed regions of the AAV-2 capsid (hoping the peptides will mediate re-targeting to unknown cellular receptors), or the creation of libraries of “shuffled” viruses, in which capsid genes from several parental viruses are mixed and recombined. Most importantly, all of these new approaches and designs remain fully compatible with already established AAV vector technology, allowing for their rapid and straightforward pre-clinical evaluation. In fact, current AAV vector production methodologies are highly advanced and permit the generation of high titer stocks (>1 × 1014 recombinant particles per batch) in a very short amount of time (~10 days) (Fig. 2). As a result, AAV vectors have entered clinical evaluation and are currently being studied in about 30 ongoing trials in human patients. What are Clinically Relevant rAAV Applications in Cancer Treatment? The sum of assets described above – safety, versatility, efficacy, specificity – makes AAV an ideal vector for multiple and diverse therapeutic applications in humans. With particular respect to cancer, the use of AAV vectors is still in its infancy, but increasing preclinical data suggest that this vector system holds

AAV. Figure 2 Streamlined protocol for rAAV production. Cultured cells are transfected with two plasmids: The vector plasmid containing the foreign gene to be packaged into the viral particles, flanked by the AAV-2 ITRs, and the helper plasmid carrying the AAV-2 rep and cap genes to supply the Rep and VP proteins, respectively. In addition, the helper contains all adenoviral (Ad) genes which encode proteins with supportive function for AAV vector production, but it does not yield Adenovirus after transfection. Helpervirus infection is thus superfluous, and the resulting AAV-2 vectors are free of contaminating Adenovirus. Following a 2-day incubation of the transfected cells, the rAAV particles are harvested, purified and quantified. Note that there are numerous modifications to this basic protocol, e.g., in the number of plasmids (1–3, depending on the arrangement of AAV and adenoviral sequences).

enormous potential also for this specific application. Thus far, the approaches can be divided into strategies that either target the tumor cell directly or that modify host mechanisms. In more detail, AAV vectors have been employed in the following major categories: Antiangiogenesis, ▶immunotherapy, tumor suppressors, suicide gene therapy, drug resistance, repair strategies, and, last but not least, purging of tumor cells. For many of those categories, a currently emerging therapeutic modality which is also still in its infancy is RNA interference or RNAi (▶RNA interference and Cancer). This term describes the natural phenomenon of gene silencing mediated by short double-stranded RNAs.

AAV

The latter can be expressed from AAV vectors and thus be used to effectively and specifically suppress, for instance, expression of cellular or virally-encoded oncogenes. RNAi will likely become a valuable and crucial aspect of AAV-based cancer therapy in the near future, and will complement or perhaps even replace many of the currently existing strategies. Anti-Angiogenesis The efficacy of ▶angiogenesis inhibitors to undermine tumor ▶neovascularization and to block cancer progression as well as formation of metastases (▶metastasis) has been established in many animal models. However, this cancer therapy requires that the inhibitors are chronically administered as recombinant proteins, which is usually associated with severe problems. Therefore, AAV vectors with their unique ability to mediate sustained gene expression should prove particularly useful for this type of tumor therapy. Especially promising will be the future combination with synthetic AAV capsids that have been evolved to target the vasculature. Thus far, mostly AAV-2-based vectors have been used to deliver and express various antiangiogenesis factors in small animals, typically mice. A first important example is angiostatin, which has been expressed from AAV-2 in multiple mouse models of human cancers, including gliomas (▶Glioblastoma Multiforme) and liver cancers (▶Liver Cancer, Molecular Biology]. In all reported cases, this led to suppression of in vivo tumor growth and to substantial improvements in tumor-free survival rates. Similarly impressive are results with the related anti-angiogenic peptide ▶endostatin, whose expression from AAV2 vectors inhibited the establishment or growth of various human cancers in mice, including liver, ovarian (▶Ovarian Cancer), pancreatic (▶Pancreas Cancer, Clinical Oncology) and colorectal (▶Colon Cancer) tumors. Even better results have been obtained with the co-expression of both angiostatin and endostatin from a single or from two separate AAV vectors, exemplifying the potential for synergistic effects from combinatorial AAV therapies. Other examples for antiangiogenic AAV therapies already evaluated include the expression of a truncated form of the ▶vascular endothelial growth factor receptor (renal tumors), or of tissue inhibitors of ▶matrix metalloproteases. Immunotherapy Failure of the immune system to recognize cancer antigens can substantially contribute to tumor manifestation and progression. Although tumors can illicit strong immune responses in the early stages, this effect is frequently lost in later phases, eventually allowing for aggressive and metastatic tumor growth. Gene transfer protocols involving AAV (or other viral) vectors have thus been developed which aim to potentiate the patient’s

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antitumor responses, by either targeting the tumor cells directly, or by transducing host-derived immune effector cells. Examples for already reported tumor celldirected therapies include AAV-mediated delivery of ▶interferon genes to ex vivo cultured cancer cells, or via intra-tumoral injection (gliomas). Likewise, AAV2 has been used to express tumor necrosis factor-related ▶apoptosis-inducing ligand (▶TRAIL) in colorectal, lung and liver tumor models, resulting in significantly inhibited tumor growth and, in some cases, even in regression. Targeting cells of the host immune system, on the other hand, is a promising alternative approach and could eventually be developed into a vaccination therapy. Already, AAV-2 vectors have been used to deliver dominant tumor epitopes to antigen-presenting cells, such as CD40 ligand which was expressed in B-cells from ▶chronic lymphocytic leukemia (CLL) patients, leading to specific proliferation of ▶HLA Class I-matched allogeneic T-cells. Another potential vaccine could be AAV vectors expressing an HPV16 (▶Human Papillomaviruses) E7 CTL (cytotoxic T-cell) epitope/heat shock fusion protein, based on reports that infected mice became immunized against E7expressing tumor cells. Last but not least, encouraging studies have identified ▶dendritic cells (DC), the most potent antigen-presenting cells, as an attractive target for AAV-based cancer immunotherapies. For instance, DCs transduced with AAV vectors encoding HPV16 E6 or E7 genes caused a stark CTL response against cervical cancer cell lines, while in another study, DCs transduced with CD80-expressing AAVs induced high levels of CD8+ T-cells. Together, these findings suggest that AAV can be used to trigger strong anti-tumor CTL responses, and that AAV-based immunotherapy has substantial clinical potential for cancer treatment. Tumor Suppressors Highly attractive targets for AAV-mediated cancer therapy are oncogenes and tumor suppressor genes, respectively, whose expression is frequently dysregulated in malignant human cancers. An important example for a tumor suppressor involved in cellular checkpoint control is p53 (▶p53 Protein, Biological and Clinical Aspects), which normally prevents passage of cells with DNA damage through the cell cycle. Consequently, expression of p53 from AAV vectors was consistently found to block the growth of cancer cells in vitro and in vivo, and to mediate apoptosis and cytotoxicity. Similar results were obtained after expression of the fragile histidine triad tumor suppressor (▶FHIT), which delayed the growth of human pancreatic tumor ▶xenografts and extended longterm animal survival. In a third example, delivery of the gene encoding the monocyte chemoattractant protein MCP-1 from AAV vectors suppressed expression of the HPV E6 and E7 proteins in cervical cancer cell lines, as well as in tumors derived from these cells.

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AAV

Suicide Gene Therapy This approach is based on the idea to bio-activate a prodrug within tumor cells to a toxic species, triggered by the tumor-directed delivery of the activating enzyme from AAV vectors. The best studied example for this category is the Herpes simplex virus-encoded enzyme thymidine kinase (tk) in combination with gancyclovir. This system has already been used successfully from AAV vectors to inhibit tumor growth in a variety of human xenograft models, including liver cancer, gliomas and ▶oral squamous carcinomas. Notably, the specificity of this approach can be enhanced by the use of tissueand/or tumor-specific promoters, such as those only active in liver or melanoma cells. Moreover, the overall efficacy of the AAV/tk vectors was shown to increase following treatment of transduced cells with irradiation or topoisomerase inhibitors, both known to enhance AAV infection (in addition to their direct effects on cells). Drug Resistance Development of multiple drug resistance (MDR) is a major issue with cancer chemotherapies and is often associated with over-expression of the ▶P-glycoprotein (an ATPase that pumps chemotherapeutic drugs out of the cancer cell). One recently reported, highly effective approach to reverse the MDR phenotype is to use double-stranded AAV vectors to express anti-Pglycoprotein short hairpin RNAs (effectors of RNAi). In human ▶breast cancer and oral cancer cells, this led to a substantial sensitization to chemotherapy, suggesting a high potential to overcome the MDR obstacle with this approach. Another application is expression of the MDR1 gene from AAV vectors in ▶hematopoietic progenitors. This should confer myeloprotection in patients undergoing high-dose chemotherapy for advanced tumors, and thus prevent myelosuppresssive effects (▶Myelosuppression) from the chemotherapeutic regimen, such as infection or hemorrhaging. However, this strategy has not been fully explored in animal models yet. Repair Strategies ▶Telomerase (the enzyme maintaining and stabilizing the integrity of telomeres, i.e., chromosome ends) is an example for a therapeutically relevant target for repair strategies. Its activity is often elevated in tumor cells, and it was shown that delivery of telomerase antisense molecules [Antisense DNA Therapy] via AAV vectors (in this particular case hybrids with adenoviral vectors) can reduce tumor cell proliferation, as well as induce apoptosis. Purging of Tumor Cells from Autologous Transplants Autologous grafts (▶Graft Acceptance and Rejection), e.g. peripheral blood progenitor cells, are used for treatment of many solid human cancers. However, they can be contaminated with tumor cells that give rise to

relapse after ▶myeloablative megatherapy and graft transplantation. There is recent evidence that following infection of such contaminated grafts with recombinant AAV-2, the contaminating tumor cells are preferentially infected, while the hematopoietic progenitors are spared. Indeed, infection of sarcoma cells with AAV/tk vectors (see above) extended the survival of transplanted mice (over non-treated controls), while the same vector was unable to transduce and kill human peripheral blood progenitors. However, it remains to be proven that this strategy can indeed be applied to selectively purge tumor cells from autologous transplants. RNAi RNA-mediated silencing of gene expression (RNAi) will clearly become a major part of anti-tumor therapies in the future, as proof-of-concept for the efficacy of this approach is already overwhelming. In combination with AAV, there have only been a few reports thus far, but this field will certainly expand. One described application is to use AAV vectors to deliver short hairpin RNAs against the hec1 gene, which is highly expressed in mitotic cells where it represents a vital component of the ▶kinetochore outer plate. Transduction of glioma cells with anti-hec1 AAV vectors resulted in selective cell death, while mitotically inactive control cells were unaffected. Likewise, infected xenografts showed lower densities and were highly fibrotic, as a result of AAV treatment. It can generally be predicted that virtually any over-expressed gene that contributes to transformation can be an AAV/RNAi target, including virally-encoded (see above, e.g., HPV E6/7) or cellular oncogenes. Future Applications With the current state-of-the-art technology, the AAV vector system is already one of the most powerful and promising toolkits for development as anti-tumor bioreagents. In the future, the versatility of this system will further increase with the discovery and creation of new natural or synthetic capsids, respectively. Likewise, the field will benefit from the engineering of novel tumor- and tissue-specific gene expression cassettes, and from the design of safer and more effective therapeutic sequences, e.g., for the induction of anti-cancer RNAi. A very important approach will be to merge the different strategies into combinatorial therapies, e.g., by mixing immunotherapies with RNAi vectors, or suicide gene expression with repair approaches. Examples for such multimodality cancer therapies with AAV vectors have already been reported, and their numbers will increase in the future. Last but not least, it will also be crucial to combine AAV (or other viral) vectors with further anti-cancer effectors, such as new classes of compounds including proteasome (▶Proteasomal Inhibitors) and histone deacetylase (▶Histone Deacetylases) inhibitors.

ABC Transporter Proteins

References 1. Grimm D (2002) Production methods for gene transfer vectors based on adeno-associated virus serotypes. Methods 28:146–157 2. Grimm D, Kay MA (2004) From virus evolution to vector revolution: use of naturally occurring serotypes of adenoassociated virus (AAV) as novel vectors for human gene therapy. Curr Gene Ther 3:281–304 3. Grimm D, Pandey K, Kay MA (2005) Adeno-associated virus vectors for short hairpin RNA expression. Methods Enzymol 392:381–405 4. Li C, Bowles DE, van Dyke T et al. (2005) Adenoassociated virus vectors: Potential applications for cancer gene therapy. Cancer Gene Ther 12:913–925 5. Warrington KH, Herzog RW (2006) Treatment of human disease by adeno-associated viral gene transfer. Hum Genet 119:571–603

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▶Fluoxetine ▶Glutathione Conjugate Transporter RLIP76 ▶Major Vault Protein ▶Vault Complex

ABC (ATP-Binding Cassette) Superfamily Definition ABC (ATP-binding cassette) superfamily contains both uptake and efflux transport systems. These proteins bind ATP and hydrolyze it to energize transport of molecules outside or inside the cells.

ABC Drug-Transporters Definition Synonym ABC Transporter, ABC (ATP-Binding Cassette) Superfamily. The adenosine-triphosphate (ATP) binding cassette (ABC) transporters form the largest family of transmembrane proteins that use ATP-derived energy to transport various substances over cell membranes. Primary-active transporters, driven by energy released from ATP by inherent ATPase activity, that export substrates from the cell against a chemical gradient, Based on the arrangement of the nucleotide binding domain and the topology of its transmembrane domains, human ABC-transporters are classified into seven distinct families (ABC-A to ABC-G), including ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (cMOAT; MRP2), ABCC4 (MRP4), and ABCG2 (ABCP; MXR; BCRP). Structural characteristics based on their ▶Walker motif (ATP binding domain) and their nucleotide-binding folds across the membrane are responsible for their classification into this superfamily. Their localization pattern over the body suggests that they have an important role in the prevention of absorption as well as the excretion of potentially toxic metabolites and xenobiotics, both on a systemic and a cellular level. ABC drug-transporters (may) show substrate-overlap. Examples of mammalian ABC transporters include ▶P-glycoprotein, MRP (▶multidrug resistance protein), ▶cystic fibrosis transmembrane conductance regulator (CFTR) and the transporter associated with antigen processing (TAP). ▶P-glycoprotein ▶Irinotecan

ABC Transporter Definition ABC transporters are a superfamily of prokaryotic and eukaryotic proteins. They are usually involved in membrane transport and share a homologous nucleotidebinding domain, ABC (ATP-binding cassette). In addition to the ABC domain, ABC transporters contain or interact with hydrophobic domains containing multiple transmembrane segments. Examples of mammalian ABC transporters include P-glycoprotein, MRP (multidrugresistance protein), cystic fibrosis transmembrane conductance regulator (CFTR) and the transporter associated with antigen processing (TAP).

ABC Transporter Proteins Definition ABC transporter proteins are a superfamily of proteins responsible for transporting a broad range compounds across membranes in cells. Structural characteristics based on their Walker domains (ATP binding domain) and their nucleotide-binding folds across the membrane are responsible for their classification into this superfamily.

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ABC-Transporters

ABC-Transporters H ERMANN L AGE Charité Campus Mitte, Institute of Pathology, Berlin, Germany

Synonyms Multidrug resistance transporters; Traffic ATPases; Permeases (for import systems)

Definition ABC (ATP-binding cassette)-transporters are membraneembedded proteins with a characteristic ABC domain that utilize the energy from ATP hydrolysis for the transport of their substrates across a cellular membrane.

Characteristics The superfamily of ABC-transporters comprises one of the most abundant protein families in nature. These transporters are believed to date back in evolutionary time more than 3 billion years and are distributed in all three kingdoms of living organisms, archaea, eubacteria, and eukaryotes. ABC-transporters have to be distinguished from ABC-proteins. Both types of proteins are defined by the presence of a highly conserved ~215 amino acids consensus sequence designated as ABC domain or nucleotide-binding domain (NBD). The domain contains two short peptide motifs, a glycine-rich Walker A and a hydrophobic Walker B motif, both involved in ATP binding and commonly present in all nucleotide-binding proteins. A third consensus sequence is named ABC signature and is unique in ABC domains. ABC-containing proteins couple the phosphate bond energy of ATP hydrolysis to many cellular processes and are not necessarily restricted to transport functions. However, the proper meaning of the term ABC-transporter is satisfied when the ABC-protein is in addition associated with a hydrophobic, integral transmembrane domain (TMD) forming a translocation path. TMDs are usually composed of at least six transmembrane (TM) α-helices. They are believed to determine the specificity for the substrate molecules transported by the ABCtransporter. The minimal structural requirement for a biological active ABC-transporter seems to be two TMDs and two NBDs [TMD-NBD]2 (Fig. 1). In fullsize transporters, this structural arrangement may be formed by a single polypeptide chain and in multiprotein complexes by more than one polypeptide chain. In prokaryota, ABC transport systems are often halfsize transporters having only one TMD fused to one NBD [TMD-NBD]. Half-size transporters probably dimerize to form a full-size transporter [TMD-NBD]2 to mediate mainly the influx of essential compounds such

ABC-Transporters. Figure 1 Schematic representation of the predicted domain arrangement of (a) half-size transporters having only one TMD fused to one NBD [TMD-NBD], e.g., ABCG2 (BCRP); and (b,c) full-size transporters [TMD-NBD]2, whereby (b) shows the predicted structure of ABCB1 (MDR1), and (c) the structure of ABCC1 (MRP1) containing an additional TMD (TMD0) of unknown function. Half-size transporters probably dimerize to form a biological active ABC-transporter. These three ABC-transporters are the most important drug extrusion pumps in multidrug-resistant cancers. TMD, transmembrane domain consisting of six α-helices; NBT, nucleotidebinding domain. It should be noted that the orientation of ABCG2 is reverse to that of ABCB1 and ABCC1.

as sugars, vitamins, and metal ions into the cell. Eukaryotic ABC-transporters commonly function as exporters mediating the efflux of compounds from the cytosol to the extracellular space or to the inside of intracellular membrane-bound compartments, i.e., endoplasmic reticulum, mitochondria, peroxisomes, or vacuoles. The range of physiologically transported compounds includes lipids and sterols, ions, diverse small molecules, oligo- and polypeptides. Human ABC-Transporters In humans, 48 ABC-transporters distributed to seven subfamilies have been identified (Table 1). Although the number of human ABC-transporters is much smaller than found in bacteria, many of them are of clinical significance. Currently, 18 human genes encoding ABC-transporters have been associated with genetic diseases. Even though the majority of the members of the human ABC-transporter family are active transporters, there are some exceptions in which the energy of ATP hydrolysis is utilized to control alternative biological processes. Thus, ABCC7 (CFTR), well known as mutated in patients suffering on ▶cystic

ABC-Transporters ABC-Transporters. Table 1

Family of human ABC-transporters

Subfamilay

HUGO-nomenclature

Common names

ABCA

ABCA1 ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7 ABCA8 ABCA9 ABCA10 ABCA12 ABCA13 ABCB1 ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 ABCB9 ABCB10 ABCB11 ABCC1 ABCC2 ABCC3 ABCC4 ABCC5 ABCC6 ABCC7 ABCC8 ABCC9 ABCC10 ABCC11 ABCC12 ABCD1 ABCD2 ABCD3 ABCD4 ABCE1 ABCF1 ABCF2 ABCF3 ABCG1 ABCG2 ABCG4 ABCG5

ABC1 ABC2 ABC3, ABCC ABCR

ABCB

ABCC

ABCD

ABCE ABCF

ABCG

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ABCX

MDR1, PGY1 TAP1 TAP2 MDR3, PGY3 MTABC3 ABC7 MABC1 TABL MTABC2 BSEP, SPGP MRP1, MRP MRP2, cMOAT MRP3 MRP4 MRP5 MRP6 CFTR SUR1 SUR2 MRP7 MRP8 MRP9 ALD, ALDP ALDL1, ALDR PXMP1, PMP70 PXMP1L, P70R RNASELI, OABP ABC50

ABC8, White BCRP, MXR White2 White3

Location 9q31.1 9q34 16p13.3 1p22.1-p21 17q24.3 17q24.3 19p13.3 17q24 17q24.2 17q24 2q34 7p12.3 7q21.1 6p21.3 6p21.3 7q21.1 7p15.3 2q36 Xq12-q13 7q36 12q24 1q42 2q24 16p13.1 10q24 17q22 13q32 3q27 16p13.1 7q31.2 11p15.1 12p12.1 6p21.1 16q12.1 16q12.1 Xq28 12q11-q12 1p22-p21 14q24.3 4q31 6p21.33 7q36 3q27.1 21q22.3 4q22 11q23.3 2p21

A Size [AA]

Function

2261 2436 1704 2273 1642 1617 2146 1581 1624 1543 2595 5058 1280 808 653 1279

Cholesterol-, PS transport

842 752 718 723/766 738 1321 1531 1545 1527 1325 1437 1503 1480 1581 1549 1464 1382 1359 745 740 659 606 402 807 623 709 638 655 627 651

Iron transport Iron-, Sulfur- cluster transport

Surfactant production N-retinylidene-PE transport

MDR Peptide transport Peptide transport PC transport

Bile salt transporter MDR, organic anion transporter MDR, organic anion transporter Organic anion transporter Organic anion transporter Organic anion transporter Chloride transport Regulation Regulation

FA-, FA AcylCoA transport FA-, FA AcylCoA transport FA-, FA AcylCoA transport FA-, FA AcylCoA transport

Cholesterol transport MDR Sterol transport

AA, amino acids; FA, fatty acids; MDR, multidrug resistance; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.

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ABC Transporters (ATP-Binding Cassette Transporters)

fibrosis, appears as a chloride ion channel; ABCC8 (SUR1) and ABCC9 (SUR2) are both regulatory subunits of the regulatory sulfonylurea receptor (SUR). Other members of the ABC-transporter family couple ATP binding and hydrolysis to the control of translation or ▶DNA repair. Although the active transporters have dedicated functions involving the transport of specific substrates, the complex physiological network of ABC-transporters may also have an important role in host detoxification and protection against xenobiotics. This general function is revealed by their tissue distribution. ABC-transporters are highly expressed in important pharmacological barriers, such as the epithelium that contributes to the blood–brain barrier (BBB), the brush border membrane of intestinal cells, the biliary canalicular membrane of hepatocytes, or the lumenal membrane in proximal tubules of the kidney. Anyway, this xenobiotics pump function is the basis for the pivotal role of ABC-transporters in ▶multidrug resistance (MDR) of cancer. ABC-Transporters and Multidrug Resistance of Cancer MDR is defined as the simultaneous resistance of a tumor against a variety of antineoplastic agents with different chemical structure and mode of action. Thus, MDR is a major obstacle in clinical management of cancer by ▶chemotherapy. Although various mechanisms have been identified to mediate a multidrugresistant phenotype to malignant diseases, the enhanced drug extrusion activity of the ABC-transporter ABCB1 or ▶P-glycoprotein (MDR1; PGY1) was the first mechanism that was demonstrated to be the reason for MDR. The substrates of ABCB1 include first and foremost natural product-derived anticancer drugs, such as ▶anthracyclines, ▶epipodophyllotoxins, ▶taxans, and ▶vinca alkaloids, but not clinically important drugs like platinum-containing compounds or ▶antimetabolites. Besides ABCB1, in particular, ABCC1 (MRP1) and ABCG2 (BCRP) were found to be associated with a multidrug-resistant phenotype, but also alternative ABCtransporters can pump drugs from the inside to the outside of a cancer cell, e.g., ABCC2 (MRP2) is a platinum drug transporter. ABCB1, ABCC1, and ABCG2 have partial overlapping but not identical substrates. ABC-Transporters as Anticancer Drug Targets Following the identification of ABCB1 as a pivotal MDR-mediating factor, tremendous efforts were undertaken to identify ABCB1-interacting agents that inhibit its pump activity and, therewith, reverse the MDR phenotype. Such drugs are commonly designated as chemosensitizers or MDR modulators. Although many compounds, e.g., ▶verapamil and ▶ciclosporin derivatives, were identified as ABCB1 inhibitors or inhibitors of alternative MDR-mediating ABCtransporters, so far all of them failed in clinical trials.

References 1. Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer:2:48–58 2. Higgins CF (1993) ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8: 7–113 3. Holland IB, Cole SPC, Kuchler K, Higgins CF (eds) (2003) ABC proteins from bacteria to man. Academic Press, an imprint of Elsevier Science, London (UK) and San Diego (CA, USA) 4. Lage H (2003) ABC-transporters: implications on drug resistance from microorganisms to human cancers. Int J Antimicrob Agents 22:188–199

ABC Transporters (ATP-Binding Cassette Transporters) Definition Primary-active transporters, driven by energy released from ATP by inherent ATPase activity, that export substrates from the cell against a chemical gradient, including P-gp, MRP, and BCRP.

ABL Definition The ABL gene encodes a nuclear tyrosine kinase that is involved in chromosomal translocations in chronic myeloid leukemia (CML). ▶BCR-ABL1 ▶Chromosomal Translocations

Ablation Definition In cancer therapy, the surgical removal or destruction of tumor tissue. ▶Photothermal Ablation

ABVD

ABLES Definition Adult Blood Lead Epidemiology and Surveillance. ▶Lead Exposure

ABVD A NAS YOUNES Department of Lymphoma and Myeloma, M.D. Anderson Cancer Center, Houston, TX, USA

Definition Doxorubicin, bleomycin, vinblastine, and dacarbazine combination chemotherapy used for the treatment of patients with Hodgkin lymphoma.

Characteristics ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) is the most widely used regimen for the treatment of early and advanced stage Hodgkin lymphoma (HL). Treatment of patients with early stage classical HL evolved over the last three decades. Radiation therapy alone as the single treatment modality is no longer practiced. Today, the most widely used approach is combined modality therapy (chemotherapy plus involved field radiation therapy). In general, 2 (for favorable early stage) to 4 (for unfavorable early stage) cycles of ABVD plus 30 Gy of involved field radiation therapy is the most widely used standard of care approach. Using this approach, more than 90% of the patients are expected to be cured of their disease. Patients with bulky stage II disease, (especially with bulky mediastinal mass), or stage II with B-symptoms are usually treated similar to those with advanced stage HL with 6–8 cycles of ABVD followed by involved field radiation therapy to the bulky area. Use of chemotherapy alone has recently been proposed for a selected group of patients with early stage classical HL. The rationale for this approach is to reduce radiation-induced morbidity and mortality, including second malignancies and cardiac complications. While this approach is appealing, it will need to be further examined after prolonged follow-up. For now, it seems appropriate to treat young female patients with non-bulky early stage classical HL (especially those with mediastinal or axillary adenopathy) with chemotherapy alone to reduce the risk for breast cancer. The risks and

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benefits of combined modality versus chemotherapy alone should be discussed with patients before making a final treatment recommendation. Based on several randomized studies comparing ABVD with other multi-drug regimens, ABVD became the most widely used combination regimen for the treatment of patients with advanced HL. Chemotherapy alone (6–8 cycles) is usually considered sufficient for treating patients with advanced stage classical HL. However, involved field radiation therapy is frequently added at the end of chemotherapy to areas of bulky disease. This combined modality approach has been recently compared with chemotherapy (MOPP/ABV) alone in a randomized trial in patients with advanced stage classical HL, and showed no survival advantage, especially in those who achieved complete remission after the completion of chemotherapy. Furthermore, meta-analysis review of fourteen clinical trials comparing chemotherapy with combined modality also showed no survival advantage for those receiving the combined modality approach. Newer treatment programs such as Stanford V and BEACOPP have shown successful results, but remain less widely used compared with ABVD. Although BEACOPP has been shown to be superior to ABVDlike regimens in large-scale randomized trials, the superiority of Stanford V over standard ABVD has not yet been established. Because ABVD may cure only 50–65% of patients with poor risk advanced stage HL, more intensive programs such as BEACOPP may add benefit, despite the increased toxicity. Patients with good risk features have a high cure rate with ABVD, so the use of more intensive and more toxic regimens in this patient population should be used with caution, and preferably within a clinical trial. In fact, a recently published randomized study demonstrated that early intensification with autologous stem cell transplantation after four cycles of ABVD-like chemotherapy did not improve the outcome in patients with advanced stage HL compared with conventional chemotherapy, perhaps because many patients did not have poor risk features as identified by the international prognostic score for HL.

References 1. Bonadonna G, Bonfante V, Viviani S et al. (2004) ABVD plus subtotal nodal versus involved-field radiotherapy in early-stage Hodgkin’s disease: long-term results. J Clin Oncol 22:2835–2841 2. Meyer RM, Gospodarowicz MK, Connors JM et al. (2005) Randomized comparison of ABVD chemotherapy with a strategy that includes radiation therapy in patients with limited-stage Hodgkin’s lymphoma: National Cancer Institute of Canada Clinical Trials Group and the Eastern Cooperative Oncology Group. J Clin Oncol 23:4634–4642 3. Straus DJ, Portlock CS, Qin J et al. (2004) Results of a prospective randomized clinical trial of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by radiation therapy (RT) versus ABVD alone for

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Accelerated Phase

stages I, II, and IIIA nonbulky Hodgkin disease. Blood 104:3483–3489 4. Canellos GP (1996) Is ABVD the standard regimen for Hodgkin’s disease based on randomized CALGB comparison of MOPP, ABVD and MOPP alternating with ABVD? Leukemia 10(Suppl 2):s68 5. Diehl V, Thomas RK, Re D (2004) Part II: Hodgkin’s lymphoma – diagnosis and treatment. Lancet Oncol 5:19–26

Acetaldehydehydrogenase Definition

The enzyme which oxidizes ▶acetaldehyde. ▶Alcohol Consumption

Accelerated Phase Definition

Occurs between chronic phase and ▶blast crisis. Characterized by 10–19% myeloblasts and >20% basophils in blood or bone marrow and cytogenetic evolution; also increasing splenomegaly, platelet count, or white blood cells if unresponsive to treatment. ▶Nilotinib

Acetylation Definition The reversible, covalent attachment of an acetyl group to the lysine residue of proteins. Acetylation and deacetylation of histones play an important role in transcriptional regulation ▶Histone Deacetylases

ACD Definition

N-Acetylcysteine

Abbreviation for Appraisal Consultation Document. ▶National Institute for Health and Clinical Excellence (NICE)

ACDC ▶Adiponectin

Acetaldehyde Definition Highly reactive chemical compound (CH3CHO) that forms following ethanol metabolism. Toxic, mutagenic and carcinogenic. ▶Alcohol Consumption ▶Hepatic Ethanol Metabolism

Definition A pharmacological agent used mainly as a mucolytic and in the management of ▶paracetamol overdose and is available in different dosage forms for different indications: solution for inhalation – inhaled for mucolytic therapy or ingested for nephroprotective effect; i.v. injection – treatment of paracetamol overdose; or oral solution – various indications. ▶Chemoprotectants

Acetylsalicylic Acid Definition ASA/aspirin; one of the most successful drugs in combating reactive species overload diseases, and the ▶chemoprevention of cancers such as ▶colon cancer. ▶Inflammation

Acinar Cells

Acetyltransferase

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inhibits tumor growth by binding the ▶nucleolin protein on the surface of cancer cells.

Definition An enzymic activity that catalyzes the transfer of an acetyl moiety from acetyl CoA to a specific amino acid on a substrate protein. CBP and p300 are lysine (K)directed acetyltransferases. ▶CBP/p300 Coactivators

Achneiform Rash Definition Is a pustular rash with usual distribution over the face, scalp, and upper trunk. ▶Erlotinib (Tarceva®)

N-Acetyltransferase Definition Is an acetyl-CoA requiring enzyme that catalyses the acetylation of ▶xenobiotics that are aromatic amines or contain a hydrazine group. It participates in the ▶detoxification of a plethora of hydrazine arylamine drugs and is also able to bioactivate several known carcinogens. (Vatsis KP, Weber WW, Bell DA et al (1995) Nomenclature for N-Acetyltransferases. Pharmacogenetics 5:1–17) ▶Detoxification

ACF

Acid Rain Definition Is formed by absorption of acidic gases as SO2 or NOx by water droplets in clouds Precipitation then increases the acidity of the soil, and affects the chemical balance of surface water bodies. ▶Xenobiotics

Acidosis

Definition

Definition

Aberrant crypt foci; Putative precursors of colon cancer ▶Preneoplastic lesions.

Is present in a tissue whenever the pH is below 7.0 (or the H+-concentration is higher than 10–7 mol/L).

▶Trefoil Factors ▶Colon Cancer ▶Conjugated Linolenic Acids

▶Oxygenation of Tumors

Acinar Cells Acharan Sulfate (AS) Definition Definition Is isolated from the giant African snail Achatina fulica, responsible for inhibitory effect of tumor growth. AS

Key cell in the exocrine pancreas. These are arranged around a central lumen and form the bulk of the pancreatic gland. They contain digestive enzymes usually found in the apical region of the cells.

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Acites

Acites G ERHILD B ECKER Department of Internal Medicine II (Gastroenterology, Hepatology, Endocrinology), University Hospital Freiburg, Freiburg, Germany

Definition

Ascites is derived from the Greek word ασκóς (gr. sack, wineskin) and is defined as accumulation of protein rich fluid in the peritoneal cavity. It occurs mainly in ▶cirrhosis of the liver, but also in heart failure, tuberculosis and malignancy. Malignant ascites occurs in association with a variety of neoplasms and is defined as the abnormal accumulation of fluid in the peritoneal cavity caused by cancer.

Characteristic Malignant ascites accounts for around 10% of all cases of ascites and occurs in association with a variety of neoplasms. Malignant effusion is the escape of fluid from the blood or vessels into tissues or cavities, and is a common problem in patients with cancer. All types of cancer can metastasize to any of the body’s serous cavities and result in malignant effusion. In the Western World the most common cause of malignant ascites is ▶ovarian cancer. Other common primary sites are the pancreas, stomach and uterus, with breast, lung, and lymphoma representing the commonest extra-abdominal sites. Up to 20% of all patients with malignant ascites have cancer of unknown primary origin (▶CUP). Except in breast and ovarian cancer, the presence of malignant ascites in patients with neoplastic disease frequently signalizes the terminal phase of cancer. The mean survival time for ovarian cancer is 30–35 weeks and for tumors of lymphatic origin 58–78 weeks, whereas for cancers of the gastrointestinal tract the mean survival is only 12–20 weeks. In patients with CUP the median survival shows a great variability ranging from 1 week to 3 months in different series. Pathophysiology Fluid accumulation in the peritoneal cavity is dependent on the amount of fluid generated and the rate at which it leaves the abdominal cavity. When fluid production exceeds its clearance, free transudate will accumulate. Under physiologic conditions, transudation of plasma through capillary membranes of the peritoneal serosa continuously produces free fluid to lubricate the serosal surfaces. This fluid production is under the influence of portal pressure, plasma oncotic pressure, sodium and water retention, hepatic lymph production, and

microvascular permeability for macromolecules. Under physiologic conditions, at least two-thirds of the peritoneal fluid reabsorbs into open-ended lymphatic channels of the diaphragm and is propelled cephalad by the negative intrathoracic pressure. This fluid proceeds through mediastinal lymph channels into the right thoracic duct and empties into the right subclavian vein. The ability of the healthy subject to resorb fluid is much greater than the fluid generated, with the result that there is normally only a small volume of approx. 50 mL of fluid in the peritoneal cavity. Ascites as an abnormal accumulation of fluid in the peritonal cavity can be induced by several causes. In principle, four types of causes can be identified. (i) Ascites due to raised hydrostatic pressure, caused by cirrhosis, congestive heart failure, inferior vena caval obstruction, or hepatic vein occlusion. (ii) Ascites due to decreased osmotic pressure, caused by protein depletion (e.g. nephrotic syndrome), reduced protein intake (malnutrition) or reduced protein production (cirrhosis of the liver). (iii) Ascites due to fluid production exceeding resorptive capacity, caused by infections or malignancies. (iv) Chylous ascites, caused by obstruction and leakage of the lymph channels draining the gut. The pathophysiology of malignant ascites is multifactorial and is yet incompletely understood. Ascites may result from obstruction of lymphatic drainage by tumor cells that prevent absorption of intraperitoneal fluid and protein as often seen in lymphomas and breast cancer. Since the ascites of many patients with malignant ascites has a high protein content, alteration in vascular permeability has been implicated in the pathogenesis of ascites production. The tumor induces increasing production of peritoneal fluid due to increased microvascular permeability of tumor vasculature and the amount of ascites production correlates with the extent of neovascularization. Aside from mechanical obstruction and cytokines, the pathophysiology of malignant ascites also consists of hormonal mechanisms. Due to decreased removal of fluid as a consequence of obstructed lymphatics, the circulating blood volume is reduced and this activates the reninangiotensin-aldosterone system, leading to sodium retention. Therefore, reduced sodium intake together with diuretics is often used to treat malignant ascites, but there is no consensus on effectiveness. Available trials considering diuretics often include different patients groups with varying dose regimens and there are no randomized controlled trials to asses the efficacy of diuretics in malignant ascites. Diagnosis In most cases, ascites can be diagnosed by careful physical examination and taking a detailed history. The main clinical symptoms of ascites include abdominal

Acites

distension, ankle edema, continuous abdominal discomfort or pain, nausea, vomiting, shortness of breath and decreased mobility. Greater quantities of ascites cause abdominal distension, bulging flanks that are dull to percussion, shifting dullness and a fluid wave. Ultrasound is able to detect free peritoneal fluid if its volume is greater than 100 mL. CT and MRI are also able to detect little quantities of ascites. Malignant diagnosis is indistinguishable by physical examination from ascites caused by non-malignant conditions. Ascites detected by ultrasound, CT or MRI in the presence of typical imaging features of a malignant tumor is strongly suggestive of a malignant ascites. Diagnosis is confirmed by positive cytology of malignant cells in the fluid. A positive cytology result has a specificity of nearly 100%, but it is not very sensitive with only about 60% of malignant aspirates being cytologically positive. Compared to ascites caused by cirrhosis, malignant ascites usually contains more white blood cells and a higher level of lactate dehydrogenase. Fibronectin, cholesterol, lactate dehydrogenase, sialic acid, proteases, and antiproteases have been studied with fibronectin performing best in differentiating between malignant and non-malignant acites in most series. However, at present there is no single test available to be used routinely to differentiate between malignant and non-malignant ascites. Tumor markers, especially ▶CEA and CA-125 can be useful in diagnosing the primary tumor in malignant ascites, although they lack specificity. In case of doubt abdominal ▶paracentesis with chemical and cytologic analysis of the ascitic fluid should be used. The cell count provides immediate information about possible bacterial infection. Samples with a predominance of a least 250 neutrophils per cubic millimeter of ascitic fluid are suggestive of infection. Gram stains and culture for bacterial, fungal, and acid fast organisms are mandatory. ▶Spontaneous bacterial peritonitis is characterized by the spontaneous infection of ascitic fluid in the absence of an intra-abdominal source of infection and involves the translocation of bacteria from the intestinal lumen to the lymph nodes, with subsequent bacteremia and infection of ascitic fluid. Third-generation cephalosporins are the treatment of choice. Ascitic fluid amylase content helps to detect pancreatic ascites and gut perforation. 80% of all cases of ascites are caused by cirrhosis of the liver. The chief factor contributing to ascites in liver cirrhosis is ▶portal hypertension. Patients with ascites caused by liver disease usually have a ▶serum-ascites albumin concentration gradient (calculated by subtracking the albumin concentration of the ascitic fluid from the albumin concentration of a serum specimen obtained on the same day) ≥1.1 g/dL. If serum albumin: ascites albumin gradient is less than 1.1g/dL, portal hypertension can be safely ruled out.

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Treatment In general, practice of managing malignant ascites seems to be influenced by the evidence obtained in the context of non-malignant ascites, especially ascites caused by liver disease. Malignant ascites only accounts for approximately 10% of all cases of ascites whereas over 80% of cases are caused by chronic liver disease. So, most evidence in treatment of ascites is obtained in the context of liver disease. In ascites caused by liver cirrhosis the most important treatments are the restriction of dietary sodium intake and the use of oral diuretics because patients with liver cirrhosis retain sodium and water as a result of the renin-angiotensinaldosterone pathway. In ascites due to liver disease there is good evidence for the efficacy of a combined therapy with the diuretics spironolactone and furosemide supported by several randomized controlled trials. Treatment options for the minority of patients who are resistant to standard therapy with diuretics are therapeutic paracentesis, ▶peritoneovenous shunting, ▶transjugular intrahepatic portosystemic shunt (TIPS), extracorporal ultrafiltration of ascetic fluid with reinfusion and liver transplantation. Of these, ▶transjugular portosystemic stent shunts, extracorporal ultrafiltration and liver transplantation are specific to liver diseases, whereas abdominal paracentesis and peritoneovenous shunting are often used in managing malignant ascites. In contrast to the treatment of underlying cancer, there is no generally accepted gold standard for the management of malignant ascites so far. There are two principle approaches in managing malignant ascites. The first attempts to treat the cancer as the underlying cause of the ascites. The main treatments are systemic or intraperitoneal chemotherapies, biological therapies like intraperitoneal α or β ▶interferon, tumor necrosis factor TNF or administration of infectious agents in non pathogenic form like corynebacterium parvum or OK-432, a penicillinand heat-treated powder of Su-strain streptococcus pyogenes A3 in peritoneal cavity. Octreotide, a somatostatin analogue known to decrease the secretion of fluid by the intestinal mucosa and to increase water and electrolyte reabsorption, was used successfully in some case reports of malignant ascites. Novel therapies are radiolabeled monoclonal antibodies and radiocolloids. In tumors associated with increased activity of ▶vascular endothelial growth factor (VEGF) like ovarian, gastric, colon, pancreatic carcinomas and omental or hepatic metastatic malignancies, a new concept is to reduce the production of ascites by the inhibition of neovascularization. This is achieved via inhibition of vascular endothelial growth factor (VEGF) or inhibition of ▶matrix metalloproteinases, which are a family of enzymes present within the normal healthy individuals, but produced in high

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Aclarubicin

concentrations by a variety of tumors. However, these concepts are comparatively new and are based on experimental results or only partially investigated in Phase I trials and have not yet been evaluated in randomized controlled trials. The second approach in managing malignant ascites is palliative and relies upon reducing the volume of fluid through a variety of approaches like paracentesis, diuretics, or peritoneovenous shunts. Paracentesis is indicated for those patients who have symptoms of increasing intra-abdominal pressure. Available data show good, although temporary relief of symptoms in most patients. Symptoms seem to be significantly relieved by drainage of up to 5 L of fluid. When removing up to 5 L, intravenous fluids seem to be not routinely required. If the patient is hypotensive, dehydrated or known to have severe renal impairment and paracentesis is still indicated, intravenous hydration should be considered. The only investigated therapy in malignant ascites is infusion of dextrose 5%. There is no evidence of concurrent albumin infusions in patients with malignant ascites. To avoid repeated paracenteses, a peritoneovenous shunting may be considered. Major complications like pulmonary edema, pulmonary emboli, infection or clinically relevant disseminated intravascular coagulation have to be expected in about 6% of patients. There are no randomized trials assessing the efficacy of diuretic therapy. The available data are controversial and there are no clear predictors to identify which patients would benefit. Therefore, the use of diuretics should be considered in all patients with malignant ascites, but has to be evaluated individually. Patients with malignant ascites due to massive hepatic metastasis seem to respond more likely to diuretics than patients with malignant ascites caused by peritoneal carcinomatosis or chylous ascites. Choice of diuretics is also not sufficiently evaluated. As available data suggest that the efficacy of diuretics in malignant ascites depends on plasma renin/aldosterone concentration, aldosterone antagonists like spironolactone should be used, either alone or in combination with a loop diuretic.

References 1. Becker G, Galandi D, Blum HE (2006) Malignant ascites: systematic review and guideline for treatment. Eur J Cancer 42(5):589–597 2. Adam RA, Adam YG (2004) Malignant ascites: past, present and future. J Am Coll Surg 198(6):999–1011 3. Smith EM, Jayson GC (2003) The current and future management of malignant ascites. Clin Oncol 15:59–72 4. Gines P, Cardenas A, Arroyo V et al. (2004) Management of cirrhosis and ascites. N Engl J Med 350(16):1646–1654 5. Parsons SL, Watson SA, Steele RJC (1995) Malignant ascites. Br J Surg 83:6–14

Aclarubicin Definition Is an anthracycline anticancer agent; Synonym aclacinomycin A. ▶Adriamycin

Acquired Immunodeficiency Syndrome Definition AIDS; A life-threatening disease caused by he human immunodeficiency virus (▶HIV) and characterized by breakdown of the body's immune defenses.

Acquired Mutation Definition A mutation (a genetic change) acquired by a somatic cell after conception. ▶Gastrointestinal Stromal Tumor

Acromegaly Definition

The name “acromegaly” comes from the Greek words for “extremities” (acro) and “great” (megaly), because one of the most common symptoms of this condition is abnormal growth of the hands and feet. A hormonal disorder that most commonly occurs in middle-aged men and women. The prevalence of acromegaly is approximately 4,676 cases per million population, and the incidence is 116.9 new cases per million per year. The symptoms of acromegaly can vary and they

Actinomycin D

develop gradually over time; therefore, a diagnosis of this condition may be difficult. Early detection is a goal in the management of acromegaly because the pathologic effects of increased growth hormone (GH) production are progressive.

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Actin Cytoskeleton The actin cytoskeleton is a dynamic structure of ▶actin bundles and networks in the cytoplasm that provides a framework to maintain cell shape, protects the cell and enables cell locomotion. It also plays an important role in intra-cellular transport.

ACRP30 ▶Adiponectin

Actin Filament Severing Definition

ACTH

The breakage of noncovalent bonds between ▶actin molecules within an actin filament. ▶Geloslin

Definition Adrencorticotropic Hormone. ▶Corticotrophin

Actinic Keratosis Definition

Actin Definition A protein that is found abundantly in all eukaryotic cells. The protein exists in monomeric (globular or Gactin) and polymeric forms (filamentous or F-actin). Filamentous actin bundles to form long fibers known as Actin Microfilaments or stress fibers. Actin is a globular structural protein that polymerizes in a helix to form an actin filament. Actin filaments determine the cell shape, stabilize the cell mechanically, enable cell movements, and participate in contraction of the cell during ▶cytokinesis. Monomers of the protein actin polymerize to form long, thin fibers about 8 nm in a globular protein found in all cells. It is a major component in microfilaments and forms the contractile filaments in muscle cells. ▶Huntingtin Interacting Protein 1 (HIP1) ▶Micronucleus Assay ▶Migration ▶Tight Junction

Scaly, erythematous patches found on the skin in sun-exposed areas. Radiation induced keratosis (hornification) of the skin. It represents a precancerous lesion also known as solar keratosis or senile keratosis. May undergo malignant progression to form squamous cell carcinoma. ▶Epidermoid Carcinoma ▶Squamous Cell Carcinoma ▶Photodynamic Therapy

Actinomycin D Definition Synonym Dactinomycin. An antineoplastic antibiotic used for Nephroblastoma, Rhabdomyosarcoma, and trophoblastic disease in women. ▶Placental Site Trophoblastic Tumor (PSTT)

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Activated Fibroblasts

Activated Fibroblasts ▶Myofibroblasts

Activated Natural Killer Cells N ORIMASA I TO, H ERBERT J. III Z EH , M ICHAEL T. L OTZE University of Pittsburgh, Departments of Surgery and Bioengineering, Pittsburgh, PA, USA

Synonyms K cells; killer cells; K lymphocyte; large granular lymphocyte; lymphokine activated killer; LAK

Definition White blood cells that kill tumor and virus-infected cells as part of the body’s immune system (Unified Medical Language System). A type of white blood cell that contains granules with enzymes that can kill tumor cells or microbial cells (National Cancer Institute). A circulating cellular biosensor, regulating immunity through release of cytokines, maturation of dendritic cells, and recognition and lysis of stressed cells, allowing sampling of cellular contents for delivery to phagocytic cells (our definition).

Characteristics Biology of NK Cells ▶Natural Killer cells comprise 10–15% of circulating lymphocytes in normal adults and are also found in peripheral tissues, including the liver, peritoneal cavity, lymph nodes, and placenta. NK cells were first reported by Wunderlich, Herberman, and Sendo and others in the early 1970s. They were first discovered on the basis of their nonspecific killer activity, disturbing attempts to generate tumor-specific, ▶MHC-restricted cytotoxic T lymphocytes (CTLs). NK cell belongs to the innate immune system, bridging ▶adaptive immunity in concert with ▶dendritic cells. NK cells play a major role in the host defense against tumors and infected cells. NK cells mediate cytolysis of cultured tumor cells, and when lymphokine activated (LAK activity), against freshly acquired tumor cells. “Natural killer” suggests the initial notion that they do not require activation in order to kill target cells. NK cells are large granular lymphocytes (LGL). The targets of NK cells are stressed cells expressing either “nonself” or “the self that changed in quality,” prompting their recognition.

NK cells, when activated, can recognize cells which fail to express cognate self MHC molecules and simultaneously express (stress-induced) ligands recognized by activating NK receptors. These ligands include MICA/ MICB ULPBs, PVR, and Nectin-2 in humans or Rae-1 in mice. NK cytolytic activity is almost nonexistent at birth, increases until 15 years of age, and then gradually reduces through old age. Natural killer cells (NK cells) lack the ability to destroy tumor cells at the time of birth, acquiring cytolytic capacity following recognition. Given their ready acquisition from the peripheral blood, multiple studies have evaluated their activity in various clinical studies; for example, chronic mental stress, fatigue, and physical exertion suppress NK activity. Reduced NK activity may be related to increasing cancer risk. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection. Many studies indicate that NK activity is reduced in patients with advanced cancer. Tumor infiltrating NK cells of pediatric cancer are significantly less in number than that observed in adult cancers, prompting the notion that this creates a major nosologic difference of adult and pediatric neoplasms. Role of NK Cells in Human Cancer NK cells induce tumor cell death when NK cells recognize tumor cells with NK cell activating receptors. NK cells produce many cytokines including ▶IFNs and ▶TNF-α and suppress proliferation of tumor and cells and drive type 1 immunity. NK cells help dendritic cells to mature into DC1. NK cells have some suppressive roles against cancer. NK cells have inhibitory receptors. They become tolerant to tumor cells when inhibitory receptors are stimulated with their ligands (Fig. 1). Markers of NK Cells NK cells express CD16 (FcγRIII), CD56, ▶CD57, CD94, or CD158a. They do not express ▶T-cell receptor (TCR) or the pan T cell marker ▶CD3 or surface immunoglobulins (Ig) B cell receptor (▶CD20). NK cells recognize specific polysaccharide on target cells with NK receptor (CD161; NKR-P1) and expression of MHC class I molecules. NK Cell Receptors There are two main types of receptors for MHC class I on NK cells including the KIR (killer cell immunoglobulin-like receptors, one of the immunoglobulin superfamily) and NKG2 receptor (CD94, type C lectin family). In both, there are activating and suppressing forms that accelerate or suppress NK activity. Two explanations for NK-cell self-tolerance have been proposed: first, NK cells from MHC-class-Ideficient hosts have a lower activation potential, owing to decreased activating-receptor expression and/or

Activated Natural Killer Cells

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Activated Natural Killer Cells. Figure 1 Role of NK cells in tumor immunity. NK cells play multiple roles in tumor immunity. They recognize stressed cells or those failing to express cognate Class I major histocompability molecules, both lysing targets and serving as a source of cytokines important in initiation and perpetuation of the inflammatory response is carried out by them. They serve as helper cells, promoting immune interaction with both T and dendritic cells, critically being required for initiation of the TH1 response. Their absence may also be important in limiting autoimmunity as revealed by their critical absence in the NOD mouse strain, susceptible to autoimmune diabetes. When lysing cells, normal cells capable of undergoing apoptotic or autophagic00128 death, Types I and II death, do so. Many virally infected or transformed cells fail to undergo such death because of block of these pathways, and when lysed, undergo necrotic cell death causing DC maturation and promoting recruitment of additional inflammatory cells. In the absence of viral or bacterial pathogen signals, such chronic necrotic cell death is associated with inhibition of immune effectors and promotion of a wound repair phenotype with angiogenesis and stromagenesis, characteristic of many tumors.

function; or second, NK cells are kept self-tolerant by interactions between non-MHC-dependent receptor– ligand pairs CD94: NKG2, a C-type lectin family receptor, is conserved in both rodents and primates and identifies nonclassical (also nonpolymorphic) MHC I molecules including HLA E. Though indirect, this is a means to survey the levels of classical (polymorphic) HLA molecules. Expression of HLA E at the cell surface is dependent upon the presence of classical MHC class I leader peptides. Ly49 is a relatively ancient, C-type lectin family receptor. Humans have only one pseudogenic Ly49; the receptor for classical MHC I molecules. KIRs belong to a multigene family of more recently evolved Ig-like extracellular domain receptors. They are present in nonrodent primates and are the primary receptors for both classical MHC I (HLA A, HLA B, HLA C) and nonclassical HLA G in primates. KIRs are specific for certain HLA subtypes. ILT or LIR (leucocyte inhibitory receptors) are recently discovered members of the Ig receptor family. ▶Carcinoembryonic antigen related cell adhesion molecule 1

(▶CEACAM1 Adhesion Molecule) is an inhibitory receptor and its ligands are CEACAM1 itself and CEACAM5, known as CEA. Sialic acid binding immunoglobulin-like lectins (SIGLECs) have a V-set immunoglobulin domain, which binds sialic acid, and varying numbers of C2-set immunoglobulin domains. IRp60, KLRG1, and LAIR1 are other inhibitory receptors recently discovered (Table 1). NK Cell and Cytokines NK cells are capable of producing many cytokines including IFN-γ (▶Interferon-f γ), IFN-α, IFN-β, and TNF-α. They suppress proliferation of tumor and virally infected cells and regulate immune responses. IFN-γ (Interferon-f γ) increases NK activity as a positive feedback mechanism. NK cytolytic activity is increased by IFN-α, IFN-β, and IFN-γ (Interferon-f γ) (produced by T and NK cells), IL-2\(produced by T cells), IL-10, IL-12, and IL-15 (produced by B cell, monocyte/ macrophage, or dendritic cells). NK cytolytic activity is inhibited by IL-4 (▶Interleukin-4). IL-15 induces

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Activated Natural Killer Cells

Activated Natural Killer Cells. Table 1 receptors

Inhibitory and Activating NK Cell Receptors and their Ligands Activating NK cell

Receptors 2B4 NKp44 NKp30 NKp46 CD16 NKG2D02396 NKp80 DNAM Inhibitory NK cell receptors ILT2 KIR3DL2 KIR3DL1 KIR2DL4 KIR2DL1,2,3 CD94 CEACAM102441 IRp60 KLRG1 LAIR1 SIGLEC7 SIGLEC9

Ligands CD48 Influenza/unknown Influenza/unknown IgG MICA, MICB CD112/CD155 MHC01094-A, B, G MHC01094-A MHC01094-B MHC01094-A, B, G MHC01094-C MHC01094-C CEACAM102441, CEACAM5 Unknown Unknown Unknown Sialic acid Sialic acid

NK cell proliferation. IL-12 induces IFN-γ (Interferonf γ) production by NK cells. IFN-α, IFN-β, IFN-γ (Interferon-f γ), and TNF-α produced by NK cells activate monocyte/macrophage, vascular endothelial cells, neutrophils, and induce a local inflammation response. Cytotoxicity of NK Cells against Tumor or Infected Cells NK cells release ▶perforin from intracellular granules when they bind to target cells, along with granules containing serine proteases known as ▶granzymes. Perforin attaches to the membrane inducing an autophagic (▶Autophagy) repair process, inducing uptake of vesicles containing granzymes and associated molecules that can target cells for lysis, with perforin allowing escape through pore formation once intracellular. Granzyme induce apoptosis to the target cells utilizing various intracellular pathways. NK cells also induce ▶apoptosis to target cells by expressing apoptosis inducing molecules such as FAS ligands or ▶TRAIL on the cell surface. The distinction between apoptosis and ▶necrosis is important in cancer immunology – necrotic cells release danger/damage associated molecular pattern molecules (DAMPs) such as high-mobility group box 1 (HMGB1) protein, whereas apoptosis leads to retention of HMGB1 within the cells or apoptotic nuclei.

NK Cells and Cancer Immunotherapy Their rapid cytolytic action and broad target range suggest that NK cells may be promising candidates for cancer cell therapy. The clinical application of ex vivo manipulated cells, including NK cells, is referred to as ▶adoptive immunotherapy (AIT). The first clinical AIT trial exploited autologous ex vivo expanded and interleukin 2 (IL-2) stimulated ▶lymphokine activated killer (LAK). Although this approach produced nearly 15–20% partial and complete responses in initial trials, subsequent studies showed that a similar antitumor effect could be achieved with administration of high dose IL-2 alone. Purification and enrichment of NK cells on a clinical scale may improve therapeutic outcomes. Alternatively, stimulation of LAK cells with IL-15 or IL-21 instead of IL-2 might increase efficacy. Myeloid ▶dendritic cells (mDCs) support the tumoricidal activity of NK cells, while cytokinepreactivated NK cells activate DCs and induce their maturation and cytokine production. NK–DC interactions promote the subsequent induction of tumorspecific responses of CD4+ and CD8+ T cells, allowing NK cells to act as nominal “helper” cells in the development of the desirable type-1 responses to cancer. NK–DC interaction provides a strong rationale for the combined use of NK cells and DCs in the immunotherapy of patients with cancer. Clinical trials that are being

Acute Lung Injury (ALI)

implemented at present should allow evaluation of the immunological and clinical efficacy of combined NK–DC therapy of melanoma and other cancers.

References 1. Arnon TI, Markel G, Mandelboim O (2006) Tumor and viral recognition by natural killer cells receptors. Semin Cancer Biol 16:348–358 2. DeMarco RA, Fink MP, Lotze MT (2005) Monocytes promote natural killer cell interferon gamma production in response to the endogenous danger signal HMGB1. Mol Immunol 42(4):433–444 3. Lotze MT, Line BR, Mathisen DJ, et al. (1980) The in vivo distribution of autologous human and murine lymphoid cells grown in T cell growth factor (TCGF): implications for the adoptive immunotherapy of tumors. J Immunol 125 (4):1487–1493 4. Ito N, Demarco RA, Mailliard RB, et al. (2007) Cytolytic cells induce HMGB1 release from melanoma cell lines. J Leukoc Biol 81(1):75–83 5. Moretta A, Bottino C, Vitale M, et al. (1996) Receptors for HLA-class I-molecules in human Natural Killer cells. Annu Rev Immunol 14:619–648

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transcription factors activate various genes critical in the initiation of DNA synthesis. In ▶mesothelioma, it is thought that the persistent induction of these transcription activators following ▶asbestos exposure enhances cell division and favors malignant growth. ▶Mesothelioma ▶Polyphenols ▶Retinoid Receptor Cross-talk ▶Simian Virus 40 ▶SV40

Active ▶Melanoma Vaccines

Active Cell Death Activated Vitamin D ▶Calcitriol

▶Apoptosis

Active Immunity Definition

Activation Loop

Immunity produced by the body in response to stimulation by a disease-causing organism or a vaccine.

Definition A 20–25-residue segment within the catalytic domain of protein kinases that functions to regulate their kinase activity. ▶B-Raf Signaling

Activator Protein-1 (AP-1) Definition A dimeric complex that contains members of the c-jun, c-fos, ATF and MAF protein families. The ▶AP-1

Acute Granulocytic Leukemia ▶Acute Myeloid Leukemia

Acute Lung Injury (ALI) Definition A distinct form of acute respiratory failure characterized by diffuse pulmonary infiltrates, progressive hypoxemia, reduced lung compliance, and normal hydrostatic

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Acute Lymphoblastic Leukemia

pressures. ALI is caused by any stimulus of local or systemic inflammation, principally sepsis. ▶Sivelestat

Acute Lymphoblastic Leukemia C HING -H ON P UI St. Jude Children’s Research Hospital, Memphis, TN, USA

Synonyms Acute lymphoblastic leukemia; ALL

Definition Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from several cooperative genetic mutations in a single B or T-lymphoid progenitor, leading to altered blast cell proliferation, survival, and maturation, and eventually to the lethal accumulation of leukemic cells. Although cases can be subclassified further according to the stages of T or B-cell maturation, these distinctions are not therapeutically useful, except for the recognition of a mature B-cell, B-cell precursor, or T-cell stage.

Characteristics ALL accounts for about 12% of all childhood and adult leukemias diagnosed in developed countries and for 60% of those diagnosed in persons younger than 20 years. It is the most common cancer in children (25% of all cases) and has a peak incidence in patients between the ages of 2 and 5 years, with a second, smaller peak in the elderly. The factors predisposing children and adults to ALL remain largely unknown. Fewer than 5% cases are associated with inherited genetic syndromes defined by chromosomal instability and defective DNA repair. Ionizing radiation and mutagenic chemicals have been implicated in some cases of ALL, but their contributions appear negligible. Nonetheless, evidence collected over the past two decades has revealed that ALL is essentially a disease of acquired genetic abnormalities. Specific genetic abnormalities are found in the leukemic cells of approximately 75% of patients with ALL to date, and in all likelihood, will be identified in all cases with the improved genetic techniques. These include chromosomal translocations and chromosomal gains or losses, resulting in ▶hyperdiploidy or ▶hypodiploidy, respectively. Chromosomal translocations often

activate transcription factor genes, which in many cases control cell differentiation, are developmentally regulated, and frequently encode proteins at the tops of critical transcriptional cascades. These “master” oncogenic transcription factors, which can exert either positive or negative control over downstream responder genes, are aberrantly expressed in leukemic cells as a single gene product or as a unique fusion protein combining elements from two different transcription factors. Recently, activating mutations of ▶NOTCH1, a gene encoding a transmembrane receptor that regulates normal T-cell development, and mutations of ▶PAX5, a gene essential for B-lineage commitment and maintenance, have been identified to be most frequent cooperative mutations in T-cell and B-cell precursor ALL, respectively. Although most leukemias begin in the bone marrow and spread to other parts of the body, some may arise in an ▶extramedullary site, such as the thymus or intestine, and subsequently invade the bone marrow. The presenting features of ALL generally reflect the degree of bone marrow failure and the extent of extramedullary spread. Common signs and symptoms are . Fever . Fatigue and lethargy . Dyspnea, angina, and dizziness (older patients mainly) . Limp, bone pain, or refusal to walk (young children) . Pallor and bleeding in the skin or mouth cavity . Enlarged liver, spleen, and lymph nodes (more pronounced in children) . Anemia, low neutrophil count, and low platelet count . Metabolic abnormalities (e.g., high serum uric acid and phosphorus levels) The diagnosis of ALL is based on a morphologic examination of bone marrow cells (Figs. 1–3) and immunophenotype of cells from the same sample.

Acute Lymphoblastic Leukemia. Figure 1 Small regular blasts with scanty cytoplasm, homogeneous nuclear chromatin, and inconspicuous nucleoli.

Acute Lymphoblastic Leukemia

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Acute Lymphoblastic Leukemia. Figure 2 Admixture of large blasts with moderate amounts of cytoplasm and smaller blasts. Such cases may be mistaken for acute myeloid leukemia, emphasizing the importance of immunophenotyping and genotyping to corroborate the differential diagnosis.

Karyotyping, fluorescence in situ hybridization (FISH), and molecular genetic analysis by RT-PCR (reverse transcriptase-polymerase chain reaction) are now routinely performed by many centers to identify subtypes of ALL with prognostic and therapeutic significance, for example: . BCR-ABL fusion gene due to the t(9;22), or ▶Philadelphia chromosome – 25% of adult cases and 3–4% of childhood cases (dismal prognosis) . ▶TEL-AML1 fusion gene due to a cryptic t(12;21) – 22% of childhood cases (favorable prognosis) . Hyperdiploidy (more than 50 chromosomes per cell) – 25% of childhood cases (favorable prognosis) . Hypodiploidy (fewer than 45 chromosomes per cell) – 1% of childhood cases and 2% of adult cases (unfavorable prognosis) Contemporary risk-directed treatment can cure 80% or more of children and up to 40% of adults with ALL. Cases are generally classified as standard or high risk in adults and as low, standard, and high risk in children. Factors used to determine the relapse hazard include the presenting leukocyte count, age at diagnosis, gender, immunophenotype, ▶karyotype, molecular genetic abnormalities, initial response to therapy, and the amount of “minimal residual leukemia” upon achieving a complete ▶remission. Multidrug remission induction regimens almost always include a glucocorticoid (prednisone, prednisolone, or dexamethasone), vincristine, and at least a third agent (L-asparaginase or anthracycline), administered for 4–6 weeks. Some treatments rely on additional agents to increase the level of cell kill, thereby reducing the likelihood of the development of drug resistance and subsequent relapse. However, several studies suggest

Acute Lymphoblastic Leukemia. Figure 3 Mature B-cell ALL blasts characterized by intensely basophilic cytoplasm, regular cellular features, prominent nucleoli, and cytoplasmic vacuolation.

that intensive remission induction therapy may not be necessary for low or standard-risk patients, provided that they receive postinduction intensification therapy. Remission induction rates now range from 97 to 99% in children and from 78 to 93% in adults. Complete clinical remission is traditionally defined as restoration of normal blood cell formation with a blast cell fraction of less than 5% by light microscopic examination of the bone marrow. With this definition, some patients in complete remission may harbor as many as 1 × 1010 leukemic cells in their body. With sensitive and specific methods developed to measure minimal residual disease, it is now recognized that most patients actually have less than 0.01% of residual leukemia after 4–6 weeks of remission induction therapy, and they have excellent treatment outcome. By contrast, patients with 1% or more leukemic cells after remission induction treatment have a poor prognosis and may be candidates for hematopoietic stem cell transplantation. To improve treatment outcome, most protocols specify an intensification (or consolidation) phase in which several effective antileukemic drugs are administered in high doses soon after the patients attain a complete remission. Reinduction treatment, essentially a repetition of the initial induction therapy administered during the first few months of remission, has become an integral component of successful ALL treatment protocol. Regardless of the intensity of induction, consolidation or reinduction therapy, all children require 2–3 years of continuation treatment, usually methotrexate and mercaptopurine, with pulses of vincristine and dexamethasone for low-risk cases, and multiagent intensive chemotherapy for standard and high-risk cases. The need for continuation therapy in adults is less clear, although in most cases it is discontinued after 2–2½ years of complete remission. The central nervous system can be a ▶sanctuary site for leukemic cells, requiring intensive,

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Acute Megakaryoblastic Leukemia

intrathecally administered chemotherapy that begins early during the remission induction phase, extending through the consolidation phase and into the continuation phase. Once considered standard treatment, cranial irradiation is now reserved for less than 10% of patients who are at very high risk of relapse in the central nervous system. For selected high-risk cases, such as patients with Philadelphia chromosome-positive ALL, and those who require extended therapy to attain initial complete remission, hematopoietic stem-cell transplantation is currently the treatment of choice. In light of the development of new therapeutics, the indications for transplantation should be continuously evaluated. For example, therapy with imatinib mesylate (Gleevec; Novartis) or second-generation tyrosine kinase inhibitors has improved the duration of remission of patients with Philadelphia chromosome-positive ALL, but whether this therapy will increase the cure rate remains to be determined. Finally, the optimal clinical management of patients with ALL requires careful attention to methods for the prevention or treatment of metabolic and infectious complications, which may otherwise be fatal.

References 1. Pui C-H, Relling MV, Downing JR (2004) Acute lymphoblastic leukemia. N Engl J Med 350:1535–1548 2. Vitale A, Guarini A, Chiaretti S et al. (2006) The changing scene of adult acute lymphoblastic leukemia. Curr Opin Oncol 18:652–659 3. Pui C-H, Evans WE (2006) Treatment of acute lymphoblastic leukemia. N Engl J Med 354:166–178 4. Pui C-H, Jeha S (2007) New therapeutic strategies for the treatment of acute lymphoblastic leukaemia. Nat Rev Drug Discov 6:149–165 5. Mullighan CG, Goorha S, Radtke I et al. (2007) Genomewide analysis of genetic alterations in acute lymphoblastic leukemia. Nature 446:758–764

Acute Megakaryoblastic Leukemia J EAN -P IERRE B OURQUIN 1 , S HAI I ZRAELI 2 1

Pediatric Oncology, University Children’s Hospital Zurich, Zurich, Switzerland 2 Pediatric Hemato-Oncology, Sheba Medical Center and Tel Aviv University, Ramat Gan, Israel

Synonyms Acute Myeloid Leukemia; Subtype AML-M7 according to the French-American-British (FAB) Classification; Myeloid Leukemia of Down Syndrome (DS-ML); WHO classification: Acute Megakaryoblastic Leukemia (M7)

Definition Acute Megakaryoblastic Leukemia (AMKL) is defined as a malignant ▶clonal proliferation of immature hematopoietic cells of the megakaryocytic lineage. AMKL is a subtype of Acute Myeloid Leukemia (AML). The biologic features of AMKL are heterogeneous and ongoing characterization of the disease pathogenesis is likely to lead to a novel clinically meaningful classification of the disease.

Characteristics Epidemiology AMKL is diagnosed in 7–10% of infants and children with AML without Down syndrome (DS). In most pediatric cases the disease occurs de novo and subgroups can be identified based on cytogenetic features or biological features as described later. In contrast, AMKL is rare in adults, occurring in 1–2% of all AML cases and is frequently associated with antecedent hematological disorder such as myelodysplastic syndrome. Children with DS have a markedly increased risk to developing AMKL and represent up to 10 % of children with AML. A large proportion of Children with DS (estimated 10%) are born with a unique transient form of AMKL, often called transient myeloproliferative disorder (TMD) or transient abnormal myelopoiesis (TAM). This congenital leukemia resolves spontaneously in most of the patients. Up to 20% of those patients will relapse with a full blown AMKL by the age of 4 years. Thus the leukemia of DS represents a unique clinical entity of multistep leukemogenesis (Fig. 1). Clinical and pathologic features Typical features at diagnosis include hepato-splenomegaly, anemia, thrombocytopenia and myelofibrosis. The fibrosis is probably caused by soluble factors (such as TGF-β) secreted from the malignant megakaryoblasts. Infants with DS may exhibit marked liver failure that sometimes may be life threatening. The liver failure is secondary to liver fibrosis caused by the infiltration of leukemic cells. ▶Flow cytometry is the preferred method for ▶immunophenotypic characterization of AMKL, although in some cases the diagnosis can only be made from bone marrow or liver biopsies due to extensive myelofibrosis. Typically, the leukemic blasts express at least one megakaryoblastic antigen [CD41(GPIIb)/ CD42b(GPIbalpha) or CD61]. Coexpression of the T-lineage marker CD7 is frequently observed, suggesting pathogenic mechanism that could lead to aberrant regulation of lymphoid genes. Expression of erythroid markers (e.g. glycophorin A) and of CD36 (thrombospondine receptor) characterize the AMKL of DS. Because AMKL blasts may display low expression levels of the panhematopoietic CD45 antigen, the distinction from metastatic solid tumours may be challenging.

Acute Megakaryoblastic Leukemia

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Acute Megakaryoblastic Leukemia. Figure 1 Multistep evolution of AMKL in Down Syndrome. Mutation in GATA1 is acquired during fetal liver hematopoiesis in cells carrying a germline trisomy 21 results in congenital clonal megakaryoblastic proliferation (TMD). In almost all patients TMD resolves spontaneously leading to cure. However in about 20% of the patients additional postnatal acquired mutations in residual cells from the resolved TMD results in the development of full blown acute megakaryocytic leukemia (AMKL) during early childhood.

Cytogenetic and Biological Features Increasing evidence suggest that distinct subtypes of AMKL can be identified based on genetic and molecular characteristics. Recurrent cytogenetic abnormalities are specifically associated with AMKL and at least in part convey a prognostic significance. The megakaryoblastic disorders associated with DS (both AMKL and TMD) are characterized by the presence of an acquired mutation in the ▶transcription factor GATA1. The mutations occur in exon 2 or in the beginning of exon 3 and uniformly results in the production of a short GATA1 protein (GATA1s) that lacks the amino-terminal of the full length GATA1. GATA1 is a major regulator of normal megakaryopiesis. GATA1s blocks terminal differentiation and enhances proliferation of immature fetal megakaryoblasts. The mutations occur during ▶fetal liver hematopoiesis. The initiation of the leukemia during fetal liver hematopoiesis explains the frequent liver dysfunction observed in DS newborns with TMD. Strikingly, GATA1 is located on chromosome X and is mutated only in AMKL with trisomy 21. The precise mechanism by which trisomy 21 promotes the survival of cells with acquired mutation in GATA1 is presently unknown. One hypothesis suggests that genes on chromosome 21 code proteins that enhance fetal megakaryopoiesis. This developmental pressure of megakaryopoiesis coupled with differentiation arresting mutation in GATA1 cause clonal accumulation of megakaryoblasts diagnosed at birth as TMD. GATA1 mutation is necessary and probably sufficient for the development TMD, but additional mutations are required for the occurrence of full blown AMKL in DS patients. Why TMD spontaneously resolves and which mutations cause further evolution to AMKL is largely unknown. There are several biological subgroups among patients with AMKL that do not have DS. The most

frequent recurrent chromosomal aberration detected in non-DS AMKL is the translocation t(1;22), which typically occurs in infants and very young children that present with hepatosplenomegaly and pronounced myelofibrosis. This translocation fuses RBM15/ OTT1, an RNA export factor to MKL1/MAL1, a cofactor of the transcription factor SRF (Serum Response Factor). Less commonly fusion translocations between the MLL gene and different partners, often AF10, have been reported in AMKL. Interestingly, a second translocation involving AF10, the translocation t(10;11) which results in the fusion of CALM (clathrin-assembly protein-like lymphoid myeloid) with AF10 was reported in several cases. This translocation was also identified in other AML subtypes and in cases of T-cell ALL. In a mouse model, infection of bone marrow cells with a retroviral vector to express CALMAF10 results in a transplantable AML, demonstrating that this fusion gene represents a fundamental leukemogenic event. By ▶gene expression profiling, at least two distinct classes of non-DS AMKL could be discriminated based on their molecular phenotype. Approximately one third of the cases display an erythroid expression pattern coupled with expression of CD36 and higher expression levels of the transcription factor GATA1 in absence of detectable mutations. Interestingly this gene expression signature is reminiscent to the increased expression of erythroid markers detected in AMKL from DS patients, which are characterized by increased expression levels of mutated GATA1s. The second subtype of non-DS AMKL samples include all cases with recurrent translocation t(1;22). Interestingly, samples that share similar expression profiles with the samples positive for the translocation t(1;22) are characterized by increased expression levels of another SRF cofactor, HOP,

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Acute Myelogenous Leukemia

suggesting that similar regulatory pathways may be involved. This second class is associated with higher levels of expression of the surface antigen CD44, which was associated with worse outcome in other type of malignancies and coexpressed on the leukemia initiating cells from patients with AML. It is currently not possible to determine if the distinction of these two classes by expression profiling has a prognostic significance due to the small numbers of patients that were treated on different therapeutic protocols. A prospective study using selected genes from the AMKL signature will be required to determine if this information could be used as prognostic marker to guide selection of treatment intensity. Prognosis and Treatment Treatment results from several international study groups, including the European AML-BFM study group and UK-MRC cooperative groups, and the north american SJCRH and CCG cooperative groups show a marked difference in treatment outcome between DS and non-DS AMKL. Reduction of treatment intensity for patients with DS resulted in a marked decrease in treatment related mortality and an excellent treatment outcome (91% event free survival at 5 years in the AMLBFM 98 study), strongly suggesting a distinct leukemia biology between DS and non-DS AMKL patients. AMKL blasts from patients with DS are extremely sensitive to the chemotherapy drug Cytosine Arabinoside (ARA-C), probably due to a decrease in its cellular degradation caused by an enzyme regulated by GATA1. The results for patients with AMKL excluding patients with DS are still poor, despite of intensification of AML treatment regimens. The 5–year event free survival (EFS) reported for the most recent treatment regimen correspond to results obtained for other AML subtypes, with EFS of 42% reported for the AML-BFM93/98 trials and of 47% reported by the UKMRC 10 and 12 trials. Further research is necessary to identify new treatment modalities and biomarkers to guide treatment intensification, including the indication for bone marrow transplantation for patients at highest risk of relapse. Recent data in mouse models suggest that targeted therapy with antibodies directed against the surface marker CD44 may be a future therapeutic.

References 1. Bourquin JP, Subramanian A, Langebrake C et al. (2006) Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. PNAS 103:3339–3344 2. Ge Y, Stout ML, Tatman DA et al. (2005) GATA1, cytidine deaminase, and the high cure rate of Down syndrome children with acute megakaryocytic leukemia. J Natl Cancer Inst 97:226–231

3. Izraeli S (2006) Down’s syndrome as a model of a preleukemic condition. Haematologica 91:1448–1452 4. Oki Y, Kantarjian HM, Zhou X et al. (2006) Adult acute megakaryocytic leukemia: an analysis of 37 patients treated at M.D. Anderson Cancer Center. Blood 107:880–884 5. Reinhardt D, Diekamp S, Langebrake C et al. (2005) Acute megakaryoblastic leukemia in children and adolescents, excluding Down’s syndrome: improved outcome with intensified induction treatment. Leukemia 19: 1495–1496

Acute Myelogenous Leukemia ▶Acute Myeloid Leukemia

Acute Myeloid Leukemia B ARBARA D ESCHLER Medical Center, Department of Hematology-Oncology, University of Freiburg, Freiburg, Germany

Synonyms Acute myelogenous leukemia; Acute nonlymphocytic leukemia; ANLL; Acute granulocytic leukemia

Definition Acute myeloid leukemia (AML) is part of a group of hematological malignancies (▶hematological malignancies) in the bone marrow involving cells committed to the ▶myeloid line of cellular development. It is defined by the malignant transformation of a bone marrow-derived, self-renewing stem cell or progenitor (▶stem cells and cancer) which demonstrates a decreased rate of self-destruction and aberrant ▶differentiation. Uncontrolled growth of such cells, named blasts, is the result of ▶clonal proliferation. Blasts accumulate in the bone marrow and other organs. As a result, mature cells of ▶hematopoiesis are suppressed. For the leukemia to be called acute, the bone marrow must include greater than 20% leukemic blasts.

Characteristics Classification The first comprehensive morphologic-histochemical classification system for AML was developed by the French-American-British (FAB) Cooperative Group. This classification system categorizes AML into eight major subtypes (M0 to M7) based on morphology and

Acute Myeloid Leukemia

immunohistochemical detection of lineage markers. This classification of AML was recently revised under the auspices of the World Health Organization (WHO) (see list 1). While elements of the older FAB classification were preserved, the WHO classification incorporates and interrelates morphology, cytogenetics, molecular genetics, immunologic markers, and clinical features in an attempt to define categories that are biologically homogeneous and that have prognostic and therapeutic relevance. The most significant difference between the WHO and FAB classifications is that the minimum blast percentage for the diagnosis of AML is at least 20% blasts in the blood or bone marrow (the FAB scheme required the blast percentage to be at least 30%). What was known as “refractory anemia with excess blasts in transformation” (RAEB-t) of myelodysplastic syndromes (▶MDS), is now included within the broader category of “AML with multilineage dysplasia” as “AML with multilineage dysplasia following a MDS.” List 1 – WHO classification of AML (the older FAB classifications are given in parentheses where appropriate): . AML with characteristic genetic abnormalities – AML with t(8;21)(q22;q22); (AML/ETO) – AML with inv(16)(p13q22) or t(16;16)(p13; q22); (CBFβ/MYH11) – Acute promyelocytic leukemia (AML with t(15;17)(q22;q12); (PML/RARα) and variants) – AML with 11q23 (MLL) abnormalities . AML with multilineage dysplasia – AML with prior MDS – AML without prior MDS . AML and MDS, therapy-related – Alkylating agent-related AML and MDS – Topoisomerase II inhibitor-related AML . AML not otherwise categorized – Acute myeloblastic leukemia, minimally differentiated (FAB M0) – Acute myeloblastic leukemia without maturation (FAB M1)

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– Acute myeloblastic leukemia with maturation (FAB M2) – Acute myelomonocytic leukemia (AMML) (FAB M4) – Acute monoblastic leukemia and acute monocytic leukemia (FAB M5a and M5b) – Acute erythroid leukemias (FAB M6a and M6b) – Acute megakaryoblastic leukemia (FAB M7) – AML/transient myeloproliferative disorder in Down syndrome – Acute basophilic leukemia – Acute panmyelosis with myelofibrosis – Myeloid sarcoma . Acute leukemias of ambiguous lineage Epidemiology AML is infrequent but highly malignant, responsible for a large number of cancer-related deaths. AML accounts for approximately 25% of all leukemias in adults in industrialized countries and, thus, is the most frequent form of leukemia. Worldwide, the incidence of AML is highest in the United States, Australia, and Western Europe. The age-adjusted incidence rate of AML in the United States in the years 1975–2003 has been relatively stable at approximately 3.4 per 100,000 persons (=2.5 per 100,000 when age-adjusted to the world standard population). The American Cancer Society estimates that 11,930 individuals will be diagnosed with AML in 2006 in the United States. Patients that are newly diagnosed with AML have a median age of 65 years. From 2000 to 2003, the U.S. incidence rate in people under the age of 65 was only 1.8 per 100,000, while the incidence rate in people aged 65 or over was 17 per 100,000 (Fig. 1). AML is thus primarily a disease of later adulthood with an age-dependent mortality of 2.7 to nearly 18 per 100,000. The incidence of AML varies to a small degree depending on gender and race. AML in adults is slightly more prevalent in males in most countries. In the US in 2000, AML was more common in Whites with 3.8 per 100,000 than in Blacks (3.2 per 100,000).

Acute Myeloid Leukemia. Figure 1 Age-specific incidence of AML (USA: 2000–2003) (Source: SEER).

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Acute Myeloid Leukemia

Acute Myeloid Leukemia. Table 1

Risk factors

Genetic disorders

Down syndrome Klinefelter syndrome Patau syndrome Ataxia telangiectasia Shwachman syndrome Kostman syndrome Neurofibromatosis Fanconi anemia Li–Fraumeni syndrome Physical and chemical Benzene exposure Drugs as pipobroman Pesticides Cigarette smoking Embalming fluids Herbicides Radiation Exposure Non-/therapeutic radiation Chemotherapy Alkylating agents topoisomerase II inhibitors Anthracyclines Taxanes

Etiology The development of AML has been associated with several risk factors summarized in Table 1. Generally, only a small number of observed cases can be traced back to known risk factors. These include age, antecedent hematological disease, genetic disorders as well as exposures to radiation, chemical or other hazardous substances (e.g., benzene), and previous chemotherapy (e.g., treatment with ▶alkylating agents). Leukemogenesis, like ▶carcinogenesis is a multistep process that requires the susceptibility of a hematopoietic progenitor cell to inductive agents at multiple stages. The different subtypes of AML may have distinct causal mechanisms, suggesting a functional link between a particular molecular abnormality or mutation and the causal agent. Most cases of AML arise without objectifiable leukemogenic exposure. Signs and Symptoms of AML AML can cause different uncharacteristic signs and symptoms such as weight loss, unusual fatigue, and fever. Many patients feel a loss of well-being. Most symptoms can be traced back to bone marrow insufficiency: Anemia, immunodeficiency caused by neutropenia, and thrombocytopenia. Diagnostic procedures and types of specimen necessary to reach the diagnosis of AML are the following:

Acute Myeloid Leukemia. Figure 2 Myeloid blasts in peripheral blood detected by light microscopy.

. Blood cell counts and microscopic blood cell examination (Fig. 2) . Bone marrow aspiration and biopsy . Routine microscopic exam of bone marrow . Flow cytometry . Immunocytochemistry . Cytogenetics . Molecular genetic studies The peripheral blood count may reveal a decreased white blood cell count (leukopenia) as well as leukocytosis (increased white blood cell count). Leukemia cells do not protect against infection and may cause congestion of blood vessels (leukostasis). Thrombocytopenia, a decrease of platelets, can lead to excessive bruising, ▶petechiae, and bleeding. When leukemia cells spread outside the bone marrow, it is called extramedullary manifestation. Small pigmented spots that look like common rashes may indicate skin involvement. A tumor-like collection of AML cells is called chloroma or granulocytic sarcoma. AML sometimes causes enlargement of the liver and spleen. Prognostic Factors AML is a curable disease; the chance of cure for a specific patient depends on a number of prognostic factors. Some of the strongest prognostic information can be obtained by ▶cytogenetic analysis. Normal cytogenetics indicates average-risk AML. Cytogenetic abnormalities that suggest a good prognosis include translocations t(8;21) and t(15;17), as well as inv(16). Patients with AML that is characterized by deletions of the long arms or monosomies of chromosomes 5 or 7; by translocations or inversions of chromosome 3, t(6;9), t(9;22); or by abnormalities of chromosome 11q23 have particularly poor prognoses. Further adverse prognostic factors include central nervous system involvement with leukemia, elevated

Acute Nonlymphocytic Leukemia

white blood cell count (>100,000/mm3), treatmentinduced AML, and a history of MDS. Leukemias in which cells express the progenitor cell antigen CD34 and/or the P-glycoprotein (MDR1 gene product) have an inferior outcome. Due to a higher relapse rate, patients with AML associated with an internal tandem duplication of the FLT3 gene (FLT3/ITD mutation) have a poorer outcome. Beyond these disease-specific factors, patient-specific parameters like comorbidities and frailty have a strong impact on the course of the disease and treatment tolerability, as reflected by the age-dependent surge in mortality. Therapy Therapeutic approaches can be differentiated as curative (aimed at long-term cure) or palliative (principally aimed at achieving best quality of life) (▶palliative therapy). Curative intensive chemotherapeutic treatment (▶chemotherapy of cancer, progress and perspectives) for AML is considered the standard procedure, usually divided in two phases, induction and consolidation (post-remission) therapy. It is traditionally based on two substances, cytarabine (cytosine arabinoside) and anthracycline. The objective of a curative treatment approach is to rapidly eliminate the cancer cells with induction chemotherapy, called remission. Complete remission occurs in 60–80% of patients. More than 15% of adults with AML (about 25% of those who attain complete remission) can be expected to survive 3 or more years and may be cured. Remission rates in adult AML are inversely related to age, with an expected remission rate of >65% for those younger than 60years. Duration of remission may be shorter in older patients. Increased morbidity and mortality during induction appear to be directly related to age. This is associated with several factors including the ability to tolerate intensive treatment approaches. Without treatment, the average life expectancy is about 3 months. Complications during treatment include relapse of the disease, severe infections, or life-threatening bleeding. During this time, supportive care consists of patient isolation to prevent infection, antibiotics to treat infections, and transfusion of blood products. After remission is achieved, further treatment is known as consolidation and is necessary in order to achieve a permanent cure. Consolidation may consist of either further chemotherapy or a bone marrow, or stem cell transplantation. The aforementioned treatments are appropriate for all subtypes of AML except for one type of AML known as ▶acute promyelocytic leukemia (APL). Newer treatments, especially for those patients not tolerating intensive chemotherapy, include monoclonal antibodies, demethylating agents, and experimental

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drugs given in clinical trials. Thus, while the diagnosis of AML in itself does not represent a therapeutic mandate for intensive chemotherapy in all cases, the latter is the only curative approach to treatment. Decisions whether to treat patients with intensive chemotherapy, new agents, or solely best ▶supportive care should be based on a sum of patient factors (including age, previous history of MDS, ▶comorbidity, frailty, and patients’ preferences), in addition to the blast count and the above-described prognostic factors. Careful consideration of these factors is especially relevant in older, multimorbid patients with AML. ▶Acute Megakaryoblastic Leukemia ▶Nucleoporin

References 1. Brunning RD, Matutes E, Harris NL et al. (2001) Acute myeloid leukaemia: introduction. In: Jaffe ES, Harris NL, Stein H et al. (eds) Pathology and genetics of tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon, France. World Health Organization Classification of Tumours, 3, pp 77–80 2. Ries LAG, Harkins D, Krapcho M et al. (eds) (2006) SEER cancer statistics review, 1975–2003. National Cancer Institute, Bethesda, MD 3. Parkin DM, Whelan SL, Ferlay J et al. (eds) (1997) Cancer incidence in five continents, vol 7. IARC Scientific publications, Lyon, France 4. Grimwade D, Walker H, Harrison G et al. (2001) The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood 98 (5):1312–1320 5. Deschler B, de Witte T, Mertelsmann R et al. (2006) Treatment decision-making for older patients with highrisk myelodysplastic syndrome or acute myeloid leukemia: problems and approaches. Haematologica 91(11): 1513–1522

Acute Myeloid Leukemia 1 ▶Runx1

Acute Nonlymphocytic Leukemia ▶Acute Myeloid Leukemia

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Acute Promyelocytic Leukemia

Characteristics

Acute Promyelocytic Leukemia L I -Z HEN H E 1,3 , LORENA L. F IGUEIREDO -P ONTES 2 , E DUARDO M. R EGO 2 , P IER PAOLO PANDOLFI 1,3 1

Memorial Sloan-Kettering Cancer Center, Weill Cornell Graduate School of Medical Sciences, NY, USA 2 Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil 3 Cancer Genetics Program, Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02155

Definition Acute promyelocytic leukemia (APL) is a distinct subtype of ▶acute myeloid leukemia (AML) characterized by the expansion of leukemic cells blocked at the promyelocytic stage of myelopoiesis. According to the French–American–British (FAB) classification of acute leukemia, APL corresponds to the M3 and M3-variant subtypes, and according to World Health Organization classification (2001) it corresponds to the subtype: AML associated with translocations involving chromosomes 15 and 17 [t(15;17)] and variants. APL accounts for 5–10% of adult AML patients in Caucasian populations and for 20–30% among patients with Latino ancestry. Invariably, APL leukemic cells harbor ▶chromosomal translocations involving the retinoic acid receptor α (RARα) gene on chromosome 17 (Table 1), which may be fused to one of five possible partner genes: promyelocytic leukemia (PML), promyelocytic leukemia zinc finger (PLZF), nucleophosmin (NPM), nuclear mitotic apparatus (NuMA), and signal transducer and activator of transcription 5B (STAT5b). This leads to the generation of fusion genes encoding distinct fusion proteins. The sensitivity of APL to the differentiating action of all-trans retinoic acid (ATRA) is differentially mediated by the various fusion proteins (see Molecular Characterization).

Acute Promyelocytic Leukemia. Table 1

Molecular genetics of acute promyelocytic leukemia

Translocation t(15;17) t(11;17) t(5;17) t(11;17) t(17;17)

Clinical and Laboratorial Presentation The symptoms of APL are similar to those of other subtypes of AML such as weight loss, fatigue, weakness, pallor, fever, and bleeding. These symptoms manifest acutely and are accompanied by petechiae, bruising, oral bleeding, or epistaxis as well as symptoms and signs related to specific bacterial infections. Patients with APL are particularly susceptible to disseminated intravascular coagulation (DIC) and extensive bleeding is common at onset. The most common sites of clinically overt extramedullary leukemic infiltration include superficial lymphonodes, liver, and spleen. The leukocyte counts are usually lower than those observed in other AML subtypes and the differential counts reveal a variable percentage of blasts in the majority of patients. In most cases, anemia and thrombocytopenia are present at diagnosis. Abnormal promyelocytes constitute more than 20% of marrownucleated cells or more than 20% of leukocytes in peripheral blood. Leukemic blasts are morphologically characterized by the presence of distinctive large cytoplasmic granules, frequent multiple Auer rods, and a folded nucleus. The hypogranular variant (M3-variant) is characterized by the expansion of blasts containing large number of small granules that may be difficult to distinguish by light microscopy, and may be wrongly classified as monoblasts. However, both in the classical and variant M3 subtypes the cells are strongly positive for myeloperoxidase staining. A more rare hyperbasophilic variant has been described. The diagnosis is usually suspected upon the morphological examination of bone marrow and peripheral blood smears. The immunophenotypic profile suggestive of APL is composed by heterogenous intensity of expression of the CD13 surface marker associated with a homogenous expression of CD33; HLA-DR is negative in the majority of cases, and the expression of CD15 and CD34 is mutually exclusive and usually dim. The genetic confirmation of gene rearrangements involving the RARα locus is mandatory and can be done by classical cytogenetics, FISH, or RT-PCR. The

Fusion proteins PML–RARα PLZF–RARα NPM–RARα NuMA–RARα STAT5b–RARα

RARα–PML RARα–PLZF RARα–NPM RARα–NuMA? RARα–STAT5b?

Response to RA Good Poor Good Good Poor?

Acute Promyelocytic Leukemia

pattern of immunofluorescence staining using an antiPML antibody is also useful for a rapid diagnosis of APL. In APL cells a nuclear microspeckled pattern is observed in contrast to other subtypes of AML in which larger and less numerous dots (nuclear bodies) are evident. DIC occurs in 75% of M3 patients accompanied by secondary fibrinolysis. The cause of coagulopathy is complex, resulting from a combination of tissue factors and cancer procoagulant-induced activation of the coagulation, exaggerated fibrinolysis due predominantly to enhanced expression of annexin II on APL blasts, and blast cell production of cytokines. Laboratory evidence of DIC (prolonged prothrombine time and partial thromboplastin time, decreased fibrinogen and increased fibrin degradation products) should be examined in all APL patients. Molecular Characterization APL has been well characterized at the molecular level and has become one of the most compelling examples of aberrant transcriptional regulation in cancer pathogenesis. Due to reciprocal translocations, the RARα gene on chromosome 17 is fused to one of five distinct partner genes (for brevity, hereafter referred as X genes; Table 1). In the vast majority of cases, RARα fuses to the PML gene (originally named myl) on chromosome 15. In a few cases RARα fuses to the PLZF gene, to the NPM gene, to the NuMA gene, or to the STAT 5B gene located on chromosomes 11, 5, 11, or 17, respectively. The various translocations result in the generation of X–RARα and RARα–X fusion genes and the coexpression of their chimeric products in the leukemic blasts. The characterization of the genetic events of APL, and the availability of techniques such as FISH and RT-PCR, render it possible to confirm the diagnosis at the molecular level and to monitor minimal residual disease. RARα is a member of the superfamily of nuclear receptors, which acts as a retinoic acid (RA)-dependent transcriptional activator in its heterodimeric form with retinoid-X-receptors (RXR). In the absence of RA, RAR/ RXR heterodimers can repress transcription through histone deacetylation by recruiting nuclear receptor corepressors (SMRT), Sin3A, or Sin3B, which in turn, form complexes with histone deacetylases (HDAC) resulting in nucleosome assembly and transcription repression. PML–RARα represses transcription not only through HDAC, but also via interactions with DNA methyltransferases (DNMTs) leading to hypermethylation at target promoters. The epigenetic changes induced by PML–RARα are stable and maintained throughout cell divisions. ATRA causes the disassociation of the corepressor complex and the recruitment of transcriptional coactivators to the RAR/RXR complex. This is

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thought to result in terminal differentiation and growth arrest of various types of cells, including normal myeloid hematopoietic cells. The X–RARα fusion proteins function as aberrant transcriptional repressors, at least in part, through their ability to form repressive complexes with corepressors such as NCoR and HDACs. PLZF–RARα can also form, via its PLZF moiety, corepressor complexes that are less sensitive to RA than the PML–RARα corepressor complexes, thus justifying the poorer response to RA-treatment observed in these patients (see also Therapeutics). The X–RARα oncoproteins retain most of the functional domains of their parental proteins and can heterodimerize with X proteins, thus potentially acting as double-dominantnegative oncogenic products on both X and RAR/RXR regulated pathways. Recently, it has been demonstrated that APL blasts present a marked defect in TGF-β signaling including Smad2/3 phosphorylation and nuclear translocation, which is similar to that in Pml null primary cells. Remarkably, RA-treatment, which induces PML–RARα degradation, resensitizes the cells to TGF-β. It is plausible that PML–RARα may inhibit TGF-β signaling through direct inhibition of the interaction between Smad3 and the cytoplasmic form of PML (cPML). Modeling APL in Mice The transgenic approach in mice has been used successfully in modeling APL and in generating faithful mouse models harboring various APL fusion genes. In vivo, transgenic mice (TM) harboring X–RARα oncoproteins develop leukemia after a long latency suggesting that the fusion proteins are necessary, but not sufficient to cause full-blown APL. In the PML–RARα TM model, mice develop a form of leukemia that closely resembles human APL, presenting blasts with promyelocytic features that are sensitive to the differentiating action of RA. A similar phenotype was observed in NuMA–RARα TM, in which leukemia was also preceded by a period of latency, but displayed a higher penetrance. On the contrary, the leukemia developed by the PLZF–RARα TM lacked the distinctive differentiation block at the promyelocytic stage, morphologically resembling more a chronic myeloid leukemia (CML) type of disease, while NPM–RARα TM developed myelomonocytic leukemia. This analysis demonstrated that the X–RARα fusion protein plays a critical role in determining leukemic phenotype as well. Moreover, it is the X moiety of the X–RARα product to determine sensitivity to ATRA, since leukemia in PML–RARα, but not in the PLZF–RARα TM, is responsive to ATRA treatment. Modeling APL in TM contributed to the understanding of the important role of the reciprocal RARα–X fusion proteins. RARα–PML

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Acute Respiratory Distress Syndrome (ARDS)

and RARα–PLZF TM do not develop overt leukemia. However, the coexpression of RARα–PML with PML– RARα increases the penetrance and the onset of leukemia development in double mutants. Strikingly, in the PLZF–RARα TM model, the coexpression of RARα–PLZF with PLZF–RARα metamorphoses the “CML-like” leukemia in PLZF–RARα TM to a leukemia with classical APL features. In addition, RARα–PLZF renders the leukemic blasts even more unresponsive to the differentiating activity of RA. At the transcriptional level, RARα–PLZF acts as an aberrant transcription factor that can interfere with the repressive ability of PLZF. Therefore, RARα–X and X–RARα fusion products act in combination to dictate the distinctive phenotypic characteristics of each APL subtype disease. Modeling of APL in the mouse is thus allowing a better comprehension of the molecular mechanisms underlying the pathogenesis of APL as well as the development of novel therapeutic strategies. Therapeutics The exquisite sensitivity of APL blasts to the differentiating action of RA makes APL a paradigm for therapeutic approaches utilizing differentiating agents. This therapeutic approach conceptually differs from the treatments involving drug and/or irradiation therapies, because instead of eradicating the neoplastic cells by killing them, it reprograms these cells to differentiate normally. The utilization of ATRA in APL patient management has reduced early death from DIC-related complications and dramatically improved the prognosis. However, treatment with ATRA alone in APL patients induces disease remission transiently and relapse is inevitable if remission is not consolidated with chemotherapy. Most contemporary therapy protocols incorporate an anthracycline (e.g., dauno or idarubicin) with ATRA during induction, followed by consolidation therapy with ATRA, anthracyclines and cytarabine, followed by maintenance therapy. Leukocyte and platelet counts at diagnosis are frequently used as risk factors for relapse: patients presenting with more than 10,000 leukocytes/μl have high risk in contrast with those with less than 10,000/μl and platelet counts higher than 40,000/μl. In the majority of cases, relapse is accompanied by RA resistance. Unlike t(15;17)/PML–RARα APL, t(11;17)/PLZF–RARα leukemias show a distinctly worse prognosis with poor response to chemotherapy and little or no response to treatment with RA, thus defining a new APL syndrome. Up to 50% of patients treated with ATRA alone develop an “ATRA syndrome” characterized by a rapid rise in circulating polymorphonuclear leucocytes and associated with weight gain, fever, occasional renal failure, and cardiopulmonary failure, which may be life threatening in some patients. The combination of ATRA and chemotherapy in the induction and

consolidation treatment phases has been proven to be an effective strategy to prevent “ATRA syndrome” and achieve long-term disease-free survival. Arsenic trioxide (As2O3), a chemical used in Chinese medicine, is also extremely effective in the treatment of APL. About 90% of APL patients treated with As2O3 alone achieve complete remission, especially in relapsed patients who are resistant to RA and/or conventional chemotherapy. RA triggers blast differentiation while As2O3 induces both apoptosis and partial differentiation of the leukemic blasts. Utilizing PML–RARα and PLZF–RARα transgenic mouse models of APL, it has been demonstrated that the association of RA and As2O3 is effective in the former but not in the latter. Considering the importance of HDAC-mediated transcriptional repression in APL pathogenesis, the utilization of histone deacetylase inhibitors (HDACIs) such as suberanilohydroxamic acid (SAHA) or sodium phenylbutyrate (SPB) in combination with RA may represent a promising experimental therapeutic approach. Preclinical studies in transgenic mouse models of APL suggest that in fact HDACIs work as growth inhibitors and inducers of apoptosis, and that these effects are potentiated by RA.

References 1. Scaglioni PP, Pandolfi PP (2007) The theory of acute promyelocytic leukemia revisited. Curr Top Microbiol Immunol 313:85–100 2. Lin H-K, Bergmann S, Pandolfi PP (2005) Deregulated TGF-β signaling in leukemogenesis. Oncogene 24:5693–5700 3. Sanz M (2006) Treatment of acute promyelocytic leukemia. Hematology Am Soc Hematol Educ Program 147–155 4. Rego EM, Ruggero D, Tribioli C et al. (2006) Leukemia with distinct phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or NPM/RAR alpha. Oncogene 25(13):1974–1979 5. Rego EM, Pandolfi PP (2002) Reciprocal products of chromosomal translocations in human cancer pathogenesis: key players or innocent bystanders? Trends Mol Med 8:396–405

Acute Respiratory Distress Syndrome (ARDS) Definition A severe lung disease caused by a variety of direct and indirect insults. It is characterized by ▶inflammation of the lung parenchyma leading to impaired gas exchange

ADAM17

with concomitant systemic release of inflammatory mediators causing inflammation, hypoxemia, and frequently resulting in multiple organ failure. ▶Sivelestat

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ADAM Definition A Disentegrin and Metalloprotease; ADAM Molecules.

Acute Toxicity Studies ADAM10 ▶Single Dose Toxicity Studies

Definition

Acyclovir Definition A deoxyguanosine analog lacking the equivalent of 2υ and 3υ hydroxyl groups; commonly used in the treatment of herpesvirus infections. ▶HSV-TK/Ganciclovir Mediated Toxicity ▶Human Herpesvirus 6

ADAM10, synonym kuzbanian, is a member of a family of zinc-dependent transmembrane metalloproteases, involved in neuronal development in vertebrates and Drosophila and expressed in all epithelial tissues. Kuzbanian ADAM10 ▶Doublecortin ▶ADAM Molecules

ADAM17 A LEKSANDRA F RANOVIC , S TEPHEN L EE

AD Definition Androgen-dependent. ▶Cyclin G-Associated Kinase

Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ONT, Canada

Synonyms TACE; Tumor necrosis factor-alpha converting enzyme; CD156b antigen

Definition

ADAbp ▶CD26/DPPIV in Cancer Progression and Spread

ADAM17 is a zinc-dependent ▶metalloprotease belonging to the ▶ADAM (A disintegrin and metalloproteinase) family of type I transmembrane proteins. ADAM17 is involved in the ectodomain shedding of a wide variety of membrane-bound ligands and cytokines that are implicated in diverse biological processes including growth and ▶inflammation.

Characteristics

ADA-CP ▶CD26/DPPIV in Cancer Progression and Spread

Structure The 50 kb ADAM17 gene, which is located at chromosome 2p25, consists of 19 exons and encodes an 824 amino acid protein. ADAM17 is synthesized as an inactive precursor protein consisting of five domains: the

A

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ADAM17

pro-, metalloprotease, cysteine-rich, transmembrane and cytoplasmic domains. Prior to ADAM17 maturation, a conserved cysteine residue within the pro-domain interacts with the active site zinc atom maintaining the enzyme biologically inert. The active site of the metalloprotease domain contains a histidine consensus sequence (HExxHxxGxxH) that coordinates zinc atoms and water required for the enzymatic processing of ADAM17 substrates. Removal of the pro-domain occurs through a ▶furin cleavage site (RVKR), by an unidentified furin or proprotein convertase, enabling the active site zinc to interact with the required histidine residues and to generate the active protease. While the structural and functional aspects of the pro- and metalloprotease domains have been studied extensively and are well defined, the precise functions of the remaining ADAM17 domains are still somewhat obscure. The cysteine-rich domain consists of two subdomains: the disintegrin and EGF-like domains. A role in cellular ▶adhesion has been proposed for the disintegrin domain. In support of this hypothesis, ADAM17 has been shown to interact with at least one ▶integrin (α5β1) and modulate cell migration as a result of this interaction. It has also been demonstrated that the cysteine-rich domain is indispensable for the ectodomain shedding of select ADAM17 substrates and thus, might function in substrate recognition through the recruitment of accessory proteins or direct contact with the substrates themselves. The transmembrane domain tethers mature ADAM17 in the cell membrane where it exerts most of its physiological functions. Finally, the cytoplasmic domain comprises several ▶Src homology 2 (SH2) and 3 (SH3) domain binding sites as well as phosphorylation sites, and is likely involved in regulatory ▶signal transduction pathways. Expression and Regulation ADAM17 mRNA is ubiquitously expressed in most adult tissues, albeit at lower levels than those observed in fetal tissues at various stages of development. The ADAM17 zymogen is synthesized in the rough endoplasmic reticulum and is processed in the late Golgi compartment to produce the mature protease lacking the inhibitory pro-domain. This maturation step seems to entail a constitutive process as the majority of cellular ADAM17 exists in its mature form. The greater part of ADAM17 protein is localized in the perinuclear area while the remaining fraction resides at the cell surface, as expected. Notably, it appears that the membrane-bound ADAM17 population is exclusively in the processed form. This surface pool of ADAM17 is relatively stable with a half-life of ~8 h. The mechanism by which ADAM17 function is regulated is not entirely clear, however, two methods by which the protease can be activated have been described. The first method involves the activation of

ADAM17 by growth factors, such as the ▶fibroblast growth factor (FGF) and the ▶platelet-derived growth factor (PDGF). ADAM17-mediated ligand shedding can also be induced by non-physiological stimuli such as phorbol esters (▶Phorbol myristate acetate). Treatment of cells with phorbol esters, such as ▶PMA, results in increased ligand shedding without affecting the quantity or localization of endogenous ADAM17 in the cell. There is conflicting evidence with respect to the mechanism by which this stimulation occurs. One study demonstrated that PMA exerts its effects by activating the ▶extracellular signal-regulated kinase (ERK) signaling pathway, which results in the phosphorylation of ADAM17 at Thr735 in its cytoplasmic tail, while another group showed that the cytoplasmic tail of ADAM17 is not required for PMA-induced ligand shedding. Although there is no evidence that phorbol esters regulate ADAM17 activity in vivo, the ▶ERK signaling pathway has also been implicated in growth factor stimulated ADAM17 activation. For this reason, the ERK signaling pathway will likely be the focus of future studies aimed at delineating the mechanisms involved in the positive regulation of ADAM17 activity. In addition to stimulating ADAM17-mediated ligand cleavage, the treatment of cells with PMA also triggers the establishment of a negative feedback mechanism. Following an increase in ADAM17 activity and ligand shedding, the protease itself is internalized and degraded in response to prolonged treatment with PMA. This negative regulatory mechanism is probably in place to prevent over-stimulation of ligand-activated signaling pathways. In attempt to identify potential regulators of ADAM17 activity, two ADAM17 binding partners were uncovered by yeast two-hybrid screens: synapse associated protein 97 (SAP97) and protein tyrosine phosphatase PTPH1. Overexpression of either molecule results in decreased ligand shedding implicating them in the negative regulation of ADAM17 activity. Whether either of these two proteins regulates ADAM17 activity in vivo remains to be seen. The only known endogenous inhibitor of ADAM17 is the tissue metalloprotease inhibitor, TIMP3. The mechanism by which TIMP3 expression results in reduced ADAM17 activity is unknown. Biological Function ADAM17 was initially identified as the secretase responsible for the cleavage of tumor necrosis factor-alpha (TNFα), a pro-inflammatory cytokine. The generation of transgenic mice expressing ADAM17 lacking the zinc-binding sequence in its metalloprotease domain (ADAM17ΔZn/ΔZn) allowed for the identification of a multitude of additional ADAM17 substrates. The vast majority of the ADAM17ΔZn/ΔZn mice die at birth as a result of severe deficiencies in skin, muscle, lung and

ADAM17

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neuronal system development that cannot be entirely attributed to loss of TNFα shedding. This indicates the existence of other biologically relevant ADAM17 substrates. Interestingly, the few animals that do survive display a phenotype that is comparable to that of ▶transforming growth factor alpha (TGFα) or ▶epidermal growth factor receptor (EGFR) knockout mice. This includes the failure of eyelids to fuse as well as defects in skin and hair follicle development. Upon further investigation it was confirmed that ▶TGFα, an ▶EGFR ligand, is in fact an ADAM17 substrate. Moreover, ADAM17 appears to be the major convertase of several ▶EGFR ligands which are involved in a variety of cellular processes including cellular proliferation, survival, migration, and differentiation. The bulk of ADAM17 substrates, including the EGFR ligands, are involved in cell development and differentiation. Other examples include the neurogenic signaling molecule ▶Notch, the neurotrophin receptor TrkA, and the EGFR-family receptor HER4. The remaining substrates can be classified as those involved in cellular immunity and regulation of immunogenic responses, like TNFα. These substrates include the TNF receptors (TNF-RI and TNF-RII), the chemokine fractalkine, and the leukocyte adhesion molecule L-selectin to name a few. While many ADAM17 substrates have been identified to date, there is no obvious sequence or structural homology between their cleavage sites. How ADAM17 achieves substrate specificity is a key question that remains to be answered. Nonetheless, it is evident that ADAM17 substrates play an important role in a broad range of fundamental cellular processes.

malignancy. Thus ADAM17 is most highly expressed in advanced tumors, suggesting that ADAM17 and its substrates play a role in tumor progression. In accordance with these observations, there is a growing amount of evidence supporting the use of antiADAM17 drugs in the treatment of cancer. Several studies have shown that inhibition of ADAM17 activity using a variety of approaches is sufficient to inhibit EGFR ligand release and to prevent the proliferation, migration, and survival of squamous cell, ▶kidney cancer, ▶bladder cancer and ▶breast cancer cell lines in vitro. It was recently demonstrated that ▶siRNAmediated silencing of ADAM17 inhibits the release of soluble TGFα in highly malignant ▶renal carcinoma cells, thereby abolishing their ability to form tumors in nude mice. This was the first in vivo evidence that ADAM17-mediated ligand cleavage is a pivotal step in the establishment of the TGFα/EGFR autocrine ▶(Autocrine signaling) growth stimulatory loop and thus in tumorigenesis. Another study revealed that targeting ADAM17, using a ▶small molecule inhibitor, prevents ▶heregulin cleavage and hence ▶HER3 activation in non-small cell lung cancer cells. Not only did this inhibition abolish tumor growth in vivo but it also enhanced the sensitivity of the cancer cells to gefitinib, an anti-EGFR based therapy. This result suggests that the concomitant inhibition of ADAM17 and EGFR should improve patient responsiveness to such agents and increase survival. Thus targeting ADAM17 is a promising new alternative to traditional EGFR-based therapies in the treatment of human cancer.

Clinical Relevance Due to its involvement in TNFα processing, ADAM17 is considered to be a central mediator in human inflammatory diseases such as rheumatoid arthritis. Direct inhibition of TNFα or ADAM17 in arthritis-affected cartilage has been shown to reduce inflammation. For these reasons ADAM17-based therapies, such as zincchelating sulfonamide hydroxamates, are in use for the treatment of such diseases. In addition to its role in inflammatory diseases, ADAM17 is becoming increasingly implicated in the development and progression of cancer as a result of its role in the processing of EGFR ligands. The upregulation of EGFR expression and signaling is a common feature in human cancer. Unfortunately, ▶EGFR inhibitors have rendered disappointing results in ▶clinical trials and there is an apparent resistance of several cancer cell lines to these agents. Importantly, ADAM17 is also overexpressed in several neoplastic tissues including ▶breast carcinomas, ▶colon carcinomas, pancreatic ductal adenocarcinomas, and ▶ovarian carcinomas. There is also a positive correlation between ADAM17 expression and the aggressiveness of the

Summary ADAM17 was originally characterized for its role in TNFα processing and the regulation of inflammatory responses. It has since been demonstrated that ADAM17 is also a physiological convertase of a wide variety of signaling molecules implicated in the development and progression of cancer. The importance of ADAM17 in these oncogenic pathways is highlighted by the finding that silencing of ADAM17 is sufficient to abolish tumor formation in vivo. These results validate ADAM17 as a rational therapeutic target and endorse the use of ADAM17 inhibitors in the treatment of human cancer.

References 1. Blobel CP (2005) ADAMS: Key components in EGFR Signalling and Development. Nat Rev Mol Cell Biol 6:32–43 2. Franovic A, Robert I, Smith K et al. (2006) Multiple acquired renal carcinoma tumor capabilities abolished upon silencing of ADAM17. Cancer Res 66:8083–8090 3. Lee DC, Sunnarborg SW, Hinkle CL et al. (2003) TACE/ ADAM17 processing of EGFR ligands indicates a role as a

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ADAM Molecules

physiological convertase. Ann New York Acad Sci 995:22–38 4. Seals DF, Courtneidge SA (2003) The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 17:7–30 5. Zhou BS, Peyton M, He B et al. (2006) Targeting ADAMmediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell 10:39–50

ADAM Molecules J O¨ RG R INGEL 1,2 , M ATTHIAS LO¨ HR 2 1

Department of Medicine A, University of Greifswald, Greifswald, Germany 2 Molecular Gastroenterology Unit, German Cancer Research Center (DKFZ E180) Heidelberg and Department of Medicine II, Mannheim Medical Faculty, University of Heidelberg, Heidelberg, Germany

Synonyms A disintegrin and metalloprotease; Disintegrin metalloproteases; Metalloprotease disintegrin cysteine-rich; MDC

Definition A disintegrin and metalloprotease (ADAM) molecules share a common domain structure: a propeptide (prodomain), a metalloproteinase domain, a disintegrin domain, a cysteine-rich region, an epidermal growth factor (EGF)-like domain, a transmembrane region, and a cytoplasmatic domain (Fig. 1). Several ADAMs exist in both membrane-bound and secreted isoforms; the functional significance of this, in most cases, is still unclear. A subset of the presently known ADAM molecules shows catalytic activity. To date, at least 40 ADAMs have been identified in a variety of species.

A large proportion (13 ADAMs) is exclusively expressed in the male reproductive system, and only a minority can be found throughout all tissues.

Characteristics ADAM molecules, with their unique potential to combine ▶adhesion, ▶proteolysis, and signaling, are involved in a variety of cellular functions. Some have been shown to play an important role in diverse biological processes such as fertilization, myogenesis, cell signaling, inflammatory response, and cell–cell/ cell–matrix interactions. However, the respective key function has remained elusive for most ADAMs. Dysregulation of ADAM molecules has been shown in various diseases. However, there is a growing amount of reports about the role of ADAM molecules in malignant tumors. Metalloprotease Function To regulate biological activity, in normal as well as in malignant cells a wide variety of proteins are synthesized as inactive precursors that are subsequently converted to their mature active forms by ADAM molecules. A well-studied member of the ADAM molecules is ADAM17/▶TACE, which was originally described as being responsible for the proteolytic cleavage of the soluble form of ▶TNF-α. Subsequent studies have shown that ADAM17/TACE is also involved in the shedding of other biologically active proteins, including growth factors (erbB4/HER-4 and ▶transforming growth factor (TGF)-α), surface molecules (L-selectin), and interleukin (IL) receptors (IL-R; IL-1R type II and IL-6R; Fig. 2). TACE cleavage function in the activation of EGF receptor (EGFR) and EGFR signaling systems, which regulate the proliferation and motility of ▶squamous cell carcinoma cells in vitro. The key role of the EGFR/ EGFR ligand system for cancer development is wellknown. In this context, the transactivation of EGFR via ADAM17/TACE is of special interest. ADAM

ADAM Molecules. Figure 1 Domain structure of ADAMs. The ADAMs consist of a propeptide domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, a EGF-like, a transmembrane domain, and a cytoplasmatic domain.

ADAM Molecules

metalloproteases such as ADAM9 and ADAM17/ TACE regulate G protein-coupled receptor induced cell proliferation and survival. Aberrant expression of a proteolytic active ADAM17/TACE has been reported in pancreas ▶cancer cells. The increasing prevalence of ADAM17/TACE expression with higher pancreatic intraepithelial neoplasia (PanIN) grade as precursor lesions underlines the role of this molecule in ductal pancreatic adenocarcinoma development. Gene silencing experiments showed a critical role of ADAM17/TACE in the invasion process of pancreatic cancer cells. The aberrant expression of proteolytically active ADAM17/TACE may result in an uncontrolled turnover of activated target molecules, such as TNF-α, TGF-α, and MUC1 (▶mucius).

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Silencing of ADAM17 in human renal carcinoma cell lines corrects critical features associated with cancer cells, including growth autonomy, tumor inflammation, and tissue invasion. In addition, these cells fail to form in vivo tumors in the absence of ADAM17. It has also been shown that ADAM17/ TACE is overexpressed in mammary cancer and other cancer types (Table 1). ADAM12, which is upregulated, for example, in breast and gastric cancer (Table 1), is expressed in two splice forms, the transmembrane ADAM12-L and the soluble ADAM12-S. In a mouse breast cancer model, ADAM12 decreased tumor cell apoptosis and increased stromal cell apoptosis. The shedding of heparin-binding EGF by ADAM12 was shown to

ADAM Molecules. Figure 2 Schematic overview about the published functions and interactions of ADAM17/TACE.

ADAM Molecules. Table 1 as published ADAM molecule ADAM2 ADAM8 ADAM9 ADAM10 ADAM11 ADAM12 ADAM15 ADAM17/TACE ADAM19 ADAM21 ADAM22 ADAM23 ADAM28 ADAM29

Overview about the aberrant expression of ADAM molecules in different human cancer types

Human cancer type Renal Brain, prostate, lung adenocarcinoma Prostate, colon, pancreas, liver, gastric, nonsmall cell lung cancer, renal Breast, colon, prostate, pheochromocytoma, neuroblastoma Glioma, breast Breast, gastric, glioblastoma, liver, aggressive fibromatosis, giant cell tumor of the bone, brain Prostate, breast, lung, ovarian, gastric, brain, bladder Pancreas, renal, breast, colon, liver, brain, squamous cell carcinoma cells Brain ADAM21-like (ADAM21-L) T-cell leukemia Brain Brain, gastric, breast (pancreas) Nonsmall cell lung carcinoma Chronic lymphocytic leukemia

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ADAM Molecules

promote human ▶glioblastoma. In addition, in liver cancers, ADAM12 and ADAM9 expression is associated with tumor aggressiveness and progression. ADAM9 is also described to shed heparin-binding EGF. Overexpression of cytoplasmatic ADAM9 in pancreatic cancer is associated with poor differentiation and shortened survival. It is of particular interest for cancer development that ADAM molecules reported to shed cell-associated adhesion molecules such as L-selectin, MUC1, and glycoprotein (Gb) 1bα. In general, the metalloprotease protease function might be involved in various processes of cancer cells and be relevant to promote cell migration and invasion. Adhesion Function ADAM molecules are potential ligands for ▶integrins due to the presence of binding sites within the disintegrin domain. Only one ADAM (ADAM15) contains the ▶RGD integrin-binding motif and it can therefore interact not only with the αvβ3 integrin but also with the αvβ5. Additional ADAM–integrin interactions have been reported: a large number of ADAMs (1, 2, 3, 9, 12, and 15) with α9β1; ADAM9 with α6β1 and αvβ5; and ADAM28 with α4β1. Considering the recently published data on the interaction of ADAM17/TACE with the α5β1 integrin in ▶HeLa cells, it is also conceivable that ADAM17/ TACE may influence the migration and invasion in other cancer types. We are beginning to gather insights into ADAM– integrin and ADAM–▶extracellular matrix (ECM) interactions. The interplay with integrins and ECM compounds might promote ADAM function in malignant cells. Thus, cell binding to ADAM12 via beta3 integrin results in the formation of focal adhesions. Furthermore, it was shown that the cystein-rich domain of ADAM12 supports tumor cell adhesion through syndecan. ADAM23 with its inactive metalloprotease domain is exclusively involved in cell-adhesion. It was demonstrated that the interaction between the disintegrin-loop of ADAM23 and the αvβ3 integrin promotes the adhesion of ▶neuroblastoma and ▶astrocytoma cells. In contrast to the described overexpression or de novo expression in various cancer types, downregulation of ADAMs might also promote cancer development. Thus, ADAM23 gene silencing in breast cancer by promoter ▶hypermethylation may result in abnormal cell–cell interactions favoring cell migration. Signaling Function Beside the involvement of ADAM molecules in the EGFR transactivation, only few data about the signaling function of ADAM molecules are known. It is

intriguing that interactions between integrins and/or ECM- and ADAM-binding domains may induce outside–in signaling. ADAM inside–out signaling pathways might regulate shedding and/or adhesion function of the molecules. However, many ADAM cytoplasmatic domains contain binding motives for the Src homology region 3 (SH3 Domain) of various intracellular proteins. Tyrosine residues could be substrates for tyrosine kinases or could act as ligands for phosphotyrosine-binding domains, when phosphorylated. A number of binding partners have been identified for the cytoplasmatic domains of various ADAM molecules. Interaction of the cytoplasmatic domain of ADAM9 and 15 with endophilin and SH3PX1are reported. ADAM12 and ADAM15 are associated with ▶Src protein-tyrosine kinases. However, the shedding of the L1 adhesion molecules in breast cancer cells might involve a Scr protein-tyrosine kinase. Furthermore, mitotic arrest-deficient-2 (▶MAD2) was found as binding partner of ADAM17/TACE and ADAM15; MAD2β is linked to ADAM9. To date, the physiological role of this interactions as well as the implication in malignancies is speculative. Other Functions Within the ADAM molecules, ADAM11 might play a special role in malignancies. ADAM11 represents a candidate tumor suppressor gene for human breast cancer. This is based on its location within a minimal region of chromosome 17q21 previously defined by tumor deletion mapping. Taken together, there are rapidly increasing data supporting a critical implication of ADAM molecules in malignancies. But there are still more questions than answers on the function of ADAMs in human cancer and cancer development.

References 1. Seals DF, Courtneidge SA (2003) The ADAMs family of metalloproteases: multidomain proteins with multiple functions. Genes Dev 17:7–30 2. Gschwind A, Hart S, Fischer OM et al. (2004) TACE cleavage of proamphiregulin regulates GPCR-induced proliferation and motility of cancer cells. EMBO J 22:2411–2421 3. Ringel J, Jesnowski R, Moniaux N et al. (2006) Aberrant expression of a disintegrin and metalloproteinase 17/tumor necrosis factor-alpha converting enzyme increases the malignant potential in human pancreatic ductal adenocarcinoma. Cancer Res 66(18):9045–9053 4. Iba K, Albrechtsen R, Gilpin BJ et al. (1999) Cysteine-rich domain of human ADAM 12 (meltrin a) supports tumor cell adhesion. Am J Pathol 54:1489–1501 5. Karan D, Lin FC, Bryan M et al. (2003) Expression of ADAMs (a disintegrin and metalloproteases) and TIMP-3 (tissue inhibitor of metalloproteinase-3) in human prostatic adenocarcinomas. Int J Oncol 23:1365–1371

Additive Effects

Adaptive Immunity

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Adaptor Proteins

Definition

Definition

Adaptive immune responses occur when the host comes into contact with immunogenic molecules or organisms. These stimulate the expansion of the antigenspecific lymphocytes, antibody secreting B cells and T cells of the cytotoxic and helper phenotypes, which recognize cells expressing foreign antigens. B cells and T cells are the effector cells of the adaptive immune response. They bear antigen specific receptors of great diversity that are generated by random rearrangement of gene segments and other mechanisms. This results in a vast array of antigen-specific receptors clonally distributed on T and B cells, which clonally expand on contact with antigen. As the immunogen is cleared these clonal populations shrink but leave behind longlived populations of memory cells that are easily recalled on subsequent exposure to the same immunogen. Unlike the innate immune response, adaptive responses are not immediate, requiring 3–5 days for clonal expansion and differentiation of effector lymphocytes. The adaptive immune system allows for a strong immune response as well as immunological memory, where a tumor antigen is “remembered.” The adaptive immune response is antigen-specific and requires the recognition of tumor antigens during a process called antigen presentation. Antigen specificity allows for the generation of responses that are tailored to cancer cells and the ability to mount these tailored responses is maintained in the body by “memory cells.” Cells of the adaptive immune system are B- and T-lymphocytes. Adaptive humoral responses are mediated by tumor specific antibodies.

lack any intrinsic enzymatic activity themselves but instead mediate specific protein-protein interactions leading to formation of protein complexes.

▶Immunoediting ▶Specific Immunity ▶Immunoprevention of Cancer ▶DNA Vaccination ▶Inflammation

▶RAS Activation

ADAR Definition

A family of “adenosine-deaminase-acting-on-RNA” enzymes that convert adenosines to inosines in double-stranded RNA substrates. This process, known as A → I RNA editing, changes the sequence of the target dsRNA molecule so that it differs from the parent DNA strand. At translation, the inosine is interpreted as a guanosine (G). ▶ALU Elements

ADCC Definition Antibody-dependent cell-mediated cytotoxicity. ▶Immunoprevention of Cancer ▶Diabody ▶EpCAM

Adaptive Response Additive Effects Definition The process of adaptation, which allows survival under adverse conditions, is called adaptive response ▶Detoxification

Definition

Additive effects mean that the effects of ▶xenobiotics simply summate.

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Adduct

Adduct Definition In biology, adduct is a complex that forms when a chemical binds to a biological molecule, such as DNA or protein. ▶Biomonitoring ▶Adducts to DNA

(▶surrogate markers). DNA adducts are mechanistically more relevant to ▶carcinogenesis than the internal dose of a carcinogen, since they take into account interindividual differences in metabolism and of DNA repair capacity (Fig. 1). Several hundred DNA adducts, many with miscoding properties, are known to be produced by some 20 classes of carcinogens and through endogenous oxidative processes. DNA adducts are used in human ▶biomonitoring as dosimeters of early biological effects and predictors of cancer risk. These ▶biomarkers also provide tools for studying disease pathogenesis, etiology and for verifying preventive measures in human cancer.

Characteristics

Adducts to DNA H ELMUT B ARTSCH Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany

Synonyms DNA-bound carcinogens

Definition

DNA-adducts reflect the amount of a ▶xenobiotic that covalently reacts with nucleic acid bases at the target site (biologically effective dose) or in surrogate tissues

Rationale for Using DNA Adducts as Biomarkers for Exposure and Adverse Effects Evidence for the biological significance of DNA adducts in carcinogenesis is supported by the following: . Over 80% of identified or suspected human carcinogens react often after metabolic activation with nucleic acids and proteins to form macromolecular adducts . Carcinogen-DNA adducts represent the initiating events leading to mutations in ▶oncogenes and ▶tumor suppressor genes, and to ▶carcinogenesis . The carcinogenic potency of a large number of carcinogens is proportional to the extent they bind to rodent liver DNA . Humans with inherited or acquired defects in ▶DNA repair have an elevated risk of developing cancer

Adducts to DNA. Figure 1 Paradigm for the multistage process of ▶carcinogenesis with DNA adducts as initiating lesions. They are used mostly as biomarkers for the biologically effective dose both of exogenous carcinogens and of DNA-reactive agents produced by endogenous processes, such as chronic oxidative stress. Over the past 40 years emphasis has been placed on the development of accurate and sensitive methods for the detection and quantitation of DNA adducts.

Adducts to DNA

43

Biological effect markers are defined as indicators of irreversible genetic damage that result from genotoxic interactions at the target site. As DNA adducts do not often cause completely irreversible lesions, because the DNA undergoes repair (which may not be complete), they are not in the strict sense biological effect markers. However, as carcinogen dosage is linked to cancer outcome, and permanent mutations can be caused by DNA adducts, they are associated with cancer risk. This has been shown for many carcinogens and their DNA adducts, when critical toxico-kinetic parameters are taken into account. These include the steady state adduct concentration, the amount of the miscoding adduct compared to others of lesser biological relevance, the adduct half-life after carcinogen exposure has stopped, the organ, cell and gene selectivity of the adduct (Fig. 2).

and/or subpopulations of non-dividing cells can survive for several months or even years. Since somatic genetic or cytogenetic effect markers are neither chemical- nor exposure-specific, only macromolecular adducts allow identification of the structure and thus the determination of the genotoxic exposure sources. Also, cytogenetic markers are more easily affected by lifestyle and environmental components (confounders) that often act as uncontrolled or uncontrollable variables in biomonitoring and molecular epidemiology studies. In addition, at equal levels of carcinogen exposure, DNA adduct levels are a measure for the host’s capability of carcinogen metabolism and adduct repair and can be used to determine the overall effect of genetic polymorphisms on DNA damage and cancer susceptibility by a given carcinogen.

Advantages and Disadvantages of DNA Adducts Compared to Other Biomarkers For human biomonitoring both DNA and protein adducts can be used for exposure assessment as long as the response in target organs versus surrogate tissue is shown to be proportional. The latter has to be determined individually for each carcinogen. The advantage of certain protein adduct measurements is that they often reflect cumulative past exposure (of several months), while the majority of DNA adducts is rapidly repaired or lost after exposure has ceased. However, a small portion of DNA adducts either with slow repair

Cellular Defense: Repair of DNA Adducts DNA repair (▶repair of DNA) systems such as ▶base and ▶nucleotide excision repair, O6-alkylguanineDNA alkyltransferase and ▶mismatch repair operate in human cells to remove adducted and oxidatively damaged DNA bases. Deficiency in nucleotide excision repair genes cause ▶Xeroderma pigmentosum (XP) and a high-rate occurrence of skin cancers, as well as a high susceptibility to UV-light and ▶polycyclic aromatic hydrocarbon-induced carcinogenesis. A defective mismatch repair system causes hereditary non-polyposis colorectal cancer (HNPCC).

Adducts to DNA. Figure 2 Measurement of carcinogen-DNA adducts in target tissue and cells or in surrogates. The predictive value of DNA adducts for disease risk increases with the proximity of measurements to critical lesions. Accordingly, from right to left, the specificity of this biomarker increases for predicting disease outcome.

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Adducts to DNA

Genetic defects in these DNA repair functions or inhibition of repair proteins may have dramatic consequences when DNA adducts, DNA mismatches and DNA loops are not repaired prior to cell replication and when damaged cells are not eliminated by apoptosis. Thus, characterization of germ-line and somatic mutations in DNA-repair genes can identify high-risk subjects who especially in the case of bi-allelic mutations suffer from functional defects of proteins that repair DNAadducts leading to genetic instability and cancer. Adduct Measurements in Disease Epidemiology Cross-sectional and longitudinal studies in cancer epidemiology assess the relationship between carcinogen exposures and biomarker (adduct) levels. Adduct measurement exposed in humans allow the detection, quantification and structural elucidation of specific DNA damage. Findings from such studies include the detection of background exposures manifested in “unexposed” populations and a significant interindividual variation in adduct levels in persons with comparable exposure. The latter is in part due to genetic variation in carcinogen metabolism and DNA-repair processes. Positive correlations between the extent of occupational and environmental exposures, adduct levels and adverse effects, e.g. mutations in oncogenes and tumor suppressor genes have been observed. For example, large-scale studies on geographical variations of ▶hepatocellular carcinoma and exposure to ▶aflatoxins have used aflatoxin-bound albumin adducts, urinary aflatoxin B1N7-guanine adducts, and mutational hotspots in the ▶TP53 gene as biomarkers. They revealed more than an additive interaction between the hepatocarcinogen and hepatitis B virus (▶hepatitis viruses) infection. ▶Case-control studies in disease epidemiology allow the evaluation of the role of biomarkers as cancer risk factors and the exploration of underlying mechanisms, but such studies cannot establish causality between biomarker response and cancer causation. This is especially the case when the latency period (between exposure and cancer) is long. Here, adduct measurements are of greater relevance for cancer risk estimation when exposure has been continuous. An optimal study design that can establish causality is a nested case-control study that uses questionnaire data and biological sample collection prior to disease manifestation. Once diagnosis of cancer has been made, cases are matched to appropriate controls and their stored samples analyzed. The predictive value in terms of specificity and sensitivity of a DNA adduct biomarker in biological samples can thus be determined. Association of DNA Adducts with Cancer Risk Not all types of DNA adducts are associated with the same cancer risk. Using alkylating agents, aflatoxins and aromatic amines (that induced 50% tumor

incidence) DNA adduct levels were compared in animal experiments. A 40- to 100-fold difference in the ability of DNA adducts to induce the same tumor incidence in target tissues was detected. Thus, it is difficult to predict the tumor induction potential of unknown DNA adducts. In the past, assays for DNA adduct determination provided mostly information on the total amount of adducts in bulk genomic DNA. However, new methods are capable of pinpointing adduct profiles in critical target genes (Fig. 2). Because of the multistage and complex nature of human carcinogenesis, carcinogenDNA adducts per se cannot precisely and quantitatively predict an individual’s cancer risk. At present risk estimation is limited to a group level. Background DNA-adduct Levels: Sources, Variations and Cancer Risk Prediction The major analytical challenge has been to detect levels of DNA adducts at a concentration of 0.1–1 adducts per 108 unmodified DNA bases using only low microgram amounts of DNA, and with high specificity and accuracy. Several methods are available including 32 P-postlabeling assays often in combination with immunopurification and liquid chromatography coupled to electrospray ionization-mass spectrometry. By using ultrasensitive detection methods background DNA adduct levels have been found in organs of unexposed humans and untreated animals. These are due to physiological lipid peroxidation (LPO) processes, whereby endproducts, such as 4-hydroxynonenal and malondialdehyde when formed in excess in the body, can react with DNA to yield background levels of a variety of exocyclic DNA adducts. These types of adducts generally increase with age, but are significantly increased in human subjects affected by risk cancer factors that induce chronic oxidative stress. These include chronic inflammatory processes and infections, nutritional imbalances, and metal storage disorders. In addition, oxidized DNA bases and LPO-derived DNA adducts occur more frequently in cells with impaired antioxidant defense. Exogenous carcinogens can also induce oxidative stress causing agent-specific DNA adducts and secondary oxidative DNA base damage. The biological relevance of both oxidative and LPOderived DNA damage is supported by the fact that these adducts are miscoding lesions which are recognized by specific DNA-repair enzymes. There is a growing evidence that both types of DNA lesions, either derived from exogenous and endogenous agents, play a role in the initiation and progression of the multistage carcinogenesis process, as well as other chronic degenerative diseases. Current research addresses some open questions: . What is the significance of endogenously formed DNA adducts in human cancer, particularly associated with chronic inflammatory conditions and also in relation to spontaneous tumors?

Adenocarcinoma

. Has the proportion of cancers that result from environmental agents been overestimated compared to those arising from endogenous DNA damaging processes? . Can one protect humans against endogenously derived DNA damage and prevent chronic degenerative diseases by administration of chemopreventive (antioxidative) agents, using DNA-adduct measurements to verify their efficacy? . Will LPO-derived DNA adducts serve as potential prognostic markers for assessing progression of chronic inflammatory cancer-prone diseases? Contributions of DNA-adduct Measurements to Disease Etiology and Pathogenesis New insights are gained since . Adduct analysis permits identification of hitherto unknown exogenous and endogenous DNA-reactive agents and of carcinogenic components in complex exposures, thus increasing the power to establish causal relationships in molecular epidemiology. . Highly exposed individuals can be more readily identified, and exposure to carcinogenic risk factors can be minimized or even avoided. . Subgroups in the population (so called pharmacogenetic variants) that are, due to genetic polymorphism of xenobiotic-metabolizing and DNA-repair enzymes, more susceptible to carcinogens, are identifiable by a combination of genotyping and DNA-adduct measurements. . Repeated applications of dosimetry methods for macromolecular adducts can evaluate the effectiveness of primary and secondary interventions, either by reduction of carcinogen exposure or through (chemo-)preventive strategies. . Incorporation of DNA-adduct measurements (and of other critical endpoints involved in carcinogenesis) can reduce (i) the enormous uncertainties currently associated with high-to-low dose and species-tospecies extrapolation and (ii) yield information on inter-individual risk assessment procedures. . The role of specific carcinogen exposures may be retrospectively implicated in cancer etiology by analyzing decades after the period of exposure, mutational fingerprints in tumors that arise from exogenous and endogenous agents after their reaction with DNA. Specific mutational signatures, detected in the tumor suppressor gene TP53, were associated with distinct past carcinogen exposures (e.g. tobacco smoke, aflatoxin B1, vinyl chloride, and UV-light) or inflammatory disease state (such as chronic inflammatory bowel diseases). . Adducts and derived mutations should allow to study pathogenesis and preventive approaches of chronic degenerative diseases other than cancer (e.g. atherosclerosis, Alzheimer disease).

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References 1. Toniolo P, Boffetta P, Shuker DEG et al. (eds) (1997) Application of biomarkers in cancer epidemiology. IARC Sci Publ 142. IARC, Lyon, pp 143–158 2. Gupta RC, Lutz WK (eds) (1999) Background DNA damage. Mutat Res 424:1–288 3. Vineis P, Perera F (2000) DNA adducts as markers of exposure to carcinogenesis and risk of cancer. Int J Cancer 88:325–328 4. Bartsch H, Nair J (2006) Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA-. damage and repair Langenbecks Arch Surg 391:499–510 5. Singh R, Farmer PB (2006) Liquid chromatographyelectrospray ionization-mass spectrometry: the future of DNA adduct detection. Carcinogenesis 27:178–196

Adenine Nucleotides Definition Energy-carrying molecules composed of the purine base adenine, the sugar ribose, and one (AMP), two (ADP) or three (ATP) covalently-attached phosphate groups. ▶Adenosine and Tumor Microenvironment

Adenocarcinoid Definition An appendiceal malignancy that contains both an epithelial neoplasm and a neuroendocrine (carcinoid) neoplasm simultaneously. ▶Appendiceal Epithelial Neoplasms ▶Carcinoid Tumors

Adenocarcinoma Definition A form of carcinoma that originates in glandular tissue. To be classified as adenocarcinoma, the cells do not necessarily need to be part of a gland, as long as they

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Adenomas

have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. The term adenocarcinoma is derived from “adeno” meaning “pertaining to a gland” and “carcinoma” which describes a cancer that has developed in the epithelial cells, i.e. cells that line the walls of various organs. This type accounts for about 40% of ▶lung cancer. It is usually found in the outer part of the lung. ▶Vanadium ▶Bile Duct Neoplasms ▶Appendiceal Epithelial Neoplasms

Adenosine and Tumor Microenvironment J ONATHAN B LAY Department of Pharmacology, Dalhousie University, Halifax, NS, Canada

Synonyms Adenosine; Adenine nucleoside; purine nucleoside; adenine-9-β-D-ribofuranoside

Definition

Adenomas ▶Colorectal Premalignant Lesions

Adenosine is a small molecule that is released into tissue at high concentrations in response to a deficiency of oxygen, which occurs characteristically in solid tumors. Adenosine has multiple effects within the tumor, including controlling cancer cell growth, locally inhibiting the immune system, and increasing blood vessel formation.

Characteristics

Adenomatous Polyposis Coli Definition Familial Adenomatous Polyposis; APC.

Adenosine (adenine-9-β-D-ribofuranoside, Fig. 1) is a small organic molecule that plays an important part in general cellular biochemistry. Chemically, it is a purine nucleoside. Adenosine is abundant within all cells, predominantly in the form of ▶adenine nucleotides (AMP, ADP and ATP) which participate widely in cellular energy metabolism and act as precursor molecules in many processes. However, adenosine itself can exist in a free form both inside and outside of cells, and extracellular adenosine is responsible for the regulation of many processes throughout the body. Adenosine becomes particularly important when tissues become deprived of oxygen (a state known as

Adenomatous Polyps ▶Colorectal Premalignant Lesions

Adenomucinosis (DPAM) Definition An appendiceal epithelial neoplasm that is noninvasive so that peritoneal surfaces are coated by increasing quantities of a mucinous neoplasm. ▶Appendiceal Epithelial Neoplasms

Adenosine and Tumor Microenvironment. Figure 1 The chemical structure of adenosine. Adenosine is composed of a purine base (adenine) linked through a glycosidic bond to a sugar (ribose). Successive phosphate groups may be added at the position indicated by the arrow to give AMP (adenosine monophosphate), ADP (adenosine diphosphate) and ATP (adenosine triphosphate).

Adenosine and Tumor Microenvironment

▶hypoxia). This can happen in certain pathological situations, including cancer. It may occur suddenly when blood flow is interrupted, as takes place in a stroke within the brain or during a heart attack. In solid tumors however, hypoxia is a chronic condition because the blood vessels that the cancer forms to nourish itself are not well made and are unable to supply the tissue with sufficient oxygen and other nutrients. For cells to be well oxygenated, they need to be within a distance of about 150 μm of a properly functioning blood vessel. Tumor vessels are typically far apart, are irregular in both size and orientation and can be so poorly regulated that the blood flow may periodically change direction. Cancer cells respond to these harsher conditions by changing their metabolism. In hypoxic cancer tissues, the balance of energy metabolism in the cells becomes altered. Specific changes in the biochemical pathways of hypoxic cells dramatically change the fate of adenosine. Free adenosine is normally formed principally from adenine nucleotides by the enzyme 5′-nucleotidase inside the cell (some tissues have another pathway that also contributes), and adjacent to the exterior of the cell membrane by a series of proteins including ▶CD39 and ▶CD73, the latter of which also has 5′-nucleotidase activity (Fig. 2) In hypoxia, the 5′-nucleotidase pathways that lead to adenosine production from adenine nucleotides are activated, while the adenosine kinase enzyme which serves to convert adenosine to AMP is inhibited. These and other changes rapidly increase the concentrations of adenosine within and outside the cell. Since adenosine can pass freely into and out of the cell through various ▶nucleoside transporters in the outer membrane, any excess adenosine in the cytoplasm escapes from the cell and further accumulates in the extracellular space. These sources of adenosine contribute to very high extracellular adenosine concentrations in hypoxic tissues. In tumor tissue the average concentration of adenosine in the extracellular space is approximately 10 μM. Such high concentrations can be found in small tumor nodules of about 2–3 mm in diameter, so are likely to be present in the extracellular fluid of early cancers even before the ▶angiogenic switch. Furthermore, because the level of hypoxia varies through the tumor depending upon the proximity of blood capillaries, local levels can be much higher. Finally, adenosine concentrations are highly regulated by ▶ecto-enzymes such as adenosine deaminase (ADA) at the cell surface (Fig. 2), so that the ultimate effects of adenosine depend heavily on events at the cell surface. In normal tissues, where the concentrations of adenosine are low (in the nanomolar range), the principle pathway through which adenosine is metabolized involves phosphorylation to AMP by adenosine kinase. At higher adenosine concentrations, as are present

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Adenosine and Tumor Microenvironment. Figure 2 Adenosine production in and around the tumor cell. Adenosine is produced in the cell principally from AMP through the action of 5′-nucleotidase (5′-NT). This pathway is more active under hypoxic conditions, such as exist in solid tumors. Hypoxia also inhibits adenylate kinase (AK), which catalyzes the reverse reaction to convert adenosine to AMP. Outside the cell, adenosine is produced from ATP that is present in the extracellular fluid, by the sequential enzyme activities of CD39 and CD73. The major factor restraining the levels of adenosine that can be reached is the activity of the enzyme adenosine deaminase (ADA), which breaks adenosine down to inosine. This is present both within the cell, and as an enzyme outside of the cell (ecto-enzyme) that is held in place by an anchoring protein, CD26.

inside a tumor, the major route through which disposal of adenosine occurs is by deamination to ▶inosine through ADA. ADA is found both within the cell and in the external milieu. The ADA that is present in the extracellular fluid does not remain free, but is largely captured by a 110-kDa binding protein present at the surface of many cells, particularly those of epithelial origin. This ▶ADA-binding protein (ADAbp) is found embedded as a dimer in the outer membrane of many cancer cells, where it functions to hold ADA. There is also evidence that some ADA can bind directly to adenosine ▶receptors of A1 and A2B subtypes. ADA held in this way is then able to modify adenosine concentrations immediately next to the cell surface (where the adenosine receptors are located). One factor that complicates our understanding of how adenosine levels may be regulated within cancer tissue is the fact that adenosine has the capacity to regulate its own levels. This interesting complication arises because ADAbp (also known as CD26 or DPPIV) can be down-regulated at the cell surface by adenosine. That reduces the capacity of the cell to bind ADA at the cell surface and therefore the local rate

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Adenosine and Tumor Microenvironment

of degradation of adenosine. This will extend the half-life of adenosine and increase the persistence of its action. As a result, in the high concentration environment of a tumor, adenosine has the capacity to suppress its own breakdown and enhance its actions still further. (See also ▶CD26/DPPIV in Cancer Progression and Spread.) Although adenosine is a common molecule and has a relatively simple structure, it is able to regulate cellular behavior by interacting with specific receptors. The different types of adenosine receptors are outlined in Table 1 There are four known types, all of which are ▶G-protein-coupled receptors with seven transmembrane segments in their structure, embedded in the outer membranes of responsive cells. Adenosine receptors may be found on any of the cell types within a tumor including the cancer cells, the supporting stromal cells, the endothelial cells within blood vessels, or inflammatory cells that are infiltrating the tumor. All four of the adenosine receptor subtypes have been shown to exist on cancer cells; indeed it is possible for a single cancer cell population to express all four forms of the receptor. However, adenosine receptor subtypes A3 and A2B are the most commonly observed in cancers. The adenosine concentrations that exist in tumors are sufficient to activate all four of the adenosine receptor subtypes. There are four different types of adenosine receptor Table 1, which differ in their affinity for adenosine and the signalling pathways to which they are linked through G proteins. All of the receptor subtypes are

Adenosine and Tumor Microenvironment. Table 1 The different types of cellular receptors for adenosine Receptor subtype

Signalling Affinity for Major Gα adenosine protein pathways used by (s) receptor

A1

High

Gi/o

A2A

High

Gs

A2B

Low

Gs, Gq/11

A3

Low

Gi/o, Gq/11

Adenylyl cyclase (↓ cAMP) Phospholipase C K+ channels Adenylyl cyclase (↑ cAMP) Phospholipase C Adenylyl cyclase (↑ cAMP) Phospholipase C Phospholipase A2 PI3K Adenylyl cyclase (↓ cAMP) Phospholipase C KATP Channels

able to act on adenylyl cyclase but may either increase or decrease the production of cAMP as shown. The receptors can also be coupled to phospholipase C (leading to calcium release and activation of protein kinase C), to phospholipase A2 (causing generation of arachidonic acid and subsequent production of eicosanoid lipid mediators), to phosphatidyl inositol 3-kinase (PI3K, leading to increased activity of the phospholipase D pathway) or in certain cell types can cause the activation of potassium (K) channels. The interaction of adenosine with its receptors on the different cell types in a tumor leads to a myriad of different cellular responses. Although it is at times difficult to extrapolate from the experimental approach to the disease itself, these are such as to generally favor the expansion and spread of the cancer (Fig. 3) There is evidence that synthetic agents which target individual receptor subtypes may have different actions to adenosine, sometimes not clearly directed through the adenosine receptor. When adenosine itself is studied at concentrations that are known to be present within the tumor extracellular fluid, it is typically shown to increase the growth of cancer cells. At very high concentrations of adenosine, cells may be triggered to undergo ▶apoptosis, although some tumor cells are resistant to this action of adenosine. In addition to effects on cancer cell growth and survival, adenosine acts on isolated cancer cell populations to increase cell motility, adhesion to the extracellular matrix, the expression of cell attachment proteins and receptors for molecules that can direct cell movement. The patchiness of hypoxia within tumor tissue leads to local areas of high adenosine concentrations that would be capable of influencing tumor cell behavior directionally in this way. While not yet proven, it is possible that within the context of the tumor itself, adenosine may have an influence on the distribution of cells within the tumor and perhaps their dissemination at the later stage of metastasis. Adenosine receptors are also found on endothelial cells, which are the flattened cells that line blood vessels and which are the major cellular component of the newly-formed vasculature that is formed to supply the expanding cell population with nutrients. Adenosine is able to promote endothelial cell division and motility, and has been shown to enhance the formation of blood vessels (▶angiogenesis) in experimental animal models. Adenosine may therefore have an ancillary role alongside other angiogenic factors such as ▶VEGF in regulating the formation of the tumor microvascular network. Probably the greatest potential role for adenosine in the context of cancer however, is as a local immunosuppressant within the tumor. It has long been known that the local tissue environment in cancer is capable of suppressing the immune response, and that this

Adenosine Deaminase

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Adenosine and Tumor Microenvironment. Figure 3 The multiple potential actions of adenosine within a tumor. This diagram summarizes the different ways in which adenosine might act to facilitate the survival and expansion of a malignant tumor. This figure is drawn based upon studies on individual tumor cell populations and other studies in vivo in which these responses have been observed.

is one of the factors that limits the capacity of our immune system to eliminate the cancer. Experimental studies have shown that a significant proportion of the immunosuppressive activity is mediated by soluble factors, that it increases in proportion to tissue bulk, and it is seen to decline substantially when the cancer tissue is removed from the animal or patient and dissociated into isolated cells. Adenosine is one of the possible factors responsible for this phenomenon of “metabolic suppression” of the anti-tumor immune response. The capacity for adenosine to act as an immunosuppressant is dramatically illustrated by a rare but well-known genetic disease involving a lack of ADA. In this disorder, levels of adenosine within lymphoid tissues rise and (through a combination of events involving both toxic metabolites and adenosine acting through its receptors) cause a severe immunodeficiency (well known because of the need to protect afflicted children from infection in “biobubble” tents). Adenosine is capable of interfering with the immune response at different levels and by acting on different cell types. It works through cell-surface adenosine receptors (principally A2A and A3 subtypes) to suppress various functions of T lymphocytes, natural killer (NK) cells, polymorphonuclear granulocytes, and phagocytic cells such as tissue macrophages that play a key role in recognizing the targets for immunological attack. In the case of T lymphocytes and NK cells, whose infiltration and activity is of key importance to the fate of the tumor and prognosis of the patient, adenosine suppresses successive stages in the evolution and function of the cells. It inhibits proliferation of the cells, the expression of key molecules on the cell surface that are needed to allow full activation, the extent of interaction with the cancer

cell, the release of toxic molecules involved in cell killing, and the overall capacity for killing of the cellular targets. Given the extensive effects of adenosine on nearly all of the cell types present in tumors, it would be appealing to attempt to use drugs that interfere with adenosine pathways as a way of interfering with the growth of the cancer cells, blocking the formation of new blood vessels to nourish the tumor, or relieving the immunosuppression that is due to adenosine. The challenge here lies in the fact that this is a primitive regulatory network in evolutionary terms, and adenosine has a role in the regulation of most organ systems in the mammal. Adenosine receptors of the four subtypes are found on cells throughout the body. Drugs that would a block adenosine’s action at its receptors (antagonists) or mimic its actions at a certain receptor subtype (selective agonists) run the risk of interfering with normal processes such as the control of blood flow or the transmission of nerve signals. Nevertheless, there is hope that careful targeting of certain receptors (particularly the A3 subtype) in cancer may prove to be a useful intervention.

Adenosine Deaminase Definition An enzyme involved in the metabolism of deoxyadenosine to deoxyinosine. Congenital deficiency of this enzyme results in the clinical syndrome of severe combined immunodeficiency.

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Adenovirus S TEFAN KOCHANEK Division of Gene Therapy, University of Ulm, Ulm, Germany

Definition Adenoviruses were originally isolated as etiologic agents for upper respiratory infections. Their name is derived from the initial observation that primary cell explants from human adenoids were found to degenerate secondary to the infection by an, at the time, unknown virus. According to the current official taxonomy, there are four adenovirus genera (Mastadenovirus, Atadenovirus, Aviadenovirus and Siadenovirus), indicating that adenovirus is widely distributed in vertebrates. More than 50 human serotypes have been identified. The individual serotypes are distinguished by different parameters such as immunological properties, tumorigenicity, and DNA sequence. Some serotypes may cause more serious infectious diseases such as epidemic keratoconjunctivitis, gastroenteritis or hemorrhagic cystitis. The adenovirus particle is composed of an outer icosahedric protein capsid with an inner linear double-stranded DNA genome of approximately 36 kilobases (kb) size. There are eleven structural proteins, seven to form the capsid, among them hexon, penton base and fiber being the major constituents of the adenoviral capsid, and four that are packaged in the core. Internalization of the viral particle during infection requires the interaction of the fiber and the penton base with surface proteins (receptors) of the cell. Several virally encoded proteins are associated with the viral DNA. Adenovirus is being used as a gene carrier for ▶gene therapy. Most adenoviral vectors (see below) are derived from the serotypes 2 and 5 (Ad2, Ad5) which are frequent causes for mild colds. During childhood most individuals will become immunized against different adenoviral serotypes by natural infection. Ad2 and Ad5 are not oncogenic in humans Adenoviruses have a good safety record based on vaccination studies that have been performed in military recruits two to three decades ago. As detailed below, during natural infection of permissive cells the adenoviral DNA is transcribed, replicated, and packaged into capsids within the nuclei of infected cells. Similar to other DNA viruses, two main phases can be distinguished during infection: . An early phase that is characterized by the expression of the ▶early virus genes E1, E2, E3, and E4 . A late phase after onset of viral replication in which the viral structural proteins are produced

Characteristics Infection and Viral Transcription A productive infectious cycle takes approximately 2–3 days and under optimal conditions more than 50,000 particles are produced in every infected cell. In the case of most human adenvirus serotypes the infection begins with the attachment of the virus particle to the cell surface via interaction of the tip of the capsid fiber protein with the membrane protein CAR (Coxsackie– Adenovirus receptor). As it is apparent from the name, CAR is also used by some coxsackie viruses as receptor for entry. Naturally, CAR plays an important role in the interaction of neighboring cells. The adenoviral particle is internalized by receptor-mediated endocytosis into clathrin-coated pits requiring a secondary interaction of the penton base with an αv-integrin. Following endocytosis the viral particle is sequentially disassembled, initally losing the fiber proteins, later most of the other viral structural proteins. Finally, the viral DNA is released as a DNA–protein complex through nuclear pores into the nucleus of the host cell. Shortly thereafter, transcriptional activation of the early genes E1A and E1B initiates a complex transcriptional program designed to first replicate the viral DNA and later to generate new infectious viral particles (Fig. 1). The activation of early and late transcription units follows a relatively well understood transcriptional pattern. The gene products of the E1A and E1B genes are involved in the activation of both viral and cellular genes. Under certain conditions, in particular if infection of a cell does not result in a productive but rather abortive infection (abortive infection = the infectious cycle is blocked at an early stage following infection of the host cell) together with the rare event of integration of the viral DNA into the chromosome, cellular transformation may be a consequence. The E2A and E2B gene products are involved in the replication of the viral genome and include the viral DNA polymerase (AdPol), the terminal protein (TP), and the DNA-binding protein (DBP). The E3 and E4 gene products have diverse functions leading to transcriptional activation of other promoters, preferential export of viral RNAs out of the nucleus of infected cells and suppression of host defenses. With the begin of replication of the viral genome approximately 6 h after infection, late phase transcription units are activated. Most of the late phase proteins are capsid proteins or proteins that are involved in the organization and packaging of the viral genome inside the viral capsid. The most active promoter at this stage is the major late promoter (MLP) that directs the transcription of a large primary RNA transcript that covers more than two thirds of the viral genome. From this transcript five families (L1-L5) of structural proteins are generated by differential splicing and polyadenylation. During the course of an infection the metabolism

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Adenovirus. Figure 1 Organization of the adenovirus genome and the different adenoviral vector types employed for gene transfer. Promoters are indicated by arrowheads, transcribed genes by arrows. The genes that are transcribed early during infection are the E1A, E1B, E2, E3, and E4 genes. The main gene products, generated late during infection, are transcribed from the Major Late Promoter (MLP), which directs a very long RNA message (MLTU = major late transcription unit). Different RNA species (L1-L5) that code for structural proteins are generated by alternative splicing and differential polyadenylation (for clarity not all adenoviral genes and gene products are indicated). First-generation adenoviral vectors are characterized by deletion of the E1 genes, second-generation adenoviral vectors by the additional deletion of the E2 and/or E4 genes. High-capacity adenoviral vectors have most of the viral genome removed and retain only the noncoding viral ends. In high-capacity adenoviral vectors, stuffer DNA is included in the vector genome for stability reasons.

of infected cells is redirected to support a predominant production and assembly of viral proteins. Adenoviral Functions and Oncogenesis Adenoviruses have played important roles as experimental tools in the discoveries of several fundamental principles in molecular biology, including RNA splicing and oncogenic transformation of cells. In fact, the 1993 Nobel Prize for Physiology or Medicine was awarded to Dr. Phillip Allen Sharp and Dr. Richard John Roberts for the discovery or RNA splicing and was based on their work with adenovirus RNA transcription. The induction of malignant tumors by injection of adenovirus type 12 in newborn hamsters was the first direct demonstration of a human virus causing malignant cellular transformation. This observation greatly stimulated the interest in using viruses as experimental systems in the study of the pathogenesis of cancer. While there is no epidemiological evidence

for an involvement of adenoviruses in the pathogenesis of human cancers, several serotypes have been shown to cause tumors in rodents. Some serotypes, such as Ad12 or Ad18 are highly oncogenic in animals, others, for example Ad4 or Ad5 have a low oncogenic potential. Based on several complementing observations cellular transformation is mediated by the viral E1A and E1B genes: In most virus-transformed cells the viral E1 genes are consistently found integrated into the cellular genome where they are expressed. Transfection of cells with the E1A and E1B genes is necessary and sufficient for cell transformation, and viruses with mutations in the E1 genes are defective for transformation. Several RNAs are transcribed from the E1A genes, the main species in Ad5 being the 12S and the 13S RNAs coding for E1A proteins of 243 and 289 amino acids. To a large extent, the E1A proteins exert their transforming activity by interaction with cellular proteins that are involved in cell cycle regulation such as the tumor

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suppressor pRB. While E1A alone is capable of immortalizing cells, cooperation with E1B functions is required to achieve a full transformation phenotype. Two main proteins are produced from the E1B gene by alternative splicing. The 21 kD E1B protein that has been shown to inhibit apoptosis, and the 55 kD E1B protein that interacts with the tumor suppressor protein p53. The expression of additional viral functions may contribute to E1 mediated tumorigenesis. For example, a 19 kD protein expressed from the E3 region can decrease MHC class I levels in transformed cells, and certain functions expressed from the E4 region can cooperate with the transforming activity of the E1B 55 kD protein. Gene Therapy: First- and Second-Generation Adenoviral Vectors First-generation adenoviral vectors do not replicate in human cells under normal conditions because the E1A and E1B genes are deleted from the vector genome (Fig. 1). These vectors are produced in complementing cell lines that express the E1A and E1B genes. Firstgeneration vectors have been used for gene transfer in cultured cells, animals and even clinical trials in humans to express a large number of genes in different cell types and tissues. So far the results of experiments performed in animals and clinical studies in humans have been relatively disappointing. Several significant disadvantages of first-generation adenoviral vectors have been acknowledged: . Because first-generation vectors still contain a nearly complete set of viral genes, toxicity and antiviral immune responses are frequently observed resulting in the clearance of transduced cells. Consequently, gene expression is only transient. Contributing factors for short-term gene expression include immune responses directed to the transgenic proteins expressed from the vector, if the organism is not tolerant to that protein. . The upper DNA packaging limit for adenoviruses is about 38 kb. Because most viral genes are retained on the vector only about 7–8 kb of nonviral DNA can be incorporated into such vectors. However, in many conditions the therapeutic cDNAs are either large, additional elements have to be included to achieve regulated gene expression, or multiple genes need to be expressed to obtain a therapeutic effect. Thus, it is clear that the size constraints in first-generation adenoviral vectors may be a limiting factor for many potential applications. In order to further decrease expression of late viral proteins, adenoviral vectors with inactivation of the E2 and/or E4 functions in addition to the deletion of the E1 region have been generated. These vectors are produced in cell lines that complement both E1 and E2 and/or E4 functions.

Currently it is controversial whether these secondgeneration adenoviral vectors have any significant advantages over first-generation vectors and lead to a longer duration of gene expression. “Gutless” Adenoviral Vectors In an attempt to address several of the problems observed with first-generation adenoviral vectors a novel adenoviral vector has been developed that will be useful for the functional analysis of genes in vivo and clinical studies. This vector has been variably called “high-capacity (HC)” adenoviral vector, “gutless”, “gutted” or “helper-dependent (HD)” adenoviral vector. Because all viral genes are deleted from this vector the capacity for the uptake of foreign DNA is more than 30 kb. The current production system involves the use of an adenoviral helper virus and takes advantage of the Cre-loxP recombination system. In this production scheme a first-generation adenoviral vector carries two loxP-recognition sequences that flank the adenoviral packaging signal. The vector is produced in E1-complementing cells that express the Cre-recombinase of bacteriophage P1. After infection of these cells both by helper virus and vector the packaging signal of the helper virus is excised. Therefore, vector and only little helper virus is packaged. From several in vivo experiments performed in different animal species it is apparent that these new vectors have clear advantages compared to earlier versions of adenoviral vectors and are considerably improved in safety and expression profiles. Their increased capacity for foreign DNA allows gene transfer of several expression cassettes, large promoters and some genes in their natural genomic context, a significant advantage over first- and second-generation adenoviral vectors. Replication-Competent Adenoviral Vectors for Cancer Gene Therapy While the above mentioned adenoviral vectors have been widely used in preclinical and, with the exception of “gutless” adenoviral vectors, also in clinical studies to express a wide variety of transgenes including cytokines, p53 and thymidine kinase (TK), it would be desirable to achieve gene transfer into all or most neoplastic cells within a tumor. This is clearly not possible with current vector technology. Recently, a new concept has been proposed that is based on the use of an adenovirus that is both replication competent and tumor-restricted in its growth. This virus is based on an Ad5 mutant virus that has an inactivating deletion within the E1B gene and does not express the E1B 55 kD protein. Initially, it was thought that replication of the virus was dependent on the p53 status of the host cell and that the virus was able to grow only in cells deficient for function p53 expression. However, more recent results indicate that the growth of this virus is

Adherens Junctions

independent of the p53 status cells and may depend on other cell cycle related factors. Although clinical studies so far have not been or only partially been successful, such a virus has been approved in 2005 in China for cancer therapy and is currently used in combination with chemotherapy and/or radiotherapy. In addition, replication-competent adenovirus vector are being developed, in which expression of essential viral genes, in particular of E1A, is under control of a tumor-specific promoter. These vectors have been named CRADs (conditionally replicating adenoviruses). Adenovirus Vectors for Genetic Vaccination One of the most promising applications of adenovirus vectors is in the area of genetic vaccination. For many common diseases including AIDS or Malaria there are currently no vaccines available. Since adenovirus vectors have been found to induce strong cellular and humoral (antibody) immune responses against expressed genes, many preclinical studies have been performed with the aim of vaccine development. In these studies adenovirus vectors have been found to belong to the strongest inducers of antigen-specific immune responses against different antigens. Therefore, clinical studies have been initiated, in which adenovirus vectors, either alone or in combination with proteins or other vectors, are evaluated for their potential as a vaccine against different infectious diseases.

α-subunits and βγ-complexes as well as by Ca2+ or protein kinase C. ▶G-Proteins ▶Signal Transduction

Adherens Junctions Definition Adherens junctions are intercellular junctional structures, most prominent in epithelial cells. In the adherens junction, the cell-cell adhesion is mediated by Ca2+dependent cell adhesion molecules, the cadherins; the cytoplasmic tail of these cadherins is indirectly linked to the ▶actin cytoskeleton. Normally are located more basally than ▶tight junctions. ▶Cell Adhesion Molecules ▶E-Cadherin ▶Exfoliation of Cells

Adherens Junctions

References 1. Berk AJ (2007) Adenoviridae: the viruses and their replication. In: Bernard N. Fields, David M. Knipe, Peter M. Howley (eds), Fields virology, 3rd edn. LippincottRaven, Philadelphia, New York, pp 2355–2394 2. Wold WSM, Horwitz (2007) Adenoviruses. In: Fields virology, 5th edn. Lippincott-Raven, Philadelphia, New York, pp 2395–2436 3. Doerfler W, Böhm P (eds) (1995) The molecular repertoire of adenoviruses. Current topics in microbiology and immunology, vol 199/I–III. Springer, Berlin 4. Imperiale MJ, Kochanek S (2004) Adenovirus vectors: biology, design, and production. Curr Top Microbiol Imunol 273:335–357

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J UN M IYOSHI 1 , YOSHIMI TAKAI 2 1

Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan 2 Osaka University Graduate School of Medicine/ Faculty of Medicine, Suita, Japan

Synonyms Zonula adherens; intermediate junction

Definition

Adenylyl Cyclase Definition Ubiquitous group of enzymes which catalyze the formation of the second messenger cAMP from ATP. Various mammalian isoforms have been identified which are differentially regulated through G-protein

Adherens junctions are specialized cell–cell attachments composed of transmembrane proteins and cytoplasmic proteins that anchor to the actin cytoskeleton (Fig. 1). Anchoring proteins are clustered with several actin-binding proteins in the cytoplasm adjacent to the junctional membranes. Adherens junctions form punctate or streak-like attachments in nonepithelial tissues, whereas they encircle the apical portion of adjacent epithelial cells below ▶tight junctions. Adherens junctions have prototypic roles in stabilizing the epithelium, establishing apical–basal polarity of epithelial cells, and

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Adherens Junctions. Figure 1 Epithelial cells joined by the apical adhesion complex. Adherens junctions are located below tight junctions near the apical end of the lateral cell interface in epithelial cells. Nectin and E-cadherin-based cell adhesions are connected via several cytoplasmic proteins into belts of actin filaments that underlie adherens junctions. Nectins are localized to adherens junctions via afadin, and they are associated with integrin αvβ3 in the extracellular space. Afadin binds to the tail of nectin cis-dimers as well as F-actin directly, interacting with Rap1. β-catenin binds to the tail of E-cadherin cis-dimers directly, and then α-catenin binds to β-catenin. The catenins can mediate interactions to F-actin through binding to several actin-binding proteins such as ZO proteins, afadin, vinculin, α-actinin, VASP, formin-1, and Arp2/3 complex. c-Src, Rac, Cdc42, and FAK play roles in regulating dynamic changes of the actin cytoskelton, facilitated by E-cadherin and nectin clustering.

facilitating cell–cell communication that regulates cell proliferation and movement. Since most human cancers are of epithelial origin, disruption of adherens junctions is one of the hallmarks of cancer cells exhibiting malignant transformation.

Characteristics Adherens junctions are sites of mechanical attachment regulated by dynamic changes in the actin cytoskeleton, and they also serve as sites of cell–cell communication. Adherens junctions are abundant in many tissues that

Adherens Junctions

are subjected to mechanical stress. In epithelial cells, adherens junctions coalesce into the mature zonula adherens. In cooperation with the zonula occludens (tight junctions), the zonula adherens defines apical–basal polarity by physically separating the membrane into apical and basolateral membrane domains. In addition, adherens junctions mediate nuclear ▶signal transduction induced by cell contact. For example, molecules clustered at adherens junctions could mediate contact-dependent inhibition of cell proliferation and movement: the arrest of the cell cycle in G1 phase that occurs when cell density increases to confluence in culture. Thus, the coupling of cell contact and signaling at adherens junctions reflects structural and functional regulations involved in establishing multicellular organisms. Cadherins and nectins are two major ▶cell adhesion molecules in the extracellular space. Cadherins are a superfamily composed of classical cadherins, which are the main components of adherens junctions, and nonclassical cadherins, which include desmosomal cadherins and protocadherins. The classical cadherins share a motif of five cadherin repeats in the extracellular domain, and they are divided into several subtypes including epithelial ▶(E) cadherin, placental (P) cadherin, neural (N) cadherin, and vascular endothelial (VE) cadherin. On the other hand, nectins are immunoglobulinlike adhesion molecules composed of four members. Adherens junctions facilitate cell–cell adhesion through homophilic binding between cadherin molecules, as well as homophilic and heterophilic bindings between nectin molecules on adjacent cells. It remains controversial whether or not the extracellular domain of Ecadherin first binds to form cis-dimers on the surface of the same cells, and then promotes cell-cell contacts by forming trans-dimers in a Ca2+-dependent manner. On the other hand, each member of nectins forms cisdimers, and then promotes homophilic or heterophilic trans-dimer formation in a Ca2+-independent manner. Heterophilic trans-interactions have been detected between nectin-2 and nectin-3, between nectin-1 and nectin-3, and between nectin-1 and nectin-4. Importantly, heterophilic trans-dimers form stronger cell–cell attachment than homophilic trans-dimers, which actually determines the type of cell–cell adhesion. Namely, cadherins exclusively promote adhesion between homotypic cells, whereas nectins have a dual role in promoting adhesion between homotypic cells and between heterotypic cells. Heterophilic engagement of nectins may thus play key roles in cell recognition and sorting in vivo. The intracellular domain of cadherins is associated with a cytoplasmic complex consisting of α-catenin and β-catenin, and forms structural links to the actin cytoskeleton. α-catenin does not act as a stable link to filamentous actin (F-actin) but possibly acts as a molecular switch that regulates actin dynamics at

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adherens junctions. The catenins could also mediate interactions with F-actin via binding to proteins such as ▶ZO protein-1, afadin, vinculin, and α-actinin. The intracellular domain of nectins directly binds to afadin that links nectins to the actin cytoskeleton. Localization of nectins to adherens junctions depends on the presence of afadin. Thus, the catenins and afadin cooperatively contribute to form adherens junctions that are strong yet easily remodeled. Nectin-based cell–cell adhesions establish adherens junctions, both independently and cooperating with cadherin-based cell–cell adhesions. In Madin–Darby canine kidney (MDCK) cells in culture, nectins first form cell–cell adhesion and then recruit cadherins to the nectin-based cell–cell adhesion sites to establish adherens junctions. Nectins further promote formation of tight junctions in MDCK cells by recruiting JAM (junctional adhesion molecule)-A, claudin-1, and occludin. On the other hand, nectins and integrin αvβ3 are physically associated through their extracellular domains to cooperatively regulate cell movement, proliferation, adhesion, and polarization. Thus, nectins play roles in establishing apical junctional complex, as well as in communication between cell–cell and cell–matrix junctions. Trans-interacting E-cadherin induces activation of Rac small ▶G-protein, which stabilizes nontransinteracting E-cadherin on the cell surface by inhibiting endocytosis through the reorganization of the actin cytoskeleton. p120 catenin (p120ctn) also plays a role for inhibiting endocytosis of E-cadherin. In contrast, E-cadherin undergoes endocytosis when adherens junctions are disrupted by the action of an extracellular signal, such as hepatocyte growth factor/ ▶scatter factor. Activated c-Src enhances endocytosis of E-cadherin by inducing the tyrosine phosphorylation and ubiquitylation of the E-cadherin complex. On the other hand, trans-interaction of nectins activates Cdc42 and Rac, which promotes the formation of adherens junctions mediated by the ▶IQGAP1-dependent actin cytoskeleton. In addition, afadin and activated ▶Rap1 complex interacts with p120ctn to strengthen the binding between p120ctn and E-cadherin. Furthermore, the cell polarity proteins Par-3, Par-6, and aPKC that form a ternary complex could be implicated in the assembly of adherens junctions. They regulate the association of afadin with nectins in MDCK cells. These cell polarity proteins and afadin could play cooperative roles in the formation of adherens junctions and tight junctions although the mechanism is largely unknown. Thus, E-cadherin and nectin trans-interactions induce elaborate interactions between peripheral proteins to establish mature adherens junctions. β-catenin is able to translocate to the nucleus, where it binds to lymphoid enhancer factor–T-cell factor (LEF/ TCF) that regulates gene transcription. β-catenin is involved in several signaling pathways including the

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wingless-type mammary virus integration-site family (▶Wnt) ▶signaling pathway. When Wnt proteins bind their receptors, they inactivate the serine/threonine kinase GSK3β that phosphorylates β-catenin and targets it for destruction in the proteosome. Mutations involving the serine/threonine residues of β-catenin that are phosphorylated by GSK3β can stabilize the β-catenin protein or increase its nuclear localization. Furthermore, tyrosine phosphorylation of β-catenin also disrupts the association between E-cadherin and β-catenin, allowing β-catenin to transduce signals to the nucleus. Necl-5, a member of nectin-like cell adhesion molecules (Necls), originally identified as a poliovirus receptor, could mediate growth arrest that has been known as contact inhibition of cell proliferation and movement. Necl-5 is overexpressed in human colon carcinoma, as well as in NIH3T3 cells transformed by ▶Ras activation. Necl-5 colocalizes with integrin αvβ3 and growth factor receptors at leading edges of migrating cells and regulates growth factor induced cell migration. When Necl-5 interacts in trans with nectin-3 at cell–cell contacts in NIH3T3 cells, Necl-5

undergoes downregulation from the cell surface, resulting in reduction of cell proliferation and movement. Thus, nectins and Necls have roles in mechanical cell–cell adhesion as well as cell–cell communication. Implications in Cancer Adherens junctions control epithelial cell polarity while other adhesion apparatus tends to inhibit cell migration, which is crucial for the differentiation and morphogenesis of many tissues. Loss of adherens junctions, as well as aberrant signaling involving the Wnt pathway, could contribute to carcinogenesis and ▶metastasis by causing cell depolarization, loss of contact-dependent inhibition of proliferation, and increased ▶motility and invasiveness (Fig. 2). Cancer cells that show migratory properties undergo ▶epithelial to mesenchymal transition (EMT), with the induction of transcriptional repressor proteins, such as ▶snail transcriptional factor, slug, and Twist, that downregulate E-cadherin gene expression. EMT is a basic mechanism that mediates disruption of epithelial polarity and disintegration of cancer cell nests.

Adherens Junctions. Figure 2 Signaling induced by loss of E-cadherin. Disruption of adherens junctions is caused by mutation or transcriptional repression of E-cadherin and growth-factor signaling. Dissociation of homophilic binding of E-cadherin promotes the endocytosis of E-cadherin and the disassembly of the catenins. p120ctn further promotes cell motility by activating Rac and Cdc42 to form lamellipodia and filopodia, and inhibits Rho activity that leads to stress-fiber formation. β-Catenin dissociated from the E-cadherin and catenin complex accumulated in the cytoplasm. Part of β-catenin translocates to the nucleus and binds to TCF to activate transcription of key genes required for survival of detached cells, while the other part of β-catenin is modified by phosphorylation and ubiquitination, leading to proteosome degradation. The Wnt pathway promotes β-catenin signaling by repressing the phosphorylation of β-catenin mediated by GSK-3β.

Adhesion

Reduced E-cadherin levels in cancer cells are accomplished by genetic events such as somatic mutation and reduced gene expression mediated by repressor proteins or by methylation of the promoter region of the E-cadherin gene. The genetic defects of E-cadherin have been found in human lobular breast carcinomas and scirrhus-type ▶gastric cancers, both of which have highly metastatic potentials. Mutations of β-catenin also promote migration and ▶invasion of cancer cells by the loss of interaction of adherens junctions with the actin cytoskelton. Distributions of E-cadherin and β-catenin tend to change depending on sites of tumor remodeling. In epithelial structures in the centre of cancer, E-cadherin and β-catenin are mostly present in adherens junctions. However, solitary cells at the invasive front of cancer plates shows no signal for E-cadherin but often produce signals for nuclear β-catenin. Thus, decreased E-cadherin expression promotes the release of solitary cancer cells at the invasive front and increases the survival of cancer cells by stimulating β-catenin signaling. Strategy for restoring adherens junctions, as well as cell–cell and cell–matrix communication may prevent cancer-cell invasiveness. Therapeutic targets might be molecules involved in pathways affecting the adhesive properties of E-cadherin and the assembly of the adherens-junction complex: c-Src and other tyrosine kinases, tyrosine phosphatases such as PTP-LAR, Rho, Rac, and Rap small G-proteins, transcriptional repressor proteins, and ▶merlin and the ▶ERM proteins. For example, c-Src regulates both disruption of adherens junctions and focal-adhesion turnover that are required for cancer cell motility. Twist is highly expressed in human cancers with reduced E-cadherin mRNA expression levels. In contrast, podoplanin promotes cancer cell invasion in the absence of EMT, suggesting cancer cells can also migrate as a mass, not necessarily as a single cell. Restoring E-cadherin-mediated cell adhesion could be means of preventing EMT in cancer and metastasis although EMT is not essentially required for cancer-cell invasion.

References 1. Christofori G (2006) New signals from the invasive front. Nature 441:444–450 2. Kobielak A, Fuchs E (2004) Alpha-catenin: at the junction of intercellular adhesion and actin dynamics. Nat Rev Mol Cell Biol 5:614–625 3. Takai Y, Nakanishi H (2003) Nectin and afadin: novel organizers of intercellular junctions. J Cell Sci 116:17–27 4. Takeichi M (1993) Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 5:806–811 5. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142

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Adhesion DARIO R USCIANO Friedrich Miescher Institute, Basel, Switzerland

Definition Cell adhesion is a dynamic process that results from specific interactions between cell surface molecules and their appropriate ligands. Adhesion can be found between adjacent cells (cell-cell adhesion) as well as between cells and the ▶extracellular matrix (ECM) (cell-matrix adhesion). Besides keeping a multicellular organism together, cell adhesion is also a source of specific signals to adherent cells; their phenotype can thus be regulated by their adhesive interactions. In fact, most of the cell adhesion receptors were found to be involved in ▶signal transduction. By interacting with growth factor receptors they are able to modulate their signaling efficiency. Therefore, gene expression, cytoskeletal dynamics and growth regulation all depend, at least partially, on cell adhesive interactions (Fig. 1).

Characteristics Cell Adhesion Receptors Cell adhesion molecules were grouped into distinct classes according to structural and/or functional homologies. The following receptors have been directly implicated in the malignant phenotype of tumor cells. . Integrins represent a family cell surface ▶glycoproteins that depend on divalent cations and are important in cell-ECM and cell-cell adhesion. The non-covalent association of an alpha and a beta subunit results in heterodimers that span the plasma membrane, enabling contacts with elements of the ▶cytoskeleton and signal transducing intermediates. . The immunoglobulin superfamily of adhesion receptors is mainly involved in cell-cell adhesion. Named after a 90–100 amino acid domain that is also present in Ig molecules, these kind of receptors can be expressed either as plasma membrane-spanning molecules. However, some of them are alternatively spliced and are anchored to the cell membrane by covalent linkage to phosphatidylinositol. . ▶Selectins represent a class of structurally related monomeric cell surface glycoproteins that bind specific carbohydrate ligands via their ▶lectin-like domains. Since the ligands are expressed in a specific way by vascular endothelial cells, selectins are important in lymphocyte trafficking and homing of malignant tumor cells. . Cell surface ▶proteoglycans consist of ▶glycosaminoglycans (GAG) attached to core proteins through

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Adhesion. Figure 1 Cell adhesion in normal (a, b) and cancer (c, d) cells. Normal mesenchymal cells show regular actin stress fibers (a: stained with phalloidin) and focal contacts (b: stained with anti-vinculin antibodies). In contrast, cancer cells (a highly motile melanoma cell is shown) often present with a completely disorganized actin cytoskeleton (c) and few focal contacts (d). Vinculin is typically arranged in patches at the periphery of the cell (d) (Confocal micrograph courtesy of Dr. Jörg Hagmann, FMI, Basel).

an O-glycosidic linkage. They can mediate cell-cell and cell-ECM adhesion. . ▶CD44 comprises a large family of proteins generated from one gene by alternative splicing. Variants of CD44 (CD44v) differ from the standard form (CD44s) by their implementation of ten variant exons in various combinations. Some variants have been causally related to the metastatic spread of some tumor cells. Among the ligands for CD44 are hyaluronic acid (HA), fibronectin and collagen, and chondroitin sulfate-modified proteins. . ▶Cadherins are surface glycoproteins involved in cell-cell interactions. They are involved in the formation of adherens-type functions between cells. Through their cytoplasmic tail they interact with catenins, which are important for the signal transducing ability of cadherins. . ▶Connexins are gap junction-forming proteins that oligomerize into specialized intercellular channels, connecting apposing plasma membranes. They allow the exchange of low molecular weight metabolites such as second messengers that are important in signal transduction (Table 1).

Adhesion and Cancer The selective adhesion of one cell to another or to the surrounding ECM, is of paramount importance during embryonic development as well as for the maintenance of normal adult tissue structure and function. Severe perturbations of these interactions can be, at the same time, cause and consequence of malignant transformation and also play a fundamental role during malignant progression and metastatic dissemination (Fig. 1). . Adhesion to the ECM through integrin receptors is important for anchorage dependent cell growth and cell survival. Normal cells that are detached from the ECM are locked in the G1 phase of the cell cycle (by loss of activity of the cyclinE/cdk2 complex) and undergo apoptosis (anoikis). Transformed cells, in which integrin signaling is altered, acquire the ability to grow in suspension and do not succumb to anoikis. . Adhesion to neighboring cells, mediated by cell-cell adhesion molecules (e.g. N-CAM and C-CAM) and by gap-junctions, inhibits growth of normal cells (what is commonly known as “contact growth inhibition”). Loss of these contacts due to the

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disrupted function of the relative adhesion molecules may result in uncontrolled proliferation. . The differentiated state of mature cells (their “identity”) is also maintained through specific adhesion to the ECM and adjacent cells: a loss of identity is thus a likely consequence if specific contacts are lost, finally resulting in the ambiguous phenotype of many tumor cells (Fig. 2).

abnormal adhesiveness that is generally displayed by tumor cells appears to contribute to their metastatic behavior. Both positive and negative regulation of cell adhesion are required in the metastatic process, since metastatic cells must break away from the primary tumor, travel in the circulation where they can interact with blood cells and then adhere to cellular and extracellular matrix elements at specific secondary sites.

Certain genes that code for cell adhesion molecules may therefore be considered as ▶tumor suppressor genes or even ▶metastasis suppressor genes since their loss or a functional mutation can strongly contribute to the acquisition of the malignant phenotype.

Adhesion within The Tumor Mass The majority of normal adult cells are restricted by compartment boundaries that are usually conserved during the early stages of development of a tumor. Therefore, the detachment of malignant cells from the primary tumor is an essential step for the initiation of the metastatic cascade. During ▶tumor progression changes on the cell surface that lead to a weakening of the cellular constraints, contribute to the release of such mutant cells

Adhesion in Metastasis Adhesive interactions play a very critical role in the process of metastatic tumor dissemination, and the Adhesion. Table 1

Adhesion receptors

Family Integrins IgG superfamily Cadherins Selectins Connexins Cell surface proteoglycans CD44

Main members

Type of adhesion

Characterized by the different α- and β-subunits Cell-ECM cell-cell ICAM-1, V-CAM, N-CAM, CD2 (LFA2), LFA3, CD4, CD8, MHC (class Cell-ECM cell-cell I and II) E, P, L Cell-cell (adherens junction) E, P, N Cell-cell 26 (tumor suppressor) 32 (liver) 43 (glial cells) Cell-cell (gap junctions) Syndecan, glypican Cell-ECM cell-cell CD44s, CD44v

Cell-ECM cell-cell

Adhesion. Figure 2 Cell adhesion and maintenance of a normal differentiated phenotype: Detachment of a normal cell from the extracellular matrix (ECM) would normally lead to apoptosis. Normal cells that keep contact with the ECM are protected from apoptosis and may migrate and grow. Normal cells tend to be organized as sheets onto the ECM, which contributes to their polarization and differentiation. Extensive intercellular contacts among cells adhered onto the ECM lead to contact-mediated growth inhibition. Tumor cells do not undergo apoptosis when detached from the ECM and may grow, migrate and invade into the matrix, to enter the circulation and give rise to distant metastases.

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from the primary tumor mass. Indeed, it was found that tumor cells separate more easily from solid tumors than normal cells from corresponding tissues. . Cadherin expression has been shown to influence intercellular cohesion in direct correlation with invasive behavior. An increased cadherin expression in tumor lines, generally causes a tighter association of tumor cells. In vitro experiments have shown that cells which do not express cadherins or in which cadherins are functionally inhibited are more invasive than cells with normal cadherin activity. In cases where E-cadherin was involved, re-introduction of a wild type copy of the gene could revert the invasive phenotype. The loss of cadherin activity however, is not sufficient to make cells invasive. ▶Invasion also requires other cellular activities, such as ▶motility and protease production. In vivo, tumors expressing low levels of cadherins tend to be less differentiated and to exhibit higher invasive potential, although they are not necessarily more metastatic. In human cancer, a reduction in cadherin activity correlates with the infiltrative ability of tumor cells, a correlation that in many tumors is also retained in distant metastasis. . A different type of cellular constraint is provided by gap junction communication. Gap junctions play an essential role in the integrated regulation of growth, differentiation and function of tissues and organs. The disruption of gap junction communication can cause irreversible damage to the integrity of the tissue and finally contribute to tumor promotion and malignant progression by favoring local cell isolation. There is experimental evidence that a loss of intercellular junction communication affects the metastatic potential of cell lines. Normal cells use gap junctions to control the growth of tumor cells. Once gap junctional communication is lost, the signaling mechanism responsible for the exertion of such growth control is also lost. Both quantitative and qualitative changes in gap junction protein (connexins) expression were found to be associated with tumor progression during multistage skin carcinogenesis in the mouse model system as well as with tumorigenesis in a rat bladder tumor cell line. Malignant Tumor Cells in the Blood Stream: Adhesion to Blood Cells and Platelets Blood-borne tumor cells undergo various homotypic and heterotypic interactions, the effect of which will also influence their metastatic behavior. Some of these interactions may be detrimental to circulating tumor cells such as tumor cell recognition by natural killer (NK) cells, or by tumor infiltrating lymphocytes (TIL). Others may provide, to a certain extent, a protective effect and/or contribute to metastatic spreading, such as interactions with platelets or, in certain cases, with leucocytes.

. De novo expression of the cell adhesion molecule ICAM-1 by melanomas might lead to heterotypic adhesion between melanoma cells and leukocytes bearing the relative receptor (LFA-1). Such interaction might thus enhance tumor cell adhesion to migratory and invasive leukocytes, thereby contributing to further dissemination of malignant tumor cells. In this regard, it has been suggested that site specificity of cancer metastasis might be, at least partially, a consequence of the formation of “multicellular metastatic units” (MSU) consisting of tumor cells, platelets and leukocytes. A subset of leukocytes within the “MSU” would be responsible for sitespecific endothelium recognition, adhesion and stable attachment, thus serving as “carrier cells” targeting the metastatic “spheroids” to specific sites of secondary tumor foci formation. . Several lines of evidence have provided strong support for the concept that tumor cell-platelet interaction significantly contributes to hematogenous metastasis. Two categories of molecules can trigger tumor cell induced platelet aggregation (TCIPA) and activation; soluble mediators and adhesion molecules. The latter are likely to be responsible for the initial contact between tumor cells and platelet cells, and might later stabilize the interaction. P-selectin and αIIbβ3 integrin on the platelet surface may bind Lex carbohydrate determinants and fibrin on the surface of tumor cells, thus triggering platelet activation. Sialylation appears to be a general requirement for TCIPA, and ▶sialoglycoconjugates present on both tumor cells and platelets have been involved in tumor cell-platelet interactions. Mechanistically, platelets may contribute to metastasis by stabilizing tumor cell arrest in the vasculature, shielding tumor cells from physical damage, providing additional adhesion mechanisms to endothelial cells and subendothelial matrix, and serving as a potential source of growth factors. If tumor cell interaction with host platelets occurs while tumor cells are circulating, an organ-specific colonization ability of blood-borne tumor cells may be influenced. In fact, the resulting embolus will be more easily arrested in the vasculature of the first organ downstream from the primary tumor site. If this organ represents a favorable milieu for tumor growth, then interaction with platelets will enhance tumor metastasis at that site; if this is not the case, it may prevent tumor cells from reaching their preferred organ and thus cause a reduction of the metastatic potential. It seems, however, that in most cases platelets are involved only after tumor cells have arrested, and platelet activation may then stabilize the initial tumor cell arrest in the microvasculature.

Adhesion

Adhesion in the Target Organ Circulating tumor cells, either as single cells or most likely as homotypic and/or heterotypic aggregates that have escaped killing by the host immune system and lysis by mechanical shear forces associated with passage in the blood stream, need now to arrest in the microvasculature and extravasate into the organ parenchyma. In fact, the survival time of tumor cells entering the circulation is very short, usually less than 60 min. Therefore those cells that can rapidly arrest and are able to get out the blood stream might have a selective advantage in giving rise to metastatic colonies. Specific adhesion in the target organ has been proposed as a critical determinant of organ specific metastasis, and experimental data indicates that malignant tumor cells preferentially adhere to organ-specific adhesion molecules. Tumor cells, for instance, adhered more efficiently to disaggregated cells or to histologic sections prepared from their preferred site of metastasis than from other organs. These type of assays, however, do not accurately mimic the physiological situation in vivo, where the first contact of circulating tumor cells happens with the luminal surface of the vascular endothelium, and, after endothelial retraction, with the subendothelial ▶basement membrane. Adhesion to Endothelial Cells (EC) The arrest of tumor cells in the capillary bed of secondary organs and their subsequent extravasation occur through interactions with the local microvascular endothelium and the subendothelial matrix. . Biochemical heterogeneity of EC is related to both the heterogeneous microenvironment within tissues and the size of the vessel. Heterogeneity is seen in the differential expression of plasma membrane glycoproteins, cytoskeletal proteins and surface receptors in microvascular endothelium of different organs. Such heterogeneity of endothelium underscores the importance of using organ-specific capillary endothelium in studying the role of organ-specific tumor cell adhesion in metastasis. . The specificity of the adhesive interactions that depends on the heterogeneity of microvascular EC and tumor cells, may favor, in a selective way, the initial adhesive events in preferred metastatic sites. As a consequence it may also facilitate metastatic dissemination to those organs, in a way that is similar to extravasation of lymphocytes from high endothelial venules of lymphoid tissues. Infact, lymphocyte “homing” represents the paradigm for organ-specific cell adhesion and it has been shown to follow specific interactions between surface “homing” receptors on lymphocytes with vascular “addressins” expressed on the high endothelial venule surface. In a similar

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way, tumor cells express various combinations of cell surface molecules that may serve as ligands for EC surface receptors, which are typically induced upon stimulation by mediators of inflammation. A local inflammatory response might thus facilitate circulating tumor cells adhesion and arrest. The relevance of this type of interaction in directing tumor metastasis has been recently demonstrated in vivo using strains of transgenic mice that constitutively express cell surface E-selectin either in all tissues or in the liver alone. Metastatic tumor cells that do not express the ligand, colonized mostly the lung. However, following the induction of ligand expression, tumor cell colonization was redirected to the liver with tremendous efficiency. Adhesion to Extracellular Matrix Components Mammalian organisms are composed by a series of tissue compartments separated from one another by two types of extracellular matrix (ECM): basement membranes and interstitial stroma. ECM consists of three general classes of macromolecules, including collagens, proteoglycans and non-collagenous glycoproteins (such as fibronectin, laminin, entactin and tenascin among others), which are expressed in a tissue-specific fashion. Malignant cells arrested in the microcirculation sometimes do not migrate further into the organ parenchyma but grow locally in an expansive fashion until they rupture the vessel wall. In most cases however, the contact between tumor cells and the endothelium results in EC retraction with exposure of the underlying basement membrane, followed by invasion of tumor cells in the tissue. The presence of specific adhesion receptors on the membrane of metastatic cells, and the peculiar composition of the extracellular matrix at a given site will influence tumor cell retention, motility and invasion, and growth at target organs. . Electron microscopy observation on the formation of pulmonary metastasis has shown that tumor cells often adhere to regions of exposed basal lamina. The exposed subendothelial matrix is usually a better adhesive substrate for tumor cells than the endothelial cell surface. . In order to move through the ECM, tumor cells must make firm contacts with matrix molecules, be able to break these adhesive contacts as they move on and respond to chemotactic molecules that direct their movement. Interactions with the ECM may fulfill all these scopes, through the signaling effect of several cytokines (growth factors, motility factors, enzymes and enzyme inhibitors) that are stored bound to ECM molecules, and released upon interaction with tumor cells. Moreover, ECM macromolecules themselves may also function as motility attractants, and have

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been shown to stimulate both ▶chemotaxis and ▶haptotaxis. Haptotactic migration over insoluble matrix components may occur predominantly during the initial stages of metastatic invasion, while at later stages partially degraded matrix proteins, derived from proteolytic processing of the matrix, could be the major determinant of directed motility. . Finally, it has to be considered that some ECM components may actually impede cell adhesion and thus might influence directional tumor cell motility by promoting the localized detachment of the trailing edge of migrating cells. ECM-associated chondroitin sulfate proteoglycans such as decorin, or the glycoprotein tenascin, have been suggested to modulate tumor cell adhesion and motility in this way.

have domains that extend into both the extracellular space and the intracellular space. The extracellular domain of a cell adhesion protein can bind to other molecules that might be either on the surface of an adjacent cell (cell-to-cell adhesion) or part of the ▶extracellular matrix (cell-to-ECM adhesion).

Adhesion and Drug Resistance The malignant phenotype of tumor cells depends, at least partially, on the weakening of cell-matrix and cellcell interactions that occurs during tumor progression. However, late stage tumors maintain some level of intercellular adhesion, or even tend to reactivate certain adhesion mechanisms, indicating that modulation of cell adhesion is a dynamic process. Given the beneficial effect of cell adhesion on apoptosis resistance, an increased level of adhesion may facilitate survival of tumor emboli, and there is evidence that it can help tumor cells to evade the cytotoxic effects of anticancer therapy.

▶Arginine-Depleting Enzyme Arginine Deiminase

▶Furin ▶Sjögren Syndrome

ADI

Adipocyte Complement-Related Protein of 30 kDa ▶Adiponectin

References 1. Rusciano D, Welch DR, Burger MM (2000) Cancer metastasis: experimental approaches. Laboratory Techniques in Biochemistry and Molecular Biology, Volume 29, Elsevier Science B.V. 2. Yamasaki H, Omori Y, Zaidan-Dagli ML et al. (1999) Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis. Cancer Detect Prev 23:273–279 3. Boudreau N, Bissell MJ (1998) Extracellular matrix signaling: integration of form and function in normal and malignant cells. Curr Opin Cell Biol 10:640–646 4. Ruohslahti E, Öbrink B (1996) Common principles in cell adhesion. Exp Cell Res 227:1–11 5. Hedrick L, Cho KR, Vogelstein B (1993) Cell adhesion molecules as tumor suppressors. Trends Cell Biol 3:36–39

Adipocyte C1q ▶Adiponectin

Adipocytes Definition Fat storing cells. ▶Signal Transduction ▶Adipose Tumors

Adhesion Molecules Definition Are transmembrane cell adhesion proteins which extend across the cell surface ▶membrane and typically

Adipocytic Tumors ▶Adipose Tumors

Adiponectin

Adipokine Definition refers to a cluster of adipocyte-secreted molecules, which consist of growth factors, metabolic hormones, and cytokines etc. Many adipokines, such as leptin, adiponectin, resistin, visfatin, and adipocyte fatty acid binding protein, play important roles in obesity and its related diseases. ▶Adiponectin

Adiponectin J ANICE B.B. L AM , Y U WANG Department of Medicine and Genome Research Center, University of Hong Kong, Hong Kong, China

Synonyms Gelatin-binding protein 28; GBP28; AdipoQ; Adipocyte complement-related protein of 30 kDa; ACRP30; Adipose most abundant gene transcript 1; apM1; Adipocyte C1q and collagen domain containing; ACDC

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Definition

Adiponectin is a major ▶adipokine secreted exclusively from adipocytes. This adipokine has been demonstrated to possess antidiabetic, antiatherogenic, antiinflammatory and, more recently, antitumorigenic properties.

Characteristics Adiponectin was originally identified as an adiposespecific gene dysregulated in ▶obesity. Human adiponectin gene is located on chromosome 3q27 and encodes a 244 amino acids polypeptide comprising of an NH2-terminal secretory signal sequence, followed by a hypervariable region, a collagenous domain, and a COOH-terminal globular domain (Fig. 1a). Circulating concentrations of adiponectin range from 3 to 30 μg/ml, which accounts for about 0.05% of total human blood proteins. Endogenous adiponectin is predominantly present as several characteristic oligomeric complexes. The basic building block of the adiponectin complex is a trimer or low molecular weight (LMW) oligomer, which is formed via hydrophobic interactions within its globular domain. Two trimers self-associate to form a disulfide-linked hexamer or middle molecular weight (MMW) oligomer, which further assembles into a bouquet-like high molecular weight (HMW) multimeric complex that consists of 12–18 monomers (Fig. 1b). Posttranslational modifications, including disulfide bond formation at a conserved cysteine residue and glycosylations occurred on several hydroxylated lysine residues within the collagenous domain, are involved in the assembly and stabilization of the

Adiponectin. Figure 1 Schematic representation of the primary structure (a) and the oligomeric complexes of adiponectin (b). Adiponectin contains a NH2-terminal signal sequence peptide and a hypervariable region, followed by a conserved collagenous domain and a COOH-terminal globular domain. A cysteine residue within the hypervariable region is involved in the disulfide bond formation. Several lysine residues located within the collagenous domain are hydroxylated and glycosylated. (b) Adiponectin exists as three oligomeric species, including the trimer (LMW), hexamer (MMW), and HMW. Disulfide bond formation and glycosylation are involved in its oligomeric formation. GG, glucosylα(1–2)galactosyl group; S−S, disulfide bonds.

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Adiponectin

oligomeric structures. Different oligomeric complexes of adiponectin activate distinct signaling pathways and possess different biological functions. Two putative adiponectin receptors, termed AdipoR1 and AdipoR2, have recently been identified. AdipoR1 is highly expressed in skeletal muscle whereas AdipoR2 is most abundantly expressed in liver. Both receptors are integral membrane proteins containing seven transmembrane spanning domains. AdipoR1/R2 mediates the effect of adiponectin on activation of ▶AMP-activated protein kinase (AMPK) and stimulation of glucose uptake and fatty acid oxidation. T-cadherin, which is highly expressed in endothelium and smooth muscle, has been identified as an adiponectin coreceptor with preference for hexameric and HMW adiponectin multimers. Both adiponectin analogues and adiponectin receptor agonists represent the potential therapeutic targets for obesity-linked diseases. Adiponectin and Carcinogenesis Although adiponectin is secreted exclusively from fat cells, the circulating adiponectin levels are paradoxically reduced in obese individuals and obesity-related pathological conditions, such as ▶insulin resistance, type 2 diabetes, and atherosclerosis. Adiponectin has been proved to have insulin-sensitizing, antidiabetic, and antiatherogenic activities. In addition, growing evidence has demonstrated adiponectin to be a potent inhibitor of carcinogenesis. Numerous clinical studies have observed an inverse association between blood adiponectin concentrations and risks of several ▶obesity-related cancers, such as ▶prostate, ▶breast, ▶endometrial, ▶gastric, and ▶colorectal cancers. Prostate Cancer Obesity is associated with prostate cancer progression, increased tumor aggressiveness, and poor prognosis. Higher plasma adiponectin is associated with a marked reduction in risk of prostate cancer, independent of other risk factors. Additionally, blood adiponectin levels in those with high-grade prostate cancer are significantly lower than those in the low-grade and intermediate-grade groups, suggesting that plasma adiponectin levels are negatively associated with the histologic grade and disease stage of prostate cancer. Adiponectin has been shown to inhibit leptin- and/ or ▶insulin-like growth factor-1 (IGF-1)-stimulated DU145 androgen independent prostate cancer cell growth and dihydrotestosterone-stimulated growth of androgen-dependent LNCaP-FGC cells at subphysiological concentrations. In addition, adiponectin enhances the inhibitory effects of the cytotoxic chemotherapy agent, doxorubicin, on prostate cancer cell growth. These data suggest that adiponectin could play an important role in the pathogenesis of prostate cancer, and may be used as a drug target for therapeutic interventions.

Breast Cancer Excess adiposity over the pre- and postmenopausal years is an independent risk factor for the development of breast cancer, and is associated with late-stage disease and poor prognosis. Clinical studies have shown that low plasma adiponectin levels are significantly associated with an increased risk for breast cancer in both pre- and postmenopausal women, particularly in a low estrogen environment. Moreover, tumors from women with low plasma adiponectin levels are more likely to show a biologically aggressive phenotype. In line with these clinical findings, experimental evidence supports the role of adiponectin as an inhibitory factor for breast cancer development. Adiponectin at physiological concentrations suppresses the proliferation and induces ▶apoptosis in the ▶estrogen receptor (ER)-negative human breast carcinoma MDA-MB-231 cells and the ERpositive human MCF7 breast cancer cells. It also inhibits insulin- and growth factors-stimulated cell growth in another ER-positive T47D human breast cancer cells. Furthermore, adiponectin replenishment therapy suppresses mammary tumorigenesis of MDA-MB-231 cells in nude mice. Endometrial Cancer Adiponectin is decreased in obesity, insulin resistance, type 2 diabetes, and polycystic ovary syndrome, all of which are well-established risk factors for endometrial cancer. Several case-control studies have demonstrated an inverse correlation between plasma levels of adiponectin and the risk of endometrial cancer, independent of other obesity-related risk factors as well as the major components of the IGF system. Moreover, a stronger inverse association is observed among obese women than among nonobese women. Further studies are needed to investigate whether adiponectin deficiency plays a causative role in the pathogenesis of endometrial cancer. Gastric and Colorectal Cancer Low plasma levels of adiponectin have been observed in patients with gastric cancer, especially in those with upper gastric cancer. Furthermore, plasma adiponectin levels tend to decrease as the tumor size, depth of invasion, and tumor stage increases. Additionally, the negative correlation is more significant in undifferentiated forms than in differentiated forms of gastric cancers. These data raise the possibility that adiponectin might play a potential role in the progression of gastric cancer, especially in the upper stomach. Although colorectal carcinogenesis is related to abdominal obesity and insulin resistance, the associations between low adiponectin levels and colorectal cancer are not conclusive. Two independent studies have suggested that men with low plasma adiponectin levels have a higher risk of colorectal cancer,

Adiponectin

whereas another report does not support this association. Moreover, adiponectin has been reported to have growth-promoting and proinflammatory actions on HT-29 colonic epithelial cancer cells. Leukemia Adiponectin inhibits cell proliferation and induces apoptosis in myelomonocytic cell lines. Decreased levels of plasma adiponectin have been found to be associated with ▶childhood acute myeloblastic leukemia (AML), but not with ▶acute lymphoblastic leukemia of B (ALL-B) or T (ALL-T) cells. Adiponectin levels are also reported to be inversely associated with ▶chronic lymphocytic leukemia and myeloproliferative diseases. However, it is worthy to note that adiponectin concentrations can be modulated by various inflammatory cytokines and interferon therapy in these conditions. Thus, whether low adiponectin level is a causal factor of leukemia, or a secondary response to ▶inflammation, needs to be further clarified. Mechanisms As summarized above, both clinical and experimental evidence support the role of adiponectin as a suppressor of tumorigenesis. Adiponectin has direct antiproliferative effects in a number of cancer cell lines. Although the underlying mechanisms remain poorly understood, adiponectin has been shown to modulate several intracellular signaling cascades involved in regulating cell proliferation and apoptosis (Fig. 2). AMPK The phosphorylation-dependent activation of ▶AMPK is the major signal transduction pathway evoked by adiponectin. AMPK mediates the insulin-sensitizing effects of adiponectin in liver and muscle. It is also

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involved in the regulatory activities of adiponectin on endothelial cell functions and cardiac remodeling. AMPK is a negative regulator on cell growth. The upstream kinase LKB1 that activates AMPK is a ▶tumor suppressor. ▶Tuberous sclerosis complex 2 (TSC2), another tumor suppressor, is downstream of AMPK and a key player in regulation of the ▶mammalian target of rapamycin (mTOR) pathway. Through inactivation of ▶mTOR, AMPK negatively regulates protein synthesis and de novo fatty acid synthesis, two essential elements for rapid cancer cell growth. In addition, AMPK controls phosphorylation and activation of the ▶P53 tumor suppressor and expression of the cell cycle inhibitor ▶p21. These molecular events might represent the potential mechanisms through which adiponectin regulate carcinogenesis. Indeed, it has been reported that adiponectin at subphysiological concentrations can induce AMPK phosphorylation and reduce the cell growth in human breast cancer MCF-7 cells. c-Jun N-Terminal Kinase (JNK) and Signal Transducer and Activator of Transcription 3 (STAT3) Both ▶JNK and STAT3 are the important regulators of cell proliferation, apoptosis, and differentiation in various physiological and pathophysiological conditions. Constitutive activation of STAT3 is crucial in malignant transformation and cancer progression. It has been reported that adiponectin stimulates the phosphorylation of JNK in prostate cancer DU145, PC-3, and LNCaP-FGC cells, as well as in hepatocellular carcinoma HepG2 cells. On the other hand, adiponectin inhibits constitutive activation of STAT3 in DU145 and HepG2 cells, suggesting that activation of JNK and inhibition of STAT3 may contribute to the suppressive effect of adiponectin on carcinogenesis. In addition, the inactivation of p42/p44

Adiponectin. Figure 2 Adiponectin elicits its antitumorigenic activities through multiple mechanisms.

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AdipoQ

MAP kinase has been implicated in the antiproliferative effects of adiponectin in human beast carcinoma MCF7 and T47D cells. Glycogen Synthase Kinase (GSK) 3b/b-Catenin Signaling Pathway Hyperactivation of the canonical ▶Wnt/β-catenin pathway is one of the most frequent signal abnormalities in many types of cancers. The central event in this pathway is the stabilization and nuclear translocation of β-catenin, where it binds to the transcription factor TCF/LEF and consequently activates a cluster of genes that ultimately establish the oncogenic phenotype. β-catenin is phosphorylated by GSK3β and then modified by ▶polyubiquitination for ▶proteasome-mediated degradation. Adiponectin could modulate the GSK3β/ β-catenin pathway in human breast cancer cells. In MDA-MB-231 cells, prolonged treatment with adiponectin markedly reduces serum-induced phosphorylation of GSK3β, decreases intracellular accumulation and nuclear translocation of β-catenin, and suppresses ▶cyclin D1 expression. These effects can be inhibited by inhibitors of GSK3β. These data suggest that the cross-talk between adipokines and the Wnt signaling pathway might represent a key mechanism underlying the development of obesity-related cancers. Other Pathways In addition to its direct suppressive effect on cancer cell proliferation, as an insulin-sensitizing hormone, adiponectin could ameliorate tumorigenesis indirectly by alleviating ▶hyperglycemia and insulin resistance, the two established risk factors for many obesityrelated cancers. Furthermore, adiponectin possesses antiinflammatory activity and can inhibit the production of a number of inflammatory factors involved in promoting tumorigenesis, such as IL6, IL8, TNFa, and MCP-1. There is also evidence supporting the anti ▶angiogenesis activity of adiponectin. It inhibits tumor neovascularization in mice, through suppression of endothelial cell proliferation, migration, and tubular formation. Moreover, adiponectin can act as a decoy for several proangiogenesis growth factors, including basic fibroblast growth factor (bFGF), platelet-derived growth factor BB (PDGF-BB), and heparin-binding epidermal growth factor (HB-EGF). In this manner, adiponectin prevents these growth factors from activating their respective receptors and effectively impedes their tumor-promoting activities. In summary, both experimental and clinical evidences support the suppressive role of adiponectin in tumorigenesis. In humans, adiponectin deficiency is closely associated with increased risks of several obesity-related cancers. Therefore, adiponectin and its agonists might represent a novel class of the anticancer agent for the treatment of these malignant

tumors. Further studies are warranted to investigate the prospective associations between plasma adiponectin levels and the risk of several obesity-related cancers, and to elucidate the detailed molecular events underlying the antitumor activities of adiponectin.

References 1. Barb D, Pazaitou-Panayiotou K, Mantzoros CS (2006) Adiponectin: a link between obesity and cancer. Expert Opin Investig Drugs 15:917–933 2. Kelesidis I, Kelesidis T, Mantzoros CS (2006) Adiponectin and cancer: a systematic review. Br J Cancer 94: 1221–1225 3. Scherer PE, Williams S, Fogliano M et al. (1995) A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270:26746–26749 4. Wang Y, Lam JB, Lam KS et al. (2006) Adiponectin modulates the glycogen synthase kinase-3β/β-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res 66:11462–11470

AdipoQ ▶Adiponectin

Adipose Most Abundant Gene Transcript 1 ▶Adiponectin

Adipose Tumors F LORENCE P EDEUTOUR , A NTOINE I TALIANO Laboratory of Solid Tumors Genetics, Nice University Hospital and CNRS UMR 6543, Faculty of Medicine, Nice, France

Synonyms Lipomatous tumors; Adipocytic tumors; Lipomas; Liposarcomas

Adipose Tumors

Definition Adipose tumors (AT) are mesenchymal neoplasms that form the largest group of human tumors. They include benign tumors, such as the very common lipomas, as well as rare malignant tumors with various degrees of clinical aggressiveness. Histologically, AT consist of adipocytic cells showing different levels of differentiation, from mature adipocytes in benign lipomas up to undifferentiated lipoblastic cells in high-grade ▶liposarcomas. The 2002 World Health Organization classification distinguishes seven entities of benign AT: lipoma, lipoblastoma/lipoblastomatosis, angiolipoma, myolipoma of soft tissue, chondroid lipoma, spindle cell/pleomorphic lipoma, and hibernoma. Malignant AT, also called liposarcomas, include three types: well differentiated liposarcoma/dedifferentiated liposarcoma, ▶myxoid/round cell liposarcoma, and pleomorphic liposarcoma. Except for the ordinary superficial lipomas, differential diagnosis between benign and malignant AT and between AT and other kinds of tumors is sometimes difficult. Studies based on tumor karyotypes have identified chromosomal abnormalities specific to benign and malignant AT and recent advances in molecular cytogenetics improved AT diagnosis. It is now possible to directly detect the genic rearrangements resulting from chromosomal alterations on interphase nuclei such as those in formalin-fixed and paraffin embedded tumor tissue sections using fluorescence in situ hybridization (FISH) (▶interphase cytogenetics) or polymerase chain reaction (PCR).

Characteristics Benign Adipose Tumors The most common benign AT are the so-called superficial “conventional lipomas.” The other types of benign AT are rare and may be the cause of diagnostic difficulties because of their clinical or histological resemblance to malignant soft tissue tumors. In most cases benign AT do not require any treatment. Surgical

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removal may be necessary in case of functional or cosmetic impairment. ▶Conventional lipomas are the most common soft-tissue neoplasm in adults. They occur mainly in the 5–7th decades of life and are generally located superficially in subcutaneous fat. They can also be situated deeply in muscles or on the surface of bones or rarely in visceral and other organ sites. Lipomas usually present as a small (200 g) with nuclear atypia, more than five mitoses on 50 high-power fields, vascular and capsular invasion, broad fibrous bands, and extensive necrosis are highly suggestive of carcinoma. Small tumors without the mentioned features are considered to be adenomas. In children, distinguishing between adenoma and carcinoma is difficult because the histologic features of small and large lesions overlap. Only about

Adrenomedullin (AM) is a member of the ▶calcitonin superfamily of peptides. It is produced in virtually every organ by many different cell types and it is secreted into the plasma where it occurs at picomolar concentrations. Over the past several years AM has increasingly received the attention of the scientific community by virtue of its implication in many normal and disease states.

Characteristics Adrenomedullin is a small peptide (52 amino acids) first isolated from a ▶pheocromocytoma in 1993. It was initially described as a hypotensive peptide although after more than a decade of research and about 2,000 published articles published, AM is now recognized as a pluripotent peptide-hormone implicated in many normal and pathological processes ranging from vascular tone and diabetes to ▶angiogenesis and embryogenesis/ ▶carcinogenesis. Adrenomedullin: Peptide and Gene Structure Adrenomedullin is generated as part of a larger precursor molecule named preproadrenomedullin (preproAM) (Fig. 1). PreproAM is 185 amino acids long and contains an N-terminal 21 amino acid signal peptide which is cleaved during the transport of the molecule across the cell membrane to produce the 164 amino acids prohormone proAM. Further processing of proAM by endopetidases generates four peptides termed proadrenomedullin N-terminal 20 peptide (PAMP), mid-regional pro-adrenomedullin (proAM 45–92), adrenomedullin (AM) and adrenotensin (proAM 153–185). From these, PAMP, ProAM 45–92 and AM are present in plasma and PAMP, AM and Adrenotensin are biologically active peptides. Both PAMP and AM peptides are produced as a ▶glycine (Gly)-extended inactive peptides which coexists in plasma with the active form generated

Adrenomedullin

upon enzymatic ▶amidation. AM shares homology with several vasoactive peptide members of the calcitonin superfamily including calcitonin, calcitonin gene related peptide (CGRP), amylin and intermedin. Members of this family share the presence of an intramolecular disulfide bond which generates a six-member ring structure and an amidated carboxy terminal, both of which are required for biological activity. In humans, the single locus of the adrenomedullin gene is located in the short arm of chromosome 11. The complete gene (2,319 bp) contains four exons and three introns which are ▶alternatively spliced during the transcription process to generate two different transcripts (Fig. 1). The shortest mRNA form includes exons 1–4 and therefore codes for a complete preprohormone which results in stoichiometric amounts of the four peptides referred above. The longest

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transcript incorporates the third intron that contains an early termination codon, resulting in a truncated preprohormone which only expresses PAMP. AM is an ancient gene that, based on our current knowledge, first appeared in the starfish with a potential dual function of neurotransmission and host defense. It shows a remarkable degree of conservation in genomic organization and peptide structure from fish to humans which supports its critical role in species survival. Signal Transduction As most soluble peptides, AM transduces its signal upon interaction with a receptor located in the cellular surface. The discovery of the AM receptor in 1998 represented a novel paradigm in the field of ▶G-protein couple receptor (GPCR) signaling. A functional receptor for AM requires physical interaction in the cellular

Adrenomedullin. Figure 1 Genomic organization of the AM gene.

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membrane of the seven transmembrane domain receptor calcitonin-receptor-like-receptor (CRLR) and either the receptor-activity-modifying protein (RAMP) 2 or RAMP3. CRLR has two alternative pharmacological profiles that are conferred by association to the accessory proteins RAMP1 (producing the CGRP receptor) and RAMP2/3 (producing the AM receptor). Therefore, the expression pattern of functional AM receptors is determined by the presence of these two components. In healthy individuals, RAMP2/3 is equally expressed among most tissues, excluding lung, female reproductive system and adipocytes which show higher levels of expression. CRLR expression although lower, parallels that of RAMP2 which suggests that the majority of CRLR signaling units in the body are complexed with RAMP2 to produce adrenomedullin receptors. Modest but robust changes in the expression of the complex CRLR-RAMP2 have been reported in certain physiological and disease states such as pregnancy, sepsis and ▶cancer. The same physiological conditions are related to high levels of AM expression. Other stimuli which result in coordinated regulation of AM, CRLR and RAMP2 include hypoxia, endocrine hormones and inflammatory cytokines. Upon binding to its receptor AM induces cAMP elevation through an adenylyl cyclase-PKA mediated pathway. While multiple reports including the seminal paper by Kitamura have consistently demonstrated cAMP mediated effects of AM, other more scarce ones have shown cAMP independent actions such as vasodilation via elevation of Ca2+ and K+-ATP, and activation of endothelial nitric oxide synthase. AM also activates Akt, ▶mitogen-activated protein kinase and focal adhesion kinase in endothelial cells which mediate its angiogenic potential. AM Serves as a Common Language Between the Different Cellular Components of the Tumor Microenvironment Many disease states have been reported to modulate the expression of AM including cancer. As we mentioned before, AM was originally isolated from an adrenal gland tumor. A wealth of subsequent studies have found that AM and its receptor are overexpressed in many human cancers and tumor cell lines establishing an ▶autocrine loop mechanism that tumor cells exploit to maintain an autonomous proliferative state. AM is intimately intertwined at several levels in the multistep process of tumor development. At the initial stage of tumor growth, rapid accumulation of malignant cells results in the establishment of an avascular nutrientdepleted ▶hypoxic environment. Low oxygen tension within and surrounding the tumor body triggers a number of survival mechanisms which allow neoplastic cells to overcome this inhospitable microenvironment. Many of these encompass the upregulation of AM’s

expression. In fact one, if not the most important driving force for AM upregulation in tumor cells is hypoxia. Cellular responses to hypoxia are mediated through a well known hypoxia inducible factor (HIF)-dependent mechanism. HIF is a heterodimeric transcription factor which is stabilized under hypoxic conditions and binds to specific DNA sequences denoted hypoxia response elements (HRE) which are present in the ▶promoter regulatory region of the AM gene (Fig. 1). Hypoxia also upregulates the expression of the AM receptor gene in many tumor types hence establishing a rational explanation behind the aforementioned autocrine growth mechanism underlying carcinogenesis (Fig. 2). As tumor derived AM is released into the microenvironment it establishes a peptide gradient which ultimately disseminates to reach a teeming collection of cell types known to be able to respond to this peptide and to be involved in further development of the tumor, including the cancer cell itself. AM not only stimulates tumor cell proliferation via its mitogenic activity but also by involving an antiapoptotic state. Although the advantageous effects of AM for the tumor cell are apparent, its actions are not restricted to this compartment within the tumor. On the contrary, AM acts as an integrative molecule allowing the crosstalk between all different compartments within the tumor microenvironment. As an example, AM is a ▶migratory factor for different inflammatory cells, including ▶mast cells. Mast cells migrate towards the tumor mass following the preestablished tumor-derived AM gradient. Hence, as mast cells approach the tumor they are exposed to increasingly higher concentrations of AM. Only when certain concentration of AM is reached in the proximity of the tumor, mast cells degranulate liberating to their immediate milieu numerous inflammatory factors (including AM) which not only enhance the tumor progression but also perpetuate the inflammatory process. AM is also implicated as a potential immune system suppressor, inhibiting macrophage function and acting as a negative regulator of the complement cascade, protective properties which help cancer cells circumvent immune surveillance. One of the most significant features distinctive of hypoxic tumors is their ability to induce angiogenesis. Tumor-induced angiogenesis is a pathological condition that results in ectopic neovascularization. Of most therapeutic interest is the finding that AM is an essential factor that regulates normal and pathological vascularization. AM was first described as a potent hypotensive peptide although its connection to the normal and pathological biology of the vascular system is much deeper than initially thought. AM is an essential factor for the normal development of vasculature as revealed by mice lacking the AM gene in mice which is embryonically lethal due to abnormal vascularization. AM also induces pathological neovascularization via

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Adrenomedullin. Figure 2 Model of the AM/tumor cell/inflammatory cells relationship in human carcinogenesis. The microenvironment around the tumor is hypoxic and stimulates expression of AM by the tumor cells. Tumorderived AM is released into the microenvironment setting up a concentration gradient of peptide that contributes to angiogenesis and attracts distal MCs to infiltrate the tumor site. Neovasculature makes possible tumor metastasis and it is used as a point of entrance for inflammatory cells (i.e. MC). As MCs migrate up the peptide gradient, higher AM concentrations are reached stimulating MC-derived angiogenic factors (AM, VEGF, bFGF, MCP-1) expression and ultimately release at the tumor site. AM mediates a paracrine tumor survival effect (direct mitogen, angiogenic factor, and anti-apoptosis), and functions as a paracrine recruitment factor drawing additional MCs to the area, thus perpetuating the inflammatory process and enhancing tumor promotion.

CRLR-RAMP2 present in the endothelial cells. Angiogenesis is a multistep process which commences with the growth of endothelial cells which is enhanced by tumor derived AM. AM also prevents hypoxiatriggered apoptosis in endothelial cells enhancing the neovascularization process. Additionally, AM participates in the remodeling of the extracellular matrix and tridimensional rearrangement of endothelial cells in the tissue which results in the establishment of the new intratumoral vasculature by stimulating migration and tube formation of endothelial cells. AM increases the permeability of the endothelial cells in the newly established vasculature which supplies the tumor with the necessary nutrients for expansion additionally it creates an access route for inflammatory cells which are attracted to the tumor site and migrate in following gradients of ▶chemoattractant and migratory factors produced by the tumor, such as AM. The same route can simultaneously be utilized by tumor cells as the entrance point to the vascular system facilitating the

metastasis process. The invasive capability of tumor cells is thus enhanced by AM.

Concluding Remarks Conclusions gleaned from the studies carried over the past fourteen years portrait AM as a molecular connector with competence to entangle and allow communication between the different cellular components of the tumor machinery which conspire under the tumor cell direction to promote cancer. It is not only the direct effect that AM has on tumor cells but also its ability to interact with all these cellular elements which makes this peptide an attractive therapeutic target for cancer. The collective research effort is shifting from trying to discern whether AM is a causative agent of cancer to better understanding its central role as a multifaceted exchange currency among the multiple cellular players involved in tumor development. Strategies utilizing blocking agents aimed at disruption of this loop might be proven successful to impede tumor growth.

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References 1. Kitamura K, Kangawa K, Kawamoto M et al. (1993) Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560 2. Zudaire E, Martinez A, Cuttitta F (2003) Adrenomedullin and cancer. Regul Pept 112:175–183 3. Cuttitta F, Portal-Nuñez S, Falco C et al. (2006) Adrenomedullin: An esoteric juggernaut of human cancers. In Kastin AJ (ed) Handbook of biologically active peptides. Elsevier

Adriamycin T SUTOMU TAKAHASHI , A KIRA N AGANUMA Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Synonyms Doxorubicin; 14-Hydroxyldaunorubicin

Definition

Adriamycin is an antineoplastic ▶anthracycline antibiotic isolated from cultures of Streptomyces peucetius var. caesius. It is widely used in the treatment of various different types of cancers. Proposed mechanisms for its antitumor activity include intercalation into DNA, inhibition of ▶topoisomerase II, and promotion of freeradical formation. However, the clinical utility of this drug is seriously limited by the development of cardiomyopathy and ▶myelosuppression.

Characteristics Chemical Properties Adriamycin is an orange–red compound, soluble in water and aqueous alcohols, moderately soluble in anhydrous methanol, and insoluble in nonpolar organic solvents. It consists of an aglycone (adriamycinone), a tetracyclic ring with adjacent quinone–hydroquinone groups in rings C-B, coupled with an amino sugar (daunosamine). It is generated by C-14 hydroxylation of its immediate precursor, ▶daunorubicin (see Fig. 1). Semisystematic derivatives of adriamycin include ▶epirubicin, an axial-to-equatorial epimer of the hydroxyl group at C-4′ in daunosamine, and ▶pirarubicin, 4′-O-tetrahydropyranyl-adriamycin, etc. Clinical Aspects Therapeutic Applications Adriamycin has a broad antitumor spectrum. It is used to treat hematopoietic malignancies such as leukemias,

lymphomas (non-Hodgkin disease, ▶Hodgkin disease) and ▶multiple myeloma, and different solid tumors (breast, thyroid, gastric, ovarian, bronchogenic, head and neck, prostate, cervical, pancreatic, uterine and hepatic carcinomas, as well as transitional cell bladder carcinomas, ▶Wilms tumor, ▶neuroblastoma, and soft tissue and bone sarcomas). Adriamycin is applied as a component of combination chemotherapy, rather than a monotherapy. Adriamycin-based combination chemotherapy regimens include ABVD (Adriamycin, Bleomycin, Vinblastine, Dacarbazine) for non-Hodgkin disease, CHOP (Cyclophosphamide, Adriamycin, Vincristine, Prednisone) for ▶Hodgkin disease, and M-VAC (Methotrexate, Vinblastine, Adriamycin, Cisplatin) for urothelial carcinoma. Pharmacokinetics Adriamycin is rapidly cleared from the plasma, quickly taken up and only slowly eliminated from organs such as the spleen, lungs, kidneys, liver, and heart. It does not cross the blood–brain barrier. Adriamycin is converted to an active metabolite, adriamycinol, through a twoelectron reduction of the side chain C-13 carbonyl moiety by NADPH-dependent cytoplasmic aldo/keto reductase or carbonyl reductase. It is converted to inactive metabolites in the liver and other tissues, and predominantly excreted in the bile. Clinical Toxicities The usual toxic side effects of adriamycin including stomatitis, nausea, vomiting, alopecia, gastrointestinal disturbance, and dermatological manifestations, are generally reversible. The dose-limiting side effects of the anthracyclines including adriamycin are myelosuppression and cardiotoxicity. Myelosuppression with leukopenia, neutropenia, and occasionally thrombocytopenia is dose-related and potentially life-threatening. Cardiotoxicity is characteristic of the anthracycline antibiotics, of which adriamycin is the most toxic. Adriamycin-induced cardiotoxicity can be acute, chronic, or delayed. The acute effect is not dose-related, and is characterized by sinus arrhythmias and/or abnormal electrocardiographic (ECG) changes (nonspecific ST-T wave change, prolongation of QT interval). Acute toxicity of this type is transient and rarely a serious problem. Chronic cardiotoxicity is a much more serious problem, being related to cumulative dose. It is irreversible and leads to dilative cardiomyopathy and congestive heart failure (CHF), usually unresponsive to cardiotonic steroids (digitalis) and β-blockers. The risk of developing CHF increases markedly at total cumulative doses in excess of 500 mg/m2. Moreover, the effects of this chronic cardiotoxicity may manifest precipitously without antecedent ECG changes. The risk of life-threatening cardiac dysfunction can be decreased by regular monitoring of endomyocardial

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Adriamycin. Figure 1 Structures of adriamycin and its analogues.

(EM) biopsy histopathological changes and left ventricular ejection fraction (LVEF) as measured by the multigated radionuclide angiography (MUGA) method and/or echocardiography (ECHO). Finally, adriamycin can also cause delayed cardiotoxicity, possibly, related to the dose. This occurs after an asymptomatic interval, mostly in people who were treated as children. Several approaches have been proposed to overcome adriamycin cardiotoxicity and that of the anthracycline antibiotics generally. Administration by slow continuous intravenous infusion (over 48–96 h) rather than the standard bolus injection decreases the likelihood of chronic cardiotoxicity. ▶Dexrazoxane (ICRF-187), an iron chelator that prevents the formation of complexes between adriamycin and iron, and subsequent production of ▶reactive oxygen species (ROS), is sometimes used as a cardioprotectant. However, it may decrease antitumor activity. Liposomal encapsulation is designed to increase safety and efficacy by decreasing cardiac and gastrointestinal toxicity through decreased exposure of these tissues to the drug, while effectively delivering it to the tumor. Polyethyleneglycol-coated (Pegylated) liposomal adriamycin (Doxil (USA), Caelyx (UK)) is currently used for treating AIDS-related ▶Kaposi sarcoma, refractory ovarian cancer, and some other solid tumors. In order to improve therapeutic efficacy and decrease side effects by promoting drug accumulation inside tumors, the water-soluble N-(2hydroxypropyl) methacrylamide (HPMA) copolymer, magnetic targeted carriers, and ▶immunoliposome

conjugates with the specificity of whole monoclonal antibodies (e.g. antibodies against CD19 or MUC-1) or FAB’ fragments have been developed as carriers of adriamycin. In further efforts to decrease the risk of developing cardiotoxicity, several derivatives of adriamycin or daunorubicin, such as epirubicin, pirarubicin, ▶idarubicin, and ▶aclarubicin have been developed. Although these agents may be less cardiotoxic than adriamycin itself, they do have a decreased antitumor activity. Pharmacological Mechanisms Mechanisms of Action Several mechanisms appear to contribute to the cytotoxic effects of adriamycin, including inhibition of DNA replication and repair; inhibition of RNA and protein synthesis via intercalation of the aglycone portion of the molecule between adjacent DNA base pairs, especially G-C base pairs; promotion of the cleavage of DNA by formation of adriamycintopoisomerase II-DNA ternary complexes; inhibition of topoisomerase I; and direct binding to the cell membrane. Formation of free radicals is another major mechanism of cytotoxicity. One-electron reduction of the quinone moiety in the C ring of adriamycin by some flavin-containing enzymes (mitochondrial NADH dehydrogenase, microsomal NADPH-cytochrome P450 reductase, and xanthine oxidase) generates adriamycinsemiquinone radicals. These rapidly react with oxygen

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to form superoxide anions, which then generate hydrogen peroxide and hydroxyl radicals in the presence of redox-active metals such as iron (III) and copper (II). The final result is DNA damage and lipid peroxidation. The semiquinone radical can be transformed into an aglycone C7-centered radical that also mediates cellular damage by DNA alkylation and lipid peroxidation. Adriamycin can bind to metal ions such as iron, copper, and manganese, by forming adriamycin–metal complexes, which may lead to generation of ROS and damage to cell membranes. Mechanisms of Resistance Development of resistance to the drug is a major obstacle in chemotherapy with adriamycin. Drug efflux pumps are important for defending cells against anticancer drugs. The acquisition of adriamycin resistance involves promotion of excretion of the drug by overexpressing the ATP-binding cassette (ABC) transporters ▶P-glycoprotein (P-gp)/ABCB1, ▶multidrug resistance-associated proteins (MRPs)/ABCC (MRP1, MRP2, and MRP6 etc.), and breast cancer resistance protein (BCRP)/ABCG2. P-gp transports hydrophobic compounds including adriamycin, while MRP1 and BCRP can extrude predominantly these glutathione conjugates. In addition, RalA-binding protein 1 (RALBP1)/Ral-interacting protein of 76 kDa (RLIP76) is a nonclassical ABC transporter involved in drug excretion. RALBP1 catalyzes ATP-dependent efflux of xenobiotics including adriamycin as well as its glutathione conjugates. In fact, the level of expression of these efflux pumps correlates with the clinical efficacy of adriamycin. ▶Glutathione-S transferases (GSTs) are a family of enzymes involved in the cellular detoxification of xenotoxins. Adriamycin and its metabolites (adriamycinol) are conjugated with glutathione by GSTs and transported by MRPs and BCRP, etc. Increased expression of GSTs, especially GSTπ, also confers adriamycin resistance by promoting detoxification. Lung resistance protein (LRP), the 110 kDa major vault protein (MVP), is a main component of vaults, which are multisubunit structures that may be involved in nucleocytoplasmic transport, and is involved in resistance to anticancer drugs including adriamycin. LRP may affect the intracellular distribution of adriamycin, but the detailed mechanisms remain unknown. Furthermore, a relationship between adriamycin resistance and qualitative and quantitative changes in the expression of topoisomerase II, a major target for adriamycin, has been reported. Mechanisms for Development of Cardiotoxicity The molecular mechanisms leading to adriamycininduced cardiotoxicity may include lipid peroxidation

by generation of ROS, abnormalities in intracellular calcium homeostasis through inhibition of sarcomeric reticulum Ca2+-ATPase (SERCA2), Na+-K+-ATPase and Na+-Ca2+ exchanger of sarcolemma, inhibition of mitochondrial creatine kinase, and interaction with cardiolipin, which is a phospholipid of the inner mitochondrial membrane in the heart. Adriamycin also promotes apoptosis by activation of p38 mitogen-activated kinases (▶MAPK) in cardiac muscle cells. Moreover, adriamycin downregulates the expression of genes for sarcomeric proteins (such as α-actin, myosin, troponin I, and myofibrillar creatine kinase) and for proteins involved in calcium homeostasis in the sarcomeric reticulum, such as SERCA2, cardiac muscle ryanodine receptor (RYR2), calsequestrin, and phospholamban, by suppression of transcription factors (e.g. MEF2C, HAND2, and GATA4) and/or activation of a the transcriptional repressor Egr-1. Adriamycinol (doxorubicinol), a secondary alcohol metabolite, may also be involved in the development of adriamycin-induced cardiotoxicity, via enhancing the inhibitory effects of SERCA2, Na+-K+-ATPase, and Na+-Ca2+ exchanger of sarcolemma. Adriamycinol also inhibits the iron-regulatory protein/ironresponsive element (IRP/IRE) system, which plays a crucial role in iron homeostasis, and may lead to cardiotoxicity.

References 1. Hortobagyi GN (1997) Anthracyclines in the treatment of cancer. An overview. Drugs 54 Suppl 4:1–7 2. Minotti G, Menna P, Salvatorelli E et al. (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56:185–229 3. Nielsen D, Maare C, Skovsgaard T (1996) Cellular resistance to anthracyclines. Gen. Pharmacol. 27:251–255 4. Awasthi S, Sharma R, Singhal SS et al. (2002) RLIP76, a novel transporter catalyzing ATP-dependent efflux of xenobiotics. Drug Metab Dispos 30:1300–1310 5. Poizat C, Sartorelli V, Chung G et al. (2000) Proteasomemediated degradation of the coactivator p300 impairs cardiac transcription. Mol Cell Biol 20:8643–8654

Adult-Onset Diabetes Definition Diabetes Type 2.

Adult Stem Cells

Adult Stem Cells R IKKE C HRISTENSEN , N EDIME S ERAKINCI Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark

Synonyms Somatic stem cells; Tissue stem cells; Postnatal stem cells

Definition An undifferentiated cell found in a differentiated tissue that can renew itself and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.

Characteristics

Adult ▶stem cells are defined as undifferentiated tissue-specific stem cells with extensive self-renewal capacity, which can proliferate to generate mature cells of the tissue of origin. The primary roles of adult stem cells are to maintain and/or regenerate the cells of damaged tissues. Adult stem cells were first described in organs and tissues characterized by high cell turnover, such as blood, gut, testis, and skin but have to date also been isolated from many other organs and tissues including brain, bone marrow, liver, heart, lung, retina, and skeletal muscle. Stem cells differ from ▶somatic cells with their different potentials and their proliferation ability. There are three kinds of stem cells: Embryonic, germinal, and adults stem cells that are classified according to their developmental potential ranging from totipotency to unipotency. The fertilized oocyte and the blastomere up to the 8-cell stage are considered as totipotent (▶totipotent stem cells) as they can differentiate to generate a complete organism. ▶Embryonic stem cells, the cells derived from the inner cell mass of the blastocyste, are pluripotent (▶pluripotent stem cells) and have the ability to differentiate into cells and tissues from all three germ layers: the endoderm, the ectoderm, and the mesoderm. ▶Germinal stem cells are also pluripotent and are derived from so-called primordial germ cells and give rise to the gametes (sperm and eggs) in adults. In contrast, adult stem cells are generally believed to be multipotent (▶multipotent stem cells) or unipotent (▶unipotent stem cells) which means that they can only give rise to progeny restricted to the tissue of origin. Hematopoietic stem cells (▶HSC), bulge stem cells in the hair follicle, and mesenchymal stem cells (▶MSC) are examples of multipotent stem cells, which can differentiate into multiple cell types of

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a single tissue, whereas epidermal stem cells, myosatellite cells of muscle, and endothelial progenitor cells are examples of unipotent stem cells, which only give rise to one mature cell type. Recently, some studies have shown that many adult tissues may contain cells with pluripotent capacity capable of generating differentiated cells from an unrelated tissue. This process is termed ▶stem cell plasticity. In most tissues/organs renewal is compensated by tissue-specific stem cells. The stem cells normally divide very rarely, but stimuli caused by damaged or injured tissue or a need to generate progeny to maintain the tissue can induce proliferation and produce daughter cells that can differentiate into the specific cell lineages of the respective tissue type. Stem cell division typically leads to the formation of committed progenitor cells with more limited self-renewal capacity as e.g., transit amplifying cells in the epidermis or lymphoid or myeloid progenitors in the bone marrow. Tissue progenitor or transit amplifying cells provides an expanded population of a proliferating tissue that differentiate into more mature and determined cells that eventually no longer proliferates and die. To maintain the balance in the adult tissues/organs, the number of progenitor/stem cells that proliferates must be equal to the number of cells that determinedly differentiate and die. If the number of proliferating cells is higher than the number of cells that maturate and die, it will give the primary feature of a cancer. Studies have shown that many of the pathways that regulate normal stem cell proliferation are dysregulated and cause neoplastic proliferation in cancer cells. Therefore, cancer may be considered a disease of dysregulated cellular self-renewal capacity. Adult stem cells reside in a special ▶microenvironment termed the stem cell niche. Stem cell niches are composed of a group of cells that provide a physical anchorage site and extrinsic factors that control stem cell proliferation and differentiation and enable them to maintain tissue homeostasis. Deregulation of the niche signals has been proposed to lead to cancer. A decrease in proliferation-inhibiting signals, or an increase in proliferation-promoting signals, may lead to excessive stem cell production and thereby development of cancer stem cells (see later). Investigation of the interaction between stem cells and their niche may reveal possible targets for cancer treatments. For example, the blocking of proliferation signals, enhancing of antiproliferative signals or induction of differentiation from the stem cell niche may be used to target the cancer stem cells. It has furthermore been suggested that targeting the stem cell niche may prevent cancer metastasis. Some cancers metastasize to sites that cannot be explained by circulation distribution, lymphatic drainage or anatomic proximity. These sites may, however, provide favorable niches that support the survival of the cancer stem cells.

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Much effort is put into the identification of ▶stem cell markers to be able to isolate the stem cells of interest. Isolation of stem cells makes it possible to enhance the knowledge of stem cell identity and to use them therapeutically. Therapeutic Potential Adult stem cell transplantation has been used in several years for the treatment of ▶hematological malignancies and lymphomas. The main purpose of stem cell transplantation in cancer treatment is to make it possible for patients to receive very high doses of chemotherapy and/or radiation. High-dose chemotherapy and radiation can severely damage or destroy the bone marrow while killing cancer cells. Before treatment, bone marrow or peripheral blood stem cells are harvested from the patient itself (for autologues transplantation) or from a donor (for allogen transplantation), frozen down, and then transplanted after the patient has received high doses of chemotherapy, radiation therapy, or both. The transplanted healthy stem cells replace the stem cells destroyed by high-dose cancer treatment and allow the bone marrow to produce healthy cells. Stem cells in general, due to their high proliferative capacity and long-term survival in comparison to somatic cells, make them very ideal candidates to use for regenerative medicine and cell replacement therapy. Lately, there has been an increasing interest in the potential use of adult stem cells in cell replacement strategies and in tissue engineering, including gene therapy. This current interest rose due to the discovery of adult stem cells with pluripotential capacity and/or transdifferentiating (▶transdifferentiation) ability, which means that cells from one tissue can differentiate into mature and functional cells of another tissue. There are reports that HSCs under certain conditions can evolve into cells of neural lineage, liver, muscle, skin, and endothelium; skeletal muscle stem cells can evolve into blood cells and neural cells; and hair follicle stem cells can evolve into neural lineage cells. Other adult stem cells that can be induced to a different cell type include MSCs, cardiac muscle stem cells, neural stem cells, and testis-derived stem cells. These cells have the advantage that they can be used as autologous transplants and have been proposed as an attractive alternative to ▶embryonic stem cells in genetic therapy. An alternative approach for therapeutic use of stem cells is to use them as cellular vehicles. It has been demonstrated that genetically modified MSCs can be used to target delivery of anticancer agents and ▶suicide gene therapy vectors to tumor cells. Upon administration, MSCs can target microscopic tumors, proliferate and differentiate, and contribute to the formation of a network of cells surrounding the tumor (tumor stroma). MSCs genetically modified to express interferon beta has, for example, been shown to inhibit

the growth of tumor cells by local production of interferon beta. MSCs are not the only stem cells that have been used as shuttle vectors for delivery of gene therapies into growing tumors. It has also been demonstrated that neural stem and progenitor cells migrate selectively to tumor loci in vivo in mice. These studies clearly suggest that a stem cell-directed prodrug therapy approach may have great use for eradicating tumors as well as to treat the residual cancer cells remaining after therapy. Genetic manipulation of adult stem cells may also be used to increase the functionality and proliferative capacity of these cells. HSCs are one of the most promising candidates for correction of single gene disorders as e.g., ▶cystic fibrosis and ▶hemoglobinopathies, due to their capability of targeting solid organs and high success rate in their isolation by using a combination of surface markers. Infants with forms of ▶severe combined immunodeficiency syndrome have successfully been treated with genetically engineered bone marrow stem cells. The stem cells were harvested from the patients, a functional gene inserted, and the genetically modified cells reintroduced to the same patient. To increase the success of chemotherapeutic treatment, drug resistant HSCs have been produced by introduction of the ▶multidrug resistance gene with the aim of limiting the myelosuppressive effects of standard chemotherapeutic agents on the stem cells. However, even though adult stem cells have shown to carry great potential to function as therapeutic agents for targeting human diseases such as cancer, degenerative and chronic diseases, they do have some restrictions such as having limited self-renewal capacity. This limitation can be overcome by the introduction of immortalizing genes that increases the cells proliferative capacity. ▶Telomerase has been, in this connection, highlighted among the numerous genes that are capable of immortalizing stem- or progenitor cells. However, suggested oncogenic potential of ▶immortalized cells release caution that before the therapeutic use of stem cells in the clinic, a thorough screen for transformation phenotype is required. Cancer Stem Cells The theory that cancer stem cells (▶cancer stem-like cells and ▶stem-like cancer cells) are involved in many types of cancer has recently gained popularity. There are many similarities between adult stem cells and cancer stem cells. Both have the ability to self-renew and differentiate into more mature diversified cells. Cancer stem cells and normal stem cells share many cell surface markers and utilize many of the same signal transduction pathways. Cancer stem cells have been identified in most types of hematopoietic malignancies, including acute myeloid leukemia, chronic myeloid leukemia, acute

Advanced Breast Cancer

lymphoblastic leukemia, and multiple myeloma. Recently, cancer stem cells have also been isolated from solid tumors such as breast, lung, and brain tumors. The cancer stem cells only represent approximately 1% of the tumor, making them difficult to detect and study. Studies have shown that cancer stem cells may cause tumors when transplanted into a secondary host indicating that the cancer stem cells can initiate and repopulate a tumor. A study of human leukemia shows that the normal hematopoetic stem cell and the neoplastic clone share common molecular mechanisms governing proliferation which is supportive of the normal hematopoetic stem cell being a target for transformation. Due to stem cells are able to divide over the lifespan of the individual they seem to allow accumulation of a number of mutations and perhaps epigenetic changes (▶epigenetics) that cause neoplastic development. In addition, it has been shown that adult stem cells can be targets for neoplastic transformation by introducing the telomerase gene into a purified stem cell. The transduced cell line showed characteristic alterations of neoplastic development such as contact inhibition, anchorage independence, and in vivo tumor formation in immunocompromised mice. All these findings give a very large support to the existence of cancer stem cells and the strong links between normal adult stem cells and cancer stem cells suggest that stem cells are targets for neoplastic transformation. Cancer stem cells may also be derived from differentiated cells. Loss of the ▶tumor suppressor genes p16Ink4 and p19Arf combined with constitutive activation of the EGF receptor (▶EGFR) caused loss of differentiation in mature brain astrocytes and the cells regained stem cell properties. The identification of cancer stem cells strongly suggests that these cells are the key targets for future therapeutic development as they fuel the replicative capacity of the cancer. Therefore, as much as understanding the nature of a cancer cell it is very crucial to understand the neoplastic potential of the stem cells. Analysis of the differences between adult stem cells and cancer stem cells is very important to be able to specifically target the cancer stem cells whilst sparing the normal stem cell population. Several studies indicate that some stem cell markers are expressed differently in normal and cancer stem cells and these may be potential targets in the development of future cancer treatments.

References 1. Pessina A, Gribaldo L (2006) The key role of adult stem cells: therapeutic perspectives. Curr Med Res Opin 22:2287–2300 2. Pan CX, Zhu W, Cheng L (2006) Implications of cancer stem cells in the treatment of cancer. Future oncol 2:723–731

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3. Lotem J, Sachs L (2006) Epigenetics and the plasticity of differentiation in normal and cancer stem cells. Oncogene 25:7663–7672 4. Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5:32–45 5. Reya T, Morrison SJ, Clarke MF et al. (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111

Adult T-cell Leukemia Definition ATL; A leukemia of mature T lymphocytes (▶T cells) developing in adults, resulting from infection with the ▶human T-cell leukemia virus (HTLV) and characterized by circulating malignant T-lymphocytes, skin lesions, lymphadenopathy (enlarged lymph nodes), hepatosplenomegaly (enlarged liver and spleen), hypercalcemia (high blood calcium), lytic (“punched out”) bone lesions, and a tendency to infection. There are four categories of ATL, based on the aggressiveness of the disease – smoldering, chronic, lymphoma, and acute.

Adult Tissue Stem Cells Definition These cells are set aside during development in order to provide a source for replenishment of tissue over time in response to damage or simply wear and tear. ▶Stem Cell Telomeres

Advanced Breast Cancer Definition Tumors of stage III or IV (larger than 5 cm and/or have metastasized). ▶Fulvestrant ▶Breast Cancer

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Aflatoxin B1

Definition

Definition

Acute erythro leukemia.

Aflatoxin B1 is a potent hepatocarcinogen produced by the mold Aspergillus flavus; ▶aflatoxins

▶ETV6

▶Detoxification ▶Carcinogenesis

Aerobic Glycolysis ▶Warburg Effect

Aflatoxins T HOMAS E. M ASSEY, K ATHERINE A. G UINDON Department of Pharmacology and Toxicology, Queen’s University, Kingston, ONT, Canada

Aesthetic and Reconstructive Surgery of the Breast

Definition

Surgical procedures that reshape the breast to improve their appearance.

Mycotoxins are contaminants of a number of agricultural products, including peanuts, corn, and other grains in warm and moist conditions. Human exposure to aflatoxins is primarily through ingestion and results in acute hepatic necrosis, marked bile duct hyperplasia, acute loss of appetite, wing weakness, and lethargy.

▶Oncoplastic Surgery

Characteristics

Definition

Afaxin Definition ▶Retinol.

Affinity-Matured IgG Response Definition Affinity is the strength of binding between a receptor, such as the antigen-binding site on an antibody and a ligand, as for example an epitope on an antigen. High titer, high affinity IgG antibodies are characteristic of an antigen-driven immune response ▶Autoantibodies

In the early 1960s, an outbreak of hepatotoxic disease in turkeys, which became known as turkey “X” disease, gained the attention of many investigators worldwide. This condition was characterized by acute hepatic necrosis, marked bile duct hyperplasia, acute loss of appetite, wing weakness, and lethargy. It was deduced that the condition was caused by consumption of peanut meal contaminated with a mycotoxin, which is a toxin of fungal origin. The culprit fungi in turkey “X” disease turned out to be strains of Aspergillus flavus, A. parasiticus, and A. nomius, and thus the term aflatoxins was coined for the toxic metabolites. More specifically, A. flavus and A. parasiticus can produce aflatoxins B1, B2, G1, G2, and M1. These mycotoxins can contaminate a number of agricultural products, including peanuts, corn, and other grains in warm and moist conditions. Human exposure to aflatoxins is primarily through ingestion. In addition to outbreaks of liver failure and gastrointestinal bleeding in Southeast Asia and Africa having been attributed to aflatoxins, ▶liver cancer incidence was observed to be elevated in regions with high endemic aflatoxin concentrations. The two major risk factors for human ▶hepatocellular carcinoma, the fifth most common cancer worldwide, are hepatitis B infection and ingestion of aflatoxins.

Aflatoxins

Aflatoxin B1 (AFB1), the most prevalent and carcinogenic of the aflatoxins, is classified as a group 1 carcinogen (carcinogenic to humans) by the International Agency for Research on Cancer. Although the majority of AFB1 research has focused on its hepatic effects, AFB1 also targets other organs, including the lung and the kidney. In the lung, exposure to inhaled AFB1, particularly from contaminated grain dusts, has been linked to ▶respiratory cancers (▶Lung cancer). Due to a significant proportion of ingested mycotoxin being excreted via the urine, the renal nephron is exposed to AFB1 and its metabolites. AFB1 accordingly alters kidney function, and is a known renal carcinogen. Biotransformation AFB1 is defined as a procarcinogen, as its bioactivation is required for carcinogenicity (Fig. 1). The initial

Aflatoxins. Figure 1 Biotransformation of AFB1.

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metabolism of AFB1 involves four types of reactions: O-dealkylation, hydroxylation, epoxidation, and ketoreduction. The enzymes responsible for the metabolism include members of the ▶cytochrome P450 family (CYPs), prostaglandin H synthase (PHS), lipoxygenase (LOX), and a cytosolic NADPH-dependent reductase. In experimental animals, CYPs involved in AFB1 bioactivation include members of 1A, 2B, 2C, and 3A subfamilies. In humans, there are multiple p450 isozymes implicated, including CYP1A2, CYP2A3, CYP2B7, CYP3A3, and CYP3A4. CYP3A4 is thought to play a predominant role in the metabolism of AFB1 in human liver; although CYP1A2 has the highest affinity for AFB1 at low concentrations, it is expressed at much lower levels than CYP3A4. PHS and LOX are involved in ▶xenobiotic bioactivation by catalyzing the oxidation of ▶arachidonic acid to produce lipid

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peroxyl radicals, which are known epoxidizing agents. Cooxidation by PHS and LOX may be a significant mechanism of AFB1 bioactivation in extrahepatic tissues such as lung, which has high PHS and LOX expression, but overall P450 activity is lower than that in the adult liver. Regardless of the enzyme catalyzing the reaction, epoxidation of AFB1 results in formation of AFB1-8,9epoxide, which can exist in both endo and exo conformations. The exo-epoxide is the isomer implicated in the ▶alkylation of DNA, with its reactivity being at least 1,000 fold greater than that of the endoepoxide. Hydroxylated metabolites of AFB1 include AFM1, AFQ1, AFP1, and AFB2a. The formation of aflatoxicol from AFB1 is reversible, and therefore aflatoxicol is considered to be a “reservoir” for AFB1 rather than a bioactivation or ▶detoxification product. The two pathways for AFB1-epoxide detoxification are glutathione conjugation and epoxide hydrolysis, with glutathione conjugation being quantitatively the most important (Fig. 1). Glutathione conjugation is catalyzed by ▶glutathione-S-transferases (GSTs), which can be highly polymorphic. Human GSTM1-1 (hGSTM1-1), which is absent in ~50% of individuals, has the highest activity towards AFB1 exo-epoxide, but the importance of this polymorphism in AFB1 carcinogenicity has not yet been clearly established. AFQ1, AFP1, and AFB2a are not highly mutagenic and therefore are considered to be detoxification products. They can form glucuronide or sulfate conjugates, which are excreted. AFM1, a metabolite of AFB1 identified in milk and urine, is less biologically active than AFB1, but regardless is a potent carcinogen. The AFM1-epoxide can also bind to DNA, forming AFM1-N7-guanine. Carcinogenesis AFB1 is considered to be a complete carcinogen, possessing activity as both an initiator and a promoter. Initiation occurs by ▶DNA damage, as well as cytotoxicity, which stimulates cell division, thus promoting tumor formation. There are many characteristics of AFB1 that makes it a useful tool for investigating ▶chemical carcinogenesis. First, the metabolites of AFB1 have been extensively investigated and their toxicity elucidated. Second, the toxicity of AFB1 is determined by a balance between bioactivation and detoxification of the AFB1-8,9-epoxide. Third, there exists multiple mechanisms of bioactivation that can be compared in terms of carcinogenic metabolites produced. Fourth, not only does the susceptibility of a species/tissue relate to ▶DNA repair capabilities (▶Repair of DNA), but AFB1 itself has effects on DNA repair activity. Fifth, the specific AFB1-DNA adduct formed can be used to predict the mutagenic responses. Finally, the parent compound and several metabolites fluoresce, facilitating detection.

The exo epoxide of AFB1 can alkylate proteins and ▶nucleic acids, with the second guanine from the 5′ end in guanine di- and trinucleotide sequences in DNA being the favored target. The major adduct formed by the exo-epoxide is 8,9-dihydro-8-(N7-guanyl)-9hydroxy AFB1, also known as AFB1-N7-Gua (Fig. 2). AFB1-N7-Gua can undergo three reactions: release of AFB1-8,9-dihydrodiol restoring guanine; depurination resulting in an apurinic site in DNA; and base-catalyzed hydrolysis to form the AFB1-formamidopyrimidine adduct (AFB1-FAPY). AFB1-FAPY, representing a significant proportion of AFB1 adducts in vivo, exists in equilibrium between two rotameric forms, designated AFB1-FAPY major and AFB1-FAPY minor. The structure of AFB1-FAPY has not been completely defined, although the proposed structure is presented in Fig. 2. It has also been shown that metabolism of AFB1 can lead to formation of 8-hydroxy-2′-deoxyguanosine in rat, duck, and woodchuck liver and in mouse lung. G to T transversion, the most frequently observed mutation induced by AFB1, results from DNA alkylation and subsequent AFB1-N7-Gua formation, and possibly by the ▶oxidative DNA damage as well. A proportion of mutations in DNA formed by AFB1 occurs at the base 5′ to the modified guanine, or even further away, due to helical distortion resulting from the AFB1 adduct. ▶P53, a ▶tumor suppressor gene considered the “guardian of the genome,” has controls on cell cycle, DNA repair, and ▶apoptosis. P53 is the most frequently targeted gene in human carcinogenesis, with a mutation frequency of 50% in most major cancers. In geographical regions with a high dietary exposure to AFB1, such as China and Sub-Saharan Africa, mutations in p53 have been implicated AFB1-induced human liver tumorigenesis. AFB1 produces mutations at the 3rd base of codon 249 in p53, causing a G→T transversion, and an amino acid substitution (arginine to serine), and thus a structural alteration of this tumor suppressor protein. This may result in deregulation of the cell cycle, and thus loss of tumor suppression by p53. The K-▶ras proto-▶oncogene, important in ▶signal transduction, is often implicated in human and mouse lung tumors. AFB1-induced point mutations at specific “hot spots” (e.g. codons 12 and 13) of the K-ras gene, which cause activation of the protein, occur in AFB1-induced mouse lung tumorigenesis and rat hepatocarcinogenesis. Repair In mammals, ▶nucleotide excision repair (NER) is important for protection against AFB1-induced carcinogenesis. NER is a DNA repair process that deals with a wide array of DNA helix-distorting lesions that affect normal base pairing, thus altering transcription and replication. In E. coli, NER is responsible for the repair of both AFB1-N7-Gua and AFB1-FAPY. In yeast, NER

Aflatoxins

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Aflatoxins. Figure 2 AFB1-exo-8,9-epoxide and DNA damage.

is also the main repair pathway, although ▶homologous recombination is also involved in the repair of AFB1induced damage. In mammals, NER is important in protection against AFB1-induced carcinogenesis. NER is the main repair mechanism for the AFB1-N7-Gua adduct. AFB1-FAPY is repaired less efficiently by mammalian NER than is AFB1-N7-Gua, an effect that is attributed to AFB1-FAPY being less distortive of DNA architecture. Apurinic sites generated by AFB1-DNA adduct formation are repaired by base excision repair (BER), although insertion of an incorrect base is a frequent occurrence. Species / Tissue Susceptibility Susceptibility to the toxic and carcinogenic effects of AFB1 varies between species, as well as between different tissue types. In humans, the liver is the main target for this toxin. In rat, duck, and trout, administration of AFB1 results in hepatocarcinogenesis, whereas this is not the case in the mouse, monkey, hamster, and mouse. The reason for this has been attributed to differences in AFB1 biotransformation and DNA repair. For example, the mouse is susceptible to pulmonary carcinogenesis by AFB1, regardless of the route of administration, but does

not develop hepatocarcinogenesis. The mouse liver expresses an alpha-class GST with high specific activity towards the exo-epoxide, and higher NER activity as compared to the rat liver. On the other hand, mouse lung has lower DNA repair activity than does liver. AFB1 is able to alter NER activity (by inhibition or elevation) in different animal species and organs, which may contribute to differential susceptibility to the mycotoxin’s carcinogenicity.

References 1. Bedard LL, Massey TE (2006) Aflatoxin B1-induced DNA damage and its repair. Cancer Lett 241(2):174–183 2. Eaton DL, Groopman JD (eds) (1994) The toxicology of aflatoxins: human health, veterinary, and agricultural significance. Academic Press, San Diego, pp3–148 3. Massey TE, Stewart RK, Daniels JM et al. (1995) Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B1 carcinogenicity. Proc Soc Exp Biol Med 208(3):213–227 4. Massey TE, Smith GBJ, Tam AS (2000) Mechanisms of aflatoxin B1 lung tumourigenesis. Exp Lung Res 26:673–683 5. Wogan GN (1973) Aflatoxin carcinogenesis. Meth Cancer Res 7:309–344

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Aflatoxin B1 Definition A potent liver carcinogen produced by fungi that infest cereal grains.

diabetic conditions. AGEs are involved in various types of disorders mainly through the interaction with its receptor RAGE. ▶Minodronate ▶Inflammation

▶Carcinogen Metabolism

Aggressive Fibromatosis AFP

▶Aggressive Fibromatosis in Children ▶Desmoid Tumor

▶Alpha-Fetoprotein ▶Alpha-Fetoprotein – Historical ▶Alpha-Fetoprotein – Modern

Aggressive Fibromatosis in Children AFP-L3

M ARRY M.

VAN DEN

H EUVEL -E IBRINK

Department of Pediatric Oncology/Hematology, ErasmusMC-Sophia Children’s Hospital, Rotterdam, The Netherlands

Definition An AFP fraction, which binds to LCA (lens culinaris agglutinin) lectin because this type of AFP has a core fucose on the N-glycan. ▶Fucosylation

Agammaglobulinemia Definition An almost total lack of immunoglobulins, or antibodies.

Synonyms Aggressive fibromatosis; Desmoid tumor

Definition

▶Aggressive fibromatosis (AF) is a rare soft tissue tumor and rare in childhood with high potential for local invasiveness and recurrence. Primary ▶surgery with ▶negative margins is the most successful primary treatment modality for ▶children with ▶AF. Positive resection margins after surgery indicate a high risk for ▶relapse. Multicenter prospective (randomized) trials are necessary to clarify the role of and best strategy for ▶treatment in pediatric AF after ▶incomplete surgery. For this purpose, ▶chemotherapy or alternatively ▶radiotherapy can be considered, each with its own potential side effects in consequence.

Characteristics

AGEs Definition

▶AGEs are advanced glycation end products. The formation and accumulation of these senescent macroprotein derivatives progress under inflammatory or

Aggressive fibromatosis (AF) (▶Supportive care) is a soft tissue tumor, which arises principally from the connective tissue of muscle and the overlying fascia (aponeurosis). The previously most used synonym is ▶desmoid tumor. The histological pattern is characterized by elongated fibroblast-like cells. Although AF is a nonmetastasizing tumor with benign histological features, it has a significant potential for local invasiveness (▶Invasion) and ▶recurrence. The overall incidence of

Aggressive Fibromatosis in Children

AF in children is 2–4 new diagnoses per 1 million a year. Childhood AF has an age distribution peak at approximately 8 years (range 0–19 years) with a slight male predominance. Clinical Presentation The typical clinical presentation of AF is a painless, slowly growing, deep-seated mass. Predilection sites are shoulder, chest wall and back, thigh, and head/neck. Children with AF of head/neck have shown to be to be younger at diagnosis than children with AF at other sites. From 1986 until 2004, ten pediatric AF case series reported a total of 206 patients. In 64 of the reviewed patients site of involvement and age at diagnosis were specified. The children with AF of head/neck had a median age of 3.6 years at diagnosis (range 0.2–9.9 years), whereas the children with AF of trunk/limb had a median age of 7.8 years (range 0.0–15.7 years) (p400 ng/ml are highly associated with HCC, not all HCC secrete AFP. It may also be elevated in patients with cirrhosis,

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viral hepatitis, drug or alcohol abuse as well as pregnancy, and may be used for screening of fetal spinal cord defects and placental disease. ▶Serum Biomarkers ▶Hepatocellular Carcinoma

Alpha-Fetoprotein – Historical K AREL K ITHIER Department of Pathology, Wayne State University School of Medicine, Detroit, MI, USA

Synonyms AFP; Tumor markers; Oncodevelopmental proteins; Carcinofetal proteins; Feto-specific proteins; Oncofetal antigens

Definition AFP or alpha-fetoprotein is a serum protein of mammalian fetuses that is hardly detectable in healthy adults. Its re-occurrence in serum of adults may often attest to specific malignancy especially in high-risk patients, such as those with hepatocellular carcinomas (▶hepatocellular carcinoma, ▶hepatoblastoma) and chronic hepatitis B or C virus infection (▶hepatitis associated hepatocellular carcinoma). It also serves in evaluation (▶serum biomarkers, ▶surrogate endpoints) of therapy and disease progress in patients with embryonal carcinomas (▶germ cell tumors, ▶platinum refractory testicular germ cell tumors).

Characteristics The studies of fetal serum proteins came from different corners: from researchers interested primarily in the development of proteins and from those studying proteins of tumor-bearing laboratory animals. These two groups were at the beginning not very aware of each other’s results. The fetal protein history began with the physicochemical and biochemical studies of serum proteins, which depended, as this often happens in the laboratory endeavor, on the development, improvement and refinement of laboratory methods. In the field of serum proteins, the electrophoretic and immunochemical techniques (▶proteomics) were of crucial importance, especially in the case of fetal proteins, where usually only minute volumes of sera were available. Studies of electrophoretic patterns of serum proteins in human fetuses showed some considerable differences when compared with the sera of adults. Thus, in 1956

Bergstrand and Czar, using filter paper electrophoresis reported on the special fetal band, (called substance X) which was located between albumin and alpha-1 globulins. Substance X was absent from maternal sera and from sera of healthy adults. Also, Halbrecht and Klibanski reported similar findings in the same year. The first immunochemical studies of the substance X were done by Muralt and by Masopust in 1961 and 1962, respectively. Using antisera to fetal serum proteins (rabbits were immunized with the human fetal sera), an additional precipitin line with alpha globulin mobility was observed on immunoelectrophoresis (IEP) of human fetal serum, however, it was not present in adult sera. This fetal component was called independently “alpha-foeto-proteine” by Muralt and “fetoprotein” by Masopust. These findings resembled older observations in large animals; in 1944 Pedersen studied bovine fetal sera by ultracentrifugation and found a distinct gradient, not present in sera of adult animals. The fraction was named fetuin. Thus, it was believed by some that the human fetoprotein was related to fetuin and the term “human fetuin” was used in some papers on human fetoprotein. Physicochemical properties of fetuin, which was found to be a typical glycoprotein, were studied by a number of workers; its physiological and pathologic properties attracted much less interest. Because fetuin and fetoprotein were present in higher concentrations in fetuses and undetectable in adults, they were sometimes called “feto-specific proteins.” Immunochemical Techniques For the detection of feto-specific proteins, the immunochemical techniques became the methods of choice in the 1960s. Antisera to these proteins were prepared by the immunization of animals, usually rabbits, with fetal sera. To obtain specific antisera to feto-specific proteins, the antisera were absorbed with the sera of adult men or animals. The absorbed antisera should contain only the antibodies directed to the feto-specific protein(s) of a given species. In some cases, the absorbed antisera showed two to three precipitin lines on IEP of fetal serum. Sometimes, in human fetal sera, two lines with the absorbed antiserum were observed. The line in alpha zone of IEP was that of human fetoprotein, the other line, in beta position, was sometimes incorrectly, without justification, called beta fetoprotein. The lines showed no antigenic relationship each to other. For this reason, the original term “fetoprotein” was changed to “alpha fetoprotein” and consequently the abbreviation of AFP came to life. The term “betafetoprotein” ceased to be used since the beta protein was later identified as fetal ferritin. AFP in Pathology In 1964, a study of a possible occurrence of AFP in sera of patients was started. The putative presence of AFP was

Alpha-Fetoprotein – Historical

tested by double radial immunodiffusion (Ouchterlony test). After hundreds of negative results, a patient was identified, who had a definitely detectable serum concentration of AFP. The diagnosis of this patient, confirmed histopathologically at the autopsy, was that of hepatocellular carcinoma. In 1966 and 1967, the occurrence of AFP in four children with a malignant growth of embryonic character was reported. One of them was a 5 year-old boy with embryonal cell carcinoma of the left testicle (▶testis cancer, ▶childhood cancer, ▶germinoma) and another patient was a 14 year-old girl with malignant teratoblastoma of the right ovary (▶ovarian cancer, ▶ovarian tumors during childhood and adolescens). Also, Abelev published in 1967 the finding of “alpha fetal globulin” in patients with embryonal testicular cancer. Several pediatric patients with non-cancerous liver diseases, such as infectious hepatitis and some unspecified hepathopathies were identified, who had detectable AFP serum levels. A highly sensitive technique, radioactive single radial immunodiffusion (employing the second, 125 Iodinelabeled antibodies to the primary antiAFP immunoglobulin fraction), enabled to quantify previously undetectable levels of AFP in various body fluids. By such means, AFP serum levels of patients with hepatocellular carcinomas were studied in a correlation with their individual histopathologic findings. A further increased sensitivity of AFP quantitation was facilitated by the development of a radioimmunoassay. This technique made the quantitation of AFP in healthy persons such as pregnant women a routine test in clinical laboratories. In the 1970s, a number of reviews on AFP were published along the studies of AFP physicochemical properties. The first studies on serum concentrations of AFP and their changes in the course of diseases were done in those years. Thus, the impact of the therapy could be evaluated and monitored in some malignancies. Fetuin versus AFP In the early years, AFP was considered by some investigators to be a protein similar to bovine fetuin and therefore called “human fetuin.” Fetuin was isolated from fetal calf serum and the antisera were prepared to fetuin, and to serum proteins of human and bovine fetuses. With the use of absorbed antiserum to calf serum, an additional protein component was detected in alpha zone of bovine fetal serum, which was not detectable in sera of adult animals. This component could be considered as a “bovine fetoprotein.” Antiserum to this protein did not react with isolated fetuin and conversely the specific antiserum to fetuin did not react in immunodiffusion experiments with the “bovine fetoprotein.” The protein was not detected in adult healthy animals; it was, however, found in sera of two, out of four, adult cows with hepatocellular carcinomas (▶comparative oncology). No antigenic relationship was observed in double

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radial immunodiffusion and the precipitin lines of fetuin and “bovine fetoprotein” crossed each other, showing thus the pattern of antigenic non-identity. AFP in laboratory Animals Rat sera were studied electrophoretically already in the 1950s. A “fetal” protein was detected by Beaton (1961) in the macroglobulin fraction of starch gel electrophoresis. This protein migrated as an alpha-2 globulin in electrophoretic media without molecular sieve effect (filter paper) and slowly in starch gel. Therefore, it was called “alpha-2 slow globulin.” The protein was found in sera of rat fetuses and newborns, as well as in pregnant rats, but not in healthy, non-pregnant adult rats. It was present, however, in sera of tumor-bearing rats and in animals with various inflammatory processes, e.g. with turpentine abscess. Another alpha globulin was found by Darcy in fetal rat sera; it was also present in sera of pregnant animals and adult rats with tumors and/or with inflammations. Protein was also detectable in much lower concentrations in healthy, non-pregnant rats. Wise in 1963, using two-dimensional electrophoresis (filter paper-starch gel) demonstrated in rat fetal sera special proteins, named “fetal postalbumins” (two electrophoretic bands), which were not present in sera of adult animals. Altogether, at least three fetal components were reported in rats. To address this question, rabbit antiserum directed to rat fetal serum proteins was prepared. The absorbed antiserum (with the serum proteins of adult, healthy, non-pregnant animals) did not react with sera of adult, healthy non-pregnant rats, or with the protein described by Darcy. It did react, however, with three different proteins on IEP of fetal rat sera; two of them located in alpha-2, and one in alpha-1 globulin zone. The antibody to the protein in alpha-2 zone could be absorbed with the serum from an adult rat with turpentine abscess. This protein was also detected immunochemically in extracted proteins from macroglobulin position in starch gel electrophoresis of fetal serum. The protein obviously corresponded to alpha-2 slow globulin of Beaton. The other precipitin line in alpha-2 globulin zone was stainable with lipid stains (Red Oil and Sudan Black B), and represented most probably a lipoprotein-esterase found by Stanislawski–Birencwajg in fetal rat serum. The precipitin line in alpha-1 zone, present in sera of fetuses, absent from sera of adult rats, either healthy or with the acute inflammation, was considered to be a typical feto-specific protein, probably related to human AFP. However, no cross- reaction was seen by immunodiffusion between human AFP and antiserum to rat fetal proteins. To prepare a monospecific antiserum to alpha Ft protein, it was important to remove the antibodies to alpha-2 slow globulin, e.g. by using sera of adult rats with some inflammatory pathology. In 1963, Abelev reported the finding of “embryonal alpha globulin” in serum of adult mouse with transplatable

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hepatoma; the globulin was also present in sera of fetal mice (▶mouse models). Much progress has been done since the early modest beginnings of AFP research. Presently, a review of AFP literature shows almost fifteen thousand papers related to the topic (▶AFP-modern).

References 1. Abelev GI (1971) Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Adv Cancer Res 14:295–358 2. Kithier K, Houstek J, Masopust J et al. (1966) Occurrence of a specific foetal protein in a primary liver carcinoma. Nature 212:414 3. Kithier K, Poulik MD (1972) Comparative studies of bovine alpha-fetoprotein and fetuin. Biochim Biophys Acta 278:505–516 4. Kithier K, Prokes J (1966) Fetal alpha-1 globulin of rat serum. Biochim Biophys Acta 127:390–399 5. Masopust J, Kithier K, Radl J et al. (1968) Occurrence of fetoprotein in patients with neoplasms and non-neoplastic diseases. Int J Cancer 3:364–373

Alpha-Fetoprotein – Modern DAVID E. K APLAN Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104

Synonyms AFP; Alpha1-fetoglobulin; Embryo-specific alphaglobulin; Embryonal serum alpha-globulin; Foetoprotein; Fetuin; Fetuin-A; α-feto-protein; α1-fetoglobulin; Embryo-specific α-globulin; Embryonal serum α-globulin

Definition Alpha-fetoprotein (AFP) is a 68.7 kDa plasma protein synthesized primarily by the fetal liver and embryonic yolk sac that is highly homologous with human albumin. Widely expressed in the fetal liver, AFP mRNA is downregulated in post-natal hepatocytes. Serum AFP levels are used clinically for detection, confirmation and follow-up of human ▶hepatocellular carcinoma (HCC) and non-seminomatous ▶germ cell tumors, although lack of sensitivity and specificity complicate its use.

Characteristics Alpha-fetoprotein (AFP) is a 590 amino-acid plasma protein that shares 40% amino acid and 40–44% nucleotide sequence homology with human serum albumin and is a member of the albumin gene superfamily. The AFP gene covers approximately

22 kB of DNA and has 15 exons and 14 introns. The human albumin gene lies 14.5 kB upstream to its AFP homologue. Regulation of AFP protein production occurs mainly at the transcriptional level. In human cells, the AFP enhancer region contains binding sites for several liver-enriched transcription factors (HNF1-4, C/EBP) which control tissue specific expression. Expression of AFP also appears to be positively regulated by NFκB, by steroids via retinoid X receptors as well as by interactions with extracellular matrix. AFP is normally expressed by villous trophoblasts in the human placenta during pregnancy and by fetal hepatoblasts. In fetal and newborn rats, AFP mRNA can be detected at low levels in the kidney, pancreas, heart and gastrointestinal tracts as well. In early postnatal life, AFP production is repressed in normal hepatocytes and silenced in non-hepatic parenchymal cells. The mechanisms for the repression or silencing of AFP expression have largely been characterized. In mice, an unlinked locus called alpha-fetoprotein regulator 1 (Afr1) on chromosome 15 appears to interact with the AFP promoter region; repression of Afr1 appears to be associated with postnatal repression of AFP expression. The AFP promoter may also interact with Ku inducing a hairpin tertiary structure that may abrogate HNF1 binding to the promoter. Postnatal repression of AFP expression in the liver has also been shown to be ▶p53- and ▶TGFβ1-dependent whereas genetic silencing primarily involves epigenetic mechanisms that concomitantly silence the upstream albumin gene. In the adult liver, AFP expression is present but repressed. In situ hybridization studies confirm the presence of minute quantities of AFP mRNA, but at levels generally below the sensitivity of immunohistochemical detection. In the setting of hepatocyte regeneration, e.g. ischemic injury, surgical resection, and chronic viral hepatitis, in ▶hepatoblastoma as well as in a subset of hepatocellular carcinoma (HCC) (and rarely ▶cholangiocarcinoma) AFP expression is de-repressed. AFP production also occurs in nonseminomatous germ cell tumors such as choriocarcinoma, mixed germ cell tumors and teratomas. In fetal and newborn rats, AFP mRNA can be detected at low levels in the kidney, pancreas, heart and gastrointestinal tracts as well. Rarely in adults, non-hepatic/non-germ cell malignancies such as ▶gastric cancer, pancreatic cancer, endometrial cancer, colon cancer, and ▶ovarian cancer are associated with loss of silencing of AFP expression. The critical activities of AFP in vivo remain poorly defined. Many cell types including vascular endothelium and T-cells express receptors for AFP. AFP administration in human cell lines has been associated with differential expression of FasL and ▶TRAIL relative to fas and ▶TRAIL receptor, leading to postulation of a role for AFP in escape from tumor immunosurveillance. AFP also appears to inhibit ▶TNF

Alternative Reading Frame

receptor 1-signalling-mediated tumor cell apoptosis. Paradoxically, some studies suggest a pro-apoptotic role for AFP in tumor cells lines via interactions with X-linked inhibitor of apoptosis protein (XIAP). Other studies postulate that AFP may mediate anti-inflammatory effects that suppress autoimmunity and anti-fetal immune responses during pregnancy, possibly via inhibition of CD4 T-cell proliferation. Serum AFP determinations has two main clinical uses. First, it is used to screen women during pregnancy for fetal developmental abnormalities. Second, AFP is used as a tumor marker for hepatocellular carcinoma (HCC) and non-seminomatous germ cell tumors. Serum AFP determinations have been used since the late 1960s to detect hepatocellular carcinoma despite limitations in its sensitivity and specificity. While AFP levels greater than 400 ng/ml are considered diagnostic of HCC, such elevations are rarely present. The sensitivity and specificity of AFP determinations also appears to be dependent on the underlying cause of liver disease that results in HCC development. Using a cutoff of 20 ng/ml, sensitivity ranges from 41% to 65% and specificity ranges from 80% to 94%. The role of serum AFP in screening programs for HCC in patients with cirrhosis remains controversial. It remains unclear if the addition of AFP determinations to routine imaging examinations, e.g. ultrasound every 6 months, provides any incremental benefit. Current guidelines from the United Network of Organ Sharing (UNOS) in the United States support the use of AFP levels greater than 400 ng/ml to confirm the presence of HCC when a hypervascular lesion on CT or MRI imaging is seen. Exception points may be petitioned from UNOS to provide the rare individual patients with AFP levels greater than 400 ng/ml but no visible tumor to increase the priority of such patients for liver transplantation. Several glycoforms (AFP-L1, -L2 and -L3) of AFP have been resolved based on differences in glycosylation groups. Lectin-reactive AFP (AFP-L3) in some studies has been associated with intrahepatic cholangiocarcinoma. In other studies a high percentage of total AFP made up of the L3 fraction has been associated with hepatocellular carcinomas. Measurement of specific glycoforms is not in routine clinical use.

Alpha-Particles Definition Helium nuclei produced as radioactive-decay products.

Alpha-SMA Definition Alpha-smooth muscle actin. ▶Stromagenesis

Alternative Lengthening of Telomeres (ALT) Definition Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism that does not involve telomerase, which probably involves recombination. It is found in a minority of cancers and immortalized cell lines. A minority of immortalized cell lines and cancers have no detectable telomerase activity and maintain their telomeres by an alternative mechanism. Although the details are not yet known, it is likely to be a recombinational mechanism in which one telomere uses another telomere (or itself via looping back) as a template for synthesis of new telomeric DNA. Cells that maintain their telomeres by ALT characteristically have very heterogeneous telomere lengths, ranging from undetectable to extremely long. ▶Senescence and Immortalization ▶Stem Cell Telomeres

References 1. Nahon JL (1987) The regulation of albumin and αfetoprotein gene expression in mammals. Biochimie 69:445–459 2. Abelev GI, Eraiser TL (1999) Cellular aspects of alpha-fetoprotein reexpression in tumors. Cancer Biol 9:95–107 3. Gupta S, Bent S, Kohlwes J (2003) Test characteristics of α-fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C. Ann Intern Med 139:46–50

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Alternative Reading Frame ▶ARF Tumor Suppressor Protein

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Alternative RNA Splicing

Alternative RNA Splicing Definition Alternative RNA splicing refers to the synthesis of different RNA molecules from a single product by differential usage of splicing junctions, which are the sequences surrounding the exon–intron boundaries. ▶Progestin

Alu Elements C HRISTINE M. M ORRIS Cancer Genetics Research Group, University of Otago at Christchurch, Christchurch, New Zealand

Definition The most abundant class of dispersed repeat elements in the human genome, and one member of the family of short interspersed repeat elements (SINEs). An estimated one million copies comprise about 10% of DNA in human cells.

Characteristics Structure Alu elements are 280 bp in length, and consist of two similar monomers that have homology to, and were originally derived from, the ▶7SL RNA gene (Fig. 1). Individual Alu elements are flanked by direct repeats, end in a 3′ A-rich tract, and the left monomer contains an internal RNA polymerase III promoter which directs

transcription initiation to the first residue of the element. Alu are ▶retrotransposable elements, and several subfamilies, mobilized from different “source” genes at different evolutionary times, can be recognized on the basis of their sequence divergence and diagnostic bases. Some of the more recently integrated subfamilies are polymorphic, occupying regions on some chromosomes that are not occupied at the same locus on others. Function The function of Alu elements has been subject to intense investigation, debate and speculation over the past two decades. Proposed roles include modulation of chromosome structure and packaging of DNA around ▶nucleosomes, initiation or switch sites for DNA replication, regulation of gene transcription through Alu-specific protein binding domains, RNA editing as preferential templates for adenosine-to inosine (A-to-I) substitution by the ▶ADAR family of enzymes, and regulation of translation by RNA transcribed from Alu elements. Although Alu expression increases in cells stressed by chemical agents or viral infection, most human Alu repeats are silent in somatic cells, with only the minor, evolutionarily younger subgroups actively transcribed. Consistent with the latter observations, CpG sites in the majority of Alu sequences are normally fully methylated in most somatic cell types, a state which is considered to suppress expression and therefore transposition. However, Alus located adjacent to CpG islands show sequence features amenable to an unmethylated state, and which may therefore have increased mobility. Alu elements are also differently methylated in male and female germ cells, with at least a subset of the recently integrated Alus being almost completely unmethylated in sperm DNA. Role in Human Cancer Alu-mediated gene rearrangement underlies several important constitutional diseases, including familial

Alu Elements. Figure 1 Alu elements have a dimeric structure that originated from 7SL RNA. Colored areas show 7SL sequences present in the Alu repeat consensus.

AME Transcription Factor

cancers. Different mechanisms for these rearrangements include recombination between homologous or non-homologous regions of Alu elements at different locations within a gene, or on the same or different chromosomes, expansion of 3′ polynucleotide tracts to form fragile sites, or disruption of coding regions of functional genes by transpositional insertion of actively transcribed Alu elements. Instability of 3′ polynucleotide tracts may also indicate a DNA ▶mismatch repair deficiency. Because of their high density in the human genome, non-random chromosomal distribution and the high degree of homology between individual elements, Alu repeats are also recognized candidates to mediate somatically acquired gene rearrangements with neoplastic potential. Specific underlying mechanisms for involvement of Alu in somatic rearrangements have only recently begun to be explored, with possibilities including promotion of DNA exchange by sequences within Alu that share homology with known recombinogenic ▶translin DNA-binding motifs or the ▶χ-like Alu core sequence, preferential recombination between DNA regions that are localized within Alu-rich clusters on the same or different chromosomes, or otherwise unknown features of individual Alu elements that predispose to recurrent recombination events associated with some ▶breakpoint cluster regions.

References 1. Hasler J, Katharin S (2006) Alu elements as regulators of gene expression. Nucleic Acids Res 34:5491–5497 2. Kolomietz E, Meyn MS, Pandita A et al. (2002) The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumours. Genes Chromosomes Cancer 35:97–112 3. Deininger PL, Batzer MA (1999) Alu repeats and human disease. Mol Genet Metab 67:183–193 4. Schmid CW (1998) Does SINE evolution preclude Alu function? Nucleic Acids Res 26:4541–4550 5. Schmid CW (1996) Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. Prog Nucl Acid Res Mol Biol 53:283–319

ALVAC-CEA Definition A cancer vaccine constructed from canary pox virus (ALVAC) and combined with the human carcinoembryonic antigen (CEA) gene. ▶Carcinoembryonic Antigen (CEA)

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Alveolar Soft Part Sarcoma Definition Rare tumor of young people comprised of large epithelioid acidophilic cells arranged in alveolar structures separated by thin vessels. The cells show light atypia and contain crystalline inclusions. There are not specific markers. ▶Uncertain or Unknown Histogenesis Tumors

AMAP1 Definition A regulator of the small GTPase Arf6, which is involved in actin cytoskeletal remodeling. AMAP1 is overexpressed in invasive carcinomas and functions in invadopodia by binding to paxillin and ▶cortactin.

AME Transcription Factor V ITALYI S ENYUK Department of Medicine (M/C 737), College of Medicine Research Building, University of Illinois at Chicago, Chicago, IL, USA

Synonyms RUNX1/MDS1/EVI1; AML1/EVI-1

Definition AME is an aggressive oncoprotein (chimeric transcription factor) associated with several types of ▶acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative disorders (MPD).

Characteristics The legendary discovery of chromosomal translocations by Janet D. Rowley in 1972 has revolutionized leukemia research and therapy by allowing biological interrogation and classification of these disorders. Several recurring translocations have been identified and the participating genes cloned and characterized at the molecular level. One such recurring abnormality is the balanced translocation between the long arms of chromosomes 3 and 21, t(3;21)(q26;q22), originally discovered in a patient with

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therapy-related chronic myelogenous leukemia (CML) which is classified as an MPD. The t(3;21) is a complicated chromosomal rearrangement that employs a mechanism of intergenic splicing to generate several ▶fusion genes of which AME is perhaps the best characterized and the most important. Among the less frequent translocations involving RUNX1 (also known as AML1, CBFA2, and PEBP2) AME is the only fusion gene that has been cloned and characterized at the molecular level. AME, obtained by in frame fusion of the truncated RUNX1 and MDS1/ EVI1 (ME) genes, is controlled by the RUNX1 promoter which becomes active during the execution of multiple steps of hematopoietic program, especially during the development of myeloid lineage. The t(3;21) is a relatively rare translocation infrequently seen in de novo leukemias. It was observed in ~1% of AML, MDS and MPD cases, and often associated with secondary leukemia that arises in patients previously treated with ▶alkylating agents or topoisomerase inhibitors for other malignancies. In particular, the t(3;21) was detected in the patients after administration of cytostatic drugs such as busulphan, teniposide, etoposide, hydroxyurea, ▶fludarabine, ▶5-fluorouracil and others. There is no unique clinical picture of t(3;21)-associated leukemias such as restriction to a certain FAB (French-American-British classification) category and it has been classified as M1, M2, M4 and M7 subtypes. The common morphologic feature of t (3;21)-positive AML is minimally differentiated blasts with prominent nucleoli and scant cytoplasm. There is no age or gender specificity for t(3;21)-associated diseases but, as for many other ▶cancers, older individuals are at higher risk. In contrast to many other translocations, the t (3;21) causes a very aggressive myeloid leukemia/▶blast crisis of CML characterized by a low response to the existing therapeutic treatments and a poor prognosis. In the largest clinical investigation of t(3;21) patients published to date, the majority of AML patients died between 1 week and 8.5 months (median 2 months) after presentation whereas MPD patients survived 1–21 months (median 6.5 months) after presentation. RUNX1 is a DNA-binding subunit of the transcription factor CBF which is essential for hematopoiesis

and is involved in several chromosomal abnormalities associated with human leukemias. RUNX1 consists of an N-terminal DNA-binding domain called Runt with homology to the product of the Drosophila segmentation gene Runt and a C-terminal activation domain. ME is a zinc-finger transcription factor related to the leukemia-associated protein ecotopic viral integration 1 site (EVI1) of unknown function. ME contains a conserved N-terminal region, called PR domain, two sets of DNA-binding zinc finger domains, a proline-rich central domain, and an acidic C-terminal domain. AME consists of the DNA-binding domain Runt of RUNX1 fused to almost the entire ME (Fig. 1). Forced expression of AME upregulates the cell cycle and blocks granulocytic differentiation of the murine hematopoietic cell line 32Dcl3, and delays the myeloid differentiation of normal murine bone marrow progenitors in vitro. The exact mechanisms of AME oncogenic activation are unknown and several possibilities exist. Also, as with many other oncoproteins, most probably AME alone is insufficient to transform a healthy normal cell into leukemic one and additional cooperating genetic abnormalities are necessary. It has been shown that the majority of AME-positive patients have, in addition to t(3;21), several other chromosomal abnormalities readily detected by cytogenetic analysis: translocations, deletions and duplications the most common of which is t(9;22)(q34;q11) found in CML patients. One of the first investigated properties of AME was its effect on a subset of target promoters regulated by both parent proteins. RUNX1 is generally considered a transcription activator through its C-terminus, which interacts with several transcription coregulators and regulates critical genes in hematopoiesis. ME is also considered a transactivator, and both parent proteins act as antagonists of AME. Therefore, it was suggested that AME could act as a bifunctional transcription factor possessing the ability to bind to and repress/deregulate both the RUNX1- and MEdependent promoters. In support of this hypothesis, it was shown that AME directly interacts with the corepressors C-terminal binding protein (CtBP) and ▶histone deacetylase 1 (HDAC1) which are often a

AME Transcription Factor. Figure 1 Diagram of ME, RUNX1 and the fusion protein AME. The Runt, PR and two zinc finger (ZnF) domains are shown. The vertical dashed line indicates the breakpoint fusion.


part of big repressor complexes transiently formed at the promoter sites. AME has distinct regions for HDAC1 and CtBP binding and, taking in consideration that both corepressors are able to dimerize and interact to each other, one AME molecule can recruit several molecules of the corepressors. AME represses the target promoters by CtBP-dependent and CtBPindependent mechanisms, probably reflecting the dual nature of this protein. In vitro CtBP enhances not only AME repression potential but also the ability of AME to upregulate growth and deregulate differentiation in murine hematopoietic cells, suggesting that AME repression is necessary for its oncogenic activity. However, the transcription properties of AME are more complicated because it also interacts with histone acetyltransferases p300/CBP-associated factor (P/CAF) and general control of amino-acid synthesis 5-like (GCN5), which are generally considered as co-activators of transcription. Both P/CAF and GCN5 efficiently acetylate the central region of AME in vivo, but the function of this modification and its role in oncogenesis are still unknown. Similar to many other fusion proteins that are activated by chromosomal translocations in human leukemia, AME is able to oligomerize and displays a complex pattern of self-interaction that involves at least three oligomerization regions, which are the proximal and the distal zinc finger domains and the Runt domain. The distal zinc finger domain is quite important in AME oligomerization because it mediates the interaction with the other two domains and an internal deletion that removes the three zinc finger motifs virtually sufficient to repair (though not completely) the self-renewal and differentiation programs of normal murine bone marrow progenitors in vitro. In vitro, this domain efficiently cooperates with CtBP in disrupting normal hematopoiesis and the internal deletion and a point mutation that abolishes CtBP binding re-establishes almost completely the hematopoietic differentiation in murine cells. Probably AME belongs to a growing group of chimeric transcription factors which inappropriately maintain high local concentration of corepressors at the specific promoter sites because of their ability to oligomerize, resulting in the deregulation of genes involved in differentiation, ▶apoptosis, and proliferation. It is highly possible that the aggressiveness of AME as an oncoprotein is in part mediated by AME ability to abrogate the growth-inhibitory effect of ▶transforming growth factor β (TGF-β) that controls cell expansion and inhibits proliferation of different cell types. The repression of TGF-beta signaling depends on the ability of the proximal zinc finger of AME directly interact with and repress ▶Smad3, an intracellular mediator of TGF-β signaling. It should be noted that in contrast to AME, ME co-operates with TGF-β and increases the sensitivity of hematopoietic cells to its stimulus.

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AME is also indirectly involved in deregulation of the hematopoietic program. It has been shown that CCAAT/enhancer-binding protein α (C/EBPα), a crucial transcription factor for normal granulopoiesis, is suppressed at translation level by more than 90% in AME-expressing U937 cells. In AML patients harboring t(3;21) the C/EBPα level is reduced even more whereas in AML patients without the t(3;21) C/EBPα is not affected. The mRNA levels remain unchanged in both cases indicating that AME does not affect C/EBPα transcription. Most probably AME acts through an intermediate effector, ▶calreticulin, a ubiquitous multifunctional calcium-binding protein, which expression is strongly correlated with both, AME expression and C/EBPα suppression. It has been shown in reporter gene assays and in Rat1 fibroblasts that AME stimulates activator protein 1 (▶AP-1) activity with dependence on the distal zinc finger domain. AP-1 activation may increase cell proliferation potentially contributing to AME oncogenic properties. A ▶mouse model of AME-positive leukemia, generated by bone marrow transplantation of AMEexpressing cells using BALB/c mice, showed that AME induces acute myeloid leukemia with a latency of 5–13 months indicating that additional genetic abnormalities are necessary for leukemogenesis. The disease was clonal in origin and resembled human acute myelomonocytic leukemia (AML FAB-M4). It has been also shown in this model that AME efficiently co-operates with breakpoint cluster region/abelson tyrosine kinase (▶BCR/ABL), a product of t(9;22) frequently seen in CML patients. Both proteins together are able to block myeloid differentiation during the pre-leukemia stage and induce AML within 1–4 months. The second mouse model for AME utilized bone marrow infection and transplantation using C57BL/6 mice. The animals displayed a variety of clinical features that are observed in essential thrombocythemia (ET) that resulted in their death after 8–16 months. The molecular etiology of ET, which is classified as an MPD, is poorly understood. Recently an activating somatic point mutation (V617F) of Janus kinase 2 (Jak2) was identified in MPD patients. Nonetheless, this mutation was not detected in ~50% of ET patients, indicating that some other molecular mechanisms exist and t(3;21) could be one of them. The differences between these two mouse models can be explained by taking into consideration that the BALB/c strain of mice is well known to have a higher tumor incidence as compared with C57BL/6 mice (because it has a mutated inhibitor of Cdk4/alternative reading frame (INK4a/ARF) locus that at least partially disables p16Ink4a, a ▶tumor suppressor protein which is frequently mutated in many cancers). A mouse model of AME knock-in has been also reported. The heterozygous mutant embryos obtained

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Amenorrhea

by breeding of AME chimeric male (ICR strain) and wild type female (C57BL/6 strain) were not viable and died of fetal liver hematopoiesis failure at around day 13.5E. Fetal liver hematopoietic progenitor cells from these mice displayed increased self-renewal capacity and impaired erythropoiesis. In addition, myeloid and megakaryocytic cells appeared dysplastic indicating that AME induces multiple defects in several myeloid lineages. Interestingly, the majority of AME chimeric mice demonstrated sudden death at the age of about 7 months without any significant signs of any disease whereas one of them developed a disease resembling megakaryoblastic leukemia at 5 months of age. Since 1987, when the t(3;21) was described for the first time, our knowledge about AME has increased vastly, however the prognosis of patients with this abnormality is still extremely poor. Hopefully, the cumulative efforts of different research groups will provide new approaches for the search of a treatment for this selected group of patients.

altered so that they are sensitive to mutagenic damage. Bacterial cell death due to mutagen is the assay readout. ▶ADMET Screen

Amidation Definition A process in which glycoxylate is removed from a precursor peptide (glycine-extended form) and an α-amide group is added. In cells this process is catalyzed by the amidation enzyme complex peptidylglycine α-amidating monooxygenase (PAM). ▶Adrenomedullin

References 1. Nucifora G, Rowley JD (1995) AMLl and the 8;21 and 3;21 translocations in acute and chronic myeloid leukemia. Blood 86:1–14 2. Nucifora G, Laricchia-Robbio L, Senyuk V (2006) EVI1 and hematopoietic disorders: history and perspectives. Gene 368:1–11 3. Rubin CM, Larson RA, Bitter MA et al. (1987) Association of a chromosomal 3;21 translocation with the blast phase of chronic myelogenous leukemia. Blood 70:1338–1342 4. Yin CC, Cortes J, Barkoh B et al. (2006) t(3;21)(q26;q22) in myeloid leukemia. Cancer 106:1730–1738

Amifostine Definition

A cytoprotective ▶adjuvant used in cancer chemotherapy involving DNA-binding chemotherapeutic agents. It is also used to decrease the cumulative nephrotoxicity associated with cisplatin and cyclophosphamide. ▶Chemoprotectants

Amenorrhea Definition Absence or cessation of menstruation. ▶Granulosa Cell Tumors ▶Prolactin

Ames Assay Definition Ames assay comprises a family of widely used bacterial assays to screen for mutagenicity. Bacteria are genetically

Amine Oxidases B RUNO M ONDOVI` 1 , PAOLA P IETRANGELI 1 , LUCIA M ARCOCCI 1 , A NTONIO T ONINELLO 2

Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2 Department of Biological Chemistry, University of Padua, Italy 1

Definition Amine oxidases (AOs) are a class of enzymes which is heterogeneous in terms of structure, catalytic mechanisms, and substrate specificity. ▶Biogenic amines, mono, di, and polyamines as well as N-acetyl amines, are oxidatively deaminated by AOs in a reaction that consumes O2 to produce the corresponding aldehydes,

Amine Oxidases

ammonium ions, and ▶hydrogen peroxide (H2O2) according to the following reaction: þ Eox þ R CH2 NHþ 3 ! Ered NH3 þ RCHO þ Ered NHþ 3 þ O2 ! Eox þ NH4 þ H2 O2

(Eox = oxidized enzyme Ered = reduced enzyme)

Characteristics Two classes of AOs can be described, which contain different prosthetic groups: the FAD-dependent AOs (FAD-AOs) containing the flavin adenin dinucleotide (FAD), and the copper-dependent AOs (Cu-AOs) containing copper and an organic cofactor produced by the copper self-catalyzed posttranslational oxidation of a tyrosine residue, i.e., TPQ (trihydroxyphenylalanine quinone). The FAD-AOs are subdivided in monoamine oxidase A and B (MAO A, MAO B), polyamine oxidase (PAO) and the recently discovered spermine oxidase (SMO). The two latter enzymes are cytosolic, catalyze the oxidation of secondary amino groups, and participate in the interconversion metabolism of polyamines. MAOs are tightly bound to a component of the mitochondrial outer membranes. Cu-AOs are often also named SSAO (semicarbazide sensitive amine oxidase) because of their inhibition by semicarbazide, which binds the organic cofactor. When strictly necessary, the name of the best substrate is used to characterize the enzymes. For instance, Cu-AOs, which oxidize diamine, histamine, and elastin, are named diamine oxidase (DAO), histaminase, and lysyl oxidase (LXAO), respectively. LXAO contains, instead of TOPA quinone, lysyl tyrosyl quinone. Sometimes, a single enzyme, such as the enzyme purified from pig kidney, may display both DAO and histaminase activities, so that the name may not imply a specific enzyme. The X-ray structure is available for several Cu-AOs, PAO, and MAO B. Functions A plethora of physiological functions, sometimes in contrast with one another, is ascribed to AOs. Although the exact molecular mechanism of their biological activity is not well-defined, a role of these enzymes in various processes through the action of either substrates or reaction products is postulated. Evidences have accumulated on the physiopathological relevance of polyamines, histamine, hydrogen peroxide, and aldehydes in cell death and ▶differentiation, ▶allergic diseases, and ▶postischemic reperfusion damage. Histamine is considered to be a main factor involved in allergic diseases. A plant Cu-AO, showing high histaminase activity, counteracts acute allergic asthma-like reaction in actively sensitized guinea pigs. The same

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enzyme modulates cardiac anaphylactic response in guinea pig. Protective effects of the plant enzyme were also observed in myocardial ischemia and reperfusion injury in in vivo rats. Bovine serum Cu-AO was shown to present an antioxidant effect, in vitro, against electrolytically induced ▶reactive oxygen species (ROS) ðO 2; 1 OH; O2 Þ . Among other physiopathological functions ascribed to AOs are, for example, the involvement of MAO in psychiatric diseases like schizophrenia, by regulating the dopamine metabolism, and of Cu-AOs in cataract, by the lens damaging effect of amine oxidation products. A primary involvement of AOs was demonstrated in cancer growth inhibition and progression, especially by means of aldehydes, H2O2, and other ROS, the AOsmediated products of biogenic amines oxidation. Aminoaldehydes were shown to interact with nucleotides or with DNA. Microinjection of Cu-AO into chick embryo fibroblast, rat cells, and glioma cells caused the inhibition of ▶DNA damage and protein synthesis. Tumor cells, with higher polyamines content than the normal controls, were more sensitive to the injected AOs. When an immobilized Cu-AO was injected into the peritoneal cavity of Swiss mice, 24 h after viable Ehrlich ascites tumor cells (▶ascites) transplantation or into a mouse (▶melanoma) model, a strong inhibition of tumor growth was observed. An induction of tumors in rat bowels (▶colon cancer) was observed on inhibition of DAO by aminoguanidine. An induction of tumors in rats was observed after carcinogenic treatment combined with PAO inhibition. Both H2O2 and aldehydes contribute to cytotoxicity, as demonstrated by incubation of Chinese hamster ovary cells with purified bovine serum AO in the presence of spermine. Catalase, the enzyme involved in H2O2 elimination, is absent in many tumor cells and thus ▶apoptosis occurs. The direct relationship between AOs, apoptosis, and cancer appears to be related to the regulation of biogenic amines and their metabolic products. H2O2 is considered to be a mediator of apoptotic cell death but the mechanism is unclear. H2O2 produced by MAO-catalyzed monoamines oxidation seems extremely important for apoptosis induction by considering the fact that MAO inhibitors are able to prevent apoptosis in human melanoma cells and that catalase inhibits the apoptosis induced by polyamines or their analogs and cathecolamines. The catalytic products of active amine oxidation are strong inducers of mitochondrial ▶membrane permeability transition (MPT). Taken together, these results indicate that active amines, operating as AO substrates, play a critical role in controlling apoptosis through their effects on MPT and the ▶respiratory chain activity by means of fluctuations in their concentrations. The conclusions of the above results may be that apoptosis is induced by polyamines through their oxidation products. Other studies exist demonstrating instead the ability of polyamines to protect

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D-Amino Acid

cells from apoptosis. This discrepancy can be explained by taking into account the protective effect of the same polyamines, probably due to a scavenging action of ROS. A crucial role of AOs in cancer promotion has also to be considered. High levels of DAO activity were occasionally found in rapidly growing tissues, while in some patients, even affected by metastatic tumors, the level of circulating DAO was unaltered. A strong correlation between serum AO activity and the factor responsible for ▶angiogenesis was recently found in non-small cell lung cancer patients. DAO activity in the small intestine mucosa was reported to increase in parallel with the degree of cell maturation, being highest in differentiated villus tip cells and lowest in the proliferative compartment. It was also found to increase in regenerating rat liver, with a peak between 16 and 48 h after partial hepatectomy. DAO activity peaks at the outset of growth and falls during the logarithmic growth phase of the cells. An increasing degree of malignancy associated with an increase of MAO A activity and decrease of MAO B and Cu-AOs activities in chemically-induced mammary cancer in the rat has been observed. Elevated activity of AO was found in skeletal metastases of prostatic cancer (▶Prostate cancer, clinical oncology). DAO and arginase, an enzyme that catalyses the synthesis of ornithine from arginine, increase in tumor tissues as compared with benign prostatic hyperplasia. A linear correlation between arginase and DAO activities was observed in patients with cancer. A high concentration of PAO and DAO was found in the cervical intraepithelial neoplasia. The rise from normal conditions seems to produce cytological changes and to play a role in the aethiology of ▶cervical cancer. DAO activity is present at high levels both in tumor tissues and in biological fluids of tumorbearing subjects. A correlation between the degree of tumor malignancy and their levels of AO activity has been observed in astrocytomas, where the activity is proportional to the degree of malignancy. The oxidation products of biogenic amines should also be carcinogenic. Acrolein, produced from the oxidation of spermine and spermidine by AOs, appears to be both carcinogenic and cytotoxic. This compound is considered to be a component of a universal cell growth regulatory system. It may act as mediator of cell transformation under oxidative stress when cells are pretreated with benzopyrene, a major carcinogenic found in cigarette smoke. The oxidation products of spermine, spermidine, and putrescine should be cofactors in the development of cervical cancer. The balance between the cell content of biogenic amine oxidizing enzymes and antioxidizing enzymes appears to be a crucial point for cancer inhibition or progression. As a general conclusion, the cancer inhibition/promotion effect of AOs might be explained by taking into consideration the full pattern of the

enzymes contained in the cells. A long-lasting imbalance of antioxidizing enzymes and AOs activity may be carcinogenic, while AOs are rapidly cytotoxic for cancer cells, because of their higher biogenic amines concentration in comparison with normal cells.

References 1. Toninello A, Pietrangeli P, De Marchi U et al. (2006) Amine oxidases in apoptosis and cancer. Biochim Biophys Acta 1765:1–13 2. Bachrach U, Eilon G (1967) Interaction of oxidized polyamine with DNA. I. Evidence of the formation of cross-links. Biochim Biophys Acta 145:418–426 3. Mondovì B (ed) (1985) Structure and functions of amine oxidases. CRC Press, Boca Raton, FL

D-Amino Acid Definition Is an optical isomer that is the mirror-image of the corresponding L amino acid. Only L amino acids are constituents of natural proteins. ▶Lactoferricin Antiangiogenesis Inhibitor

Amino Terminal End Definition The amino terminus (N-terminus) is the end of a polypeptide chain that carries an unreacted amino group. A ribosome synthesizes a polypeptide in the direction from the amino terminal end to the carboxyl terminal end. ▶AAMP

Amino-Bisphosphonate ▶Minodronate

AML1/MTG8

Aminoflavone Definition 5-Amino-2,3-fluorophenyl-6,8-difluoro-7-methyl-4H1-benzopyran-4-one. New cytostatic drug entering clinical trials. It requires bioactivation by ▶cytochromes P450 and sulfotransferases (SULTs). Its growth-inhibiting activity in 60 human tumor cell lines was primarily determined by the level of expression of SULT1A1. ▶Sulfotransferases

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AML1 ▶Runx1

AML1/ETO ▶Chromosomal Translocation t(8;21)

5-Aminolevulinic Acid Definition Is the precursor of protoporphyrin IX in the heme synthetic pathway. It is used as a photodiagnostic agent. ▶Fluorescence Diagnostics

d-Aminolevulinic Acid Definition A member of the heme biosynthesis pathway and a precursor of the photosensitizer protoporphyrin IX (PpIX). PpIX is the last reaction step before heme and its photochemical and fluorescent properties are used in photodynamic therapy/▶fluorescence diagnosis for treatment/diagnosis of malignant and nonmalignant diseases. ▶Photodynamic Therapy

AML-1/ETO/CBFb/TEL in Chromosomal Translocations Definition AML-1 has been renamed RUNX1. ETO is also known as MTG8 and is part of the RUNX1-MTG8 translocation, previously known as AML1-ETO. CBFβ binds RUNX1. TEL is also known as ETV6 and occurs in the t(12;21) translocation with RUNX1. ▶RUNX1 ▶Chromosome Translocation ▶Acute Myecloid Leukemia

AML1/EVI-1 ▶AME Transcription Factor

AML Definition

▶Acute myeloid leukemia. ▶ETV6 ▶Childhood Cancer

AML1/MTG8 ▶Chromosomal Translocation t(8;21)

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AMN107

AMN107 ▶Nilotinib

Amosite Definition

Is an amphibole form of ▶asbestos. The term refers to Asbestos Mines of South Africa. This mineral contains 31% iron; when inhaled, it is highly carcinogenic.

Amphiphysin-like ▶Bin1

Amphiregulin M AT I A S A. AVI LA , C AR M EN B ERASAIN Division of Hepatology and Gene Therapy, CIMA, University of Navarra, Pamplona, Spain

Synonyms Schwannoma-derived growth factor; SDGF

Definition

Amph II ▶Bin1

Amphibian Gastrin-Releasing Peptide

Amphiregulin (AR) is a growth factor that belongs to the ▶epidermal growth factor receptor (EGFR) family of ligands. AR was originally described as a regulator of cell growth present in the conditioned media of MCF-7 breast tumor cells. AR has been implicated in different physiologic processes including mammary gland and bone development, lung and kidney branching morphogenesis, and trophoblast growth. The expression of AR is upregulated in a variety of cancerous tissues, and signaling triggered by AR is believed to be important in tumorigenesis.

Characteristics ▶Gastrin-Releasing Peptide

Amphipathic Definition Amphipathic molecules contain both a hydrophilic and a hydrophobic moiety.

Amphiphysin II ▶Bin1

The AR human gene spans 10 kb in the genomic DNA and it is composed of six exons, upon transcription it produces a 1.4 kb mRNA. AR gene shows broad constitutive expression, being more prevalent in human ovary and placenta although it is also expressed in pancreas, cardiac muscle, testis, colon, breast, lung, spleen, and kidney, whereas it is undetectable in liver. Transactivation of AR promoter and AR gene expression can be induced by the ▶Wilms’ tumor suppressor and through the activation of the ▶protein kinase c (PKC), mitogen associated protein kinase (▶MAPK), and ▶cyclic AMP/protein kinase A (cAMP/PKA) pathways (Fig. 1). AR is synthesized as a 252-amino acid transmembrane glycoprotein, also known as transmembrane precursor or pro-form (Pro-AR) (Fig. 1). Pro-AR consists of a hydrophilic extracellular N-terminus (or ectodomain), a hydrophobic transmembrane domain (TM), and a hydrophilic cytoplasmic C-terminus (CT-tail) (Fig. 1). In the extracellular N-terminus we can distinguish an N-terminal pro-region containing glycosylation sites followed by a heparin-binding domain and an EGF-like region (Fig. 1). The EGF-like region is

Amphiregulin

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Amphiregulin. Figure 1 Transcription of the AR gene can be activated in response to the WT1 protein and the PKC, cAMP/PKA or MAPK signaling pathways. AR is synthesized as a membrane-anchored precursor (Pro-AR) encompassing an EGF-like domain, a heparin-binding domain (HB), a transmembrane region, and a carboxy-terminal cytosolic tail (CT-tail). Upon digestion by the protease TACE/ADAM17, soluble AR forms are shed from the cell surface and can interact with the EGFR in an autocrine or paracrine fashion, or bind to heparan-sulfate proteoglycans (HSPG) in the extracellular millieu. Alternatively, the yuxtacrine interaction of membrane-anchored Pro-AR with the EGFR is also possible. Shedding of AR by TACE/ADAM17 can be enhanced in response to activation of ▶G-protein coupled receptors (GPCRs). Binding and activation of the EGFR by AR triggers growth and survival signals essential for the tumor cell.

shared by other members of the ▶EGF family of ligands. At the plasma membrane Pro-AR undergoes proteolytic cleavage to release the mature soluble factor in a process known as “ectodomain shedding.” Cleavage of Pro-AR at two N-terminal sites gives rise to two major soluble forms of ~19 and ~21 kDa. Alternatively, Pro-AR cleavage can produce a larger 43-kDa soluble protein corresponding to the entire extracellular domain. Cleavage of Pro-AR at the cell surface can be mediated by tumor necrosis factor-α converting enzyme (TACE), a member of the disintegrin and metalloproteinase (ADAM) family also known as ▶ADAM17 (Fig. 1). Shedding of AR allows the autocrine or paracrine interaction of the mature ligand with its cognate receptor, the ▶EGFR (also known as ErbB1), a transmembrane protein endowed with tyrosine kinase activity, although

yuxtacrine interaction between membrane-bound ProAR and the EGFR has also been observed (Fig. 1). Binding of AR to EGFR triggers key intracellular signaling pathways, such as the mitogenic MAPK and survival ▶PI3K/Akt pathways, which have been demonstrated to participate in the transduction of AR effects (Fig. 1). Amphiregulin Structure, Expression, and Function AR was originally identified as a factor capable of inhibiting the growth of certain carcinoma cell lines, while stimulating the proliferation of normal cells, a fact that motivated its denomination. In fact, depending on its concentration and the nature of the target cell AR promotes the growth and survival of most cell types, both normal and transformed. AR gene overexpression

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has been frequently demonstrated in cancerous tissues like colon, breast, bladder, prostate, pancreas, lung, ovary, squamous cell carcinomas, hepatocarcinoma, and myeloma cells. Besides changes in AR gene expression, different stimuli can also influence the availability of this growth factor through the stimulation of Pro-AR cleavage at the cell membrane. This is achieved by the activation of TACE/ADAM17 in response to agonists acting through GPCRs in a process termed ▶EGFR transactivation (Fig. 1). The existence of EGFR transactivation involving the release of AR has been demonstrated in a variety of cancer cells, suggesting that AR could be an important mediator between diverse stimuli acting on GPCRs and the activation of protumorigenic signals conveyed through the EGFR. Interference with AR production by means of specific antisense RNAs or ▶siRNAs, or treatment with AR neutralizing antibodies, has been shown to revert many of the neoplastic phenotypic traits of cancer cells in vitro, even though the expression of other EGFR ligands was preserved in these cells. This suggests that AR plays a nonredundant role in carcinogenesis. Observations performed in vivo also lend support to a role for AR in the initiation and maintenance of the neoplastic properties of tumor cells. For instance, tissue-specific transgenic overexpression of AR in pancreas results in enhanced cell cycle progression, and in mice older than 1 year it induces dysplastic changes and premalignant alterations. Although, so far most of the evidences that support a role for AR in cancer development and progression have been gathered under experimental conditions, there are also clinical studies that point in the same direction. In this regard, a significant correlation has been established between elevated tumor tissue AR mRNA levels and poor survival in bladder carcinoma patients, or elevated serum AR concentrations and increased mortality in non-small cell lung cancer patients. In summary, the current knowledge on AR in cancer suggests that increased availability of this growth factor can provide transformed cells with a selective advantage. Targeted inhibition of AR expression or action may therefore represent a useful therapeutic strategy for a wide variety of cancers. ▶Epidermal Growth Factor (EGF)-Like Ligands

3. Fischer OM, Hart S, Gschiwnd A et al. (2003) EGFR signal transactivation in cancer cells. Biochem Soc Trans 31:1203–1208 4. Sanderson MP, Dempsey PJ, Dunbar AJ (2006) Control of ErbB signaling through metalloprotease mediated ectodomain shedding of EGF-like factors. Grwoth Factors 24:121–136 5. Berasain C, Castillo J, Perugorria MJ et al. (2007) Amphiregulin: a new growth factor in hepatocarcinogenesis. Cancer Lett 254:30–41

Amphitropic Proteins Definition

▶Peripheral Membrane Proteins.

AMPK Definition

Synonym ▶5’AMP-activated protein kinase is a fuelsensing enzyme that plays a central regulatory role in cellular energy metabolism. It stimulates fatty acid oxidation and glucose uptake, inhibits cholesterol and triglyceride synthesis, and modulates cell growth and death. ▶Adiponectin ▶Autophagy

AMPL ▶Bin1

References 1. Lee DC, Hinkle CL, Jackson LF et al. (2003) EGF family ligands. In: Thomson AW, LotzeMT (eds) The cytokine handbook. Academic Press, London, pp 959–987 2. Normanno N, De Luca A, Bianco C et al. (2006) Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366:2–16

Amplaxin ▶Cortactin

Amplification

Amplification M ANFRED S CHWAB Tumour Genetics (B030), German Cancer Research Center, DKFZ, Heidelberg, Germany

Definition Amplification is the selective increase in DNA copy number either intracellularly, as a local genomic change, or experimentally, by polymerase chain reaction (▶PCR). Increase in the level of mRNA or protein should not be referred to as amplification.

Characteristics Intracellular amplification results in a selective increase in gene copy number with the consequence of elevated gene expression. Gene amplification has been seen in three different settings . Scheduled amplification as part of a developmental gene expression program, e.g. chorion genes in ovaries of the fruitfly Drosophila melanogaster or actin genes during myogenesis in the chicken . Unscheduled amplification during acquisition of cellular ▶drug resistance. For example; amplification of the

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gene encoding dihydrofolate reductase (DHFR) can result in up to 1,000 gene copies per cell with the consequence of cellular resistance against methotrexate. . Unscheduled amplification of cellular genes involved in growth control (▶oncogenes) during ▶tumor progression. Amplification of oncogenes can result in up to several hundred gene copies and enhanced gene expression. Usually large DNA stretches (from 100 Kb up to several Mb) are amplified, and therefore ▶syntenic genes in addition to the particular oncogene can be co-amplified due to their close linkage to the oncogene. Alternatively, different ▶non-syntenic oncogenes can amplify independently in the same cell. The prototypic human cancer with oncogene amplification is ▶neuroblastoma. Here, the amplified gene, MYCN, is a ▶biomarker for patient management. Amplified DNA can be visualized cytogenetically as a ▶homogeneously staining region within chromosomes (▶HSR), as ▶double minutes (▶DM) or as ▶C-bandless chromosomes (CM) (Fig. 1). Cellular Regulation Amplification can follow different pathways, the “onion skin model” and “breakage fusion-bridge” (BFB) cycles (Fig. 2) both fit experimental observations.

Amplification. Figure 1 Cytogenetics of MYCN amplification in neuroblastoma cells. Chromosomal fluorescence in situ hybridization (FISH). High-level MYCN amplification appears in human neuroblastoma cells as two alternative cytogenetic manifestations: (a). Double minutes (DMs) (left), this tumor cell has in addition to amplified MYCN (red) amplification of another oncogene MDM2 (green). The two oncogenes are non-syntenic (2p24, and 12q13–14, respectively), and the amplification is the result of two independent genetic events. (b). Homogeneously staining region (HSR) (right), multiple copies are amplified in an HSR on chromosome 12 (with strong signal), while single copy gene is retained on the two parental chromosomes (arrows). The retention of MYCN at 2p24 indicates that not the original MYCN gene but rather a copy, presumably the result of extra-replication, has been amplified. Note also the strong signal in interphase nuclei which allows detection of amplified MYCN in tumor biopsies when chromosomes cannot be prepared.

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Amplification

Little is known about genomic or environmental elements involved in amplification. Unscheduled amplification presumably is a sporadic event that can become stabilized under selective pressures, i.e. cytostatic drugs or if cells acquire a growth advantage within a certain tissue architecture.

Clinical Relevance Resistance against cytostatic drugs poses a big problem in cancer therapy. Amplified oncogenes contribute to tumor progression, many different oncogenes have been found amplified (e.g. ▶RAS, ▶MYC, ▶MYCN, ▶MYCL, HER-2 [▶HER-2/neu], ▶ABL, etc.), in some

Amplification. Figure 2 Breakage-fusion-bridge (BFB) cycles in early stages of amplification. (a). BFB cycles start from common ▶fragile sites, where a DNA break can occur in both ▶sister chromatids. DNA repair systems will be recruited to the break and may join the free DNA ends of the two sister chromatids to form a dicentric chromosome, one that has two centromers. At anaphase, where sister chromatids are moved to the daughter cells, the dicentric chromosome at some point will break. Of the two daughter cells, one will carry a deletion, the other an inverted duplication of DNA, which is equivalent to a low-level amplification. By subsequent BFB cycles, the level of amplification can increase. (b). Low level amplification as the result of BFB cycles. FISH image, where each color shows the position and copy-number of a particular DNA sequence.

Amplified in Breast Cancer 1

tumor types the oncogene status provides information about patient prognosis: Amplified MYCN indicates poor prognosis for stage 1–3 neuroblastoma; and amplified HER-2 indicates unfavorable outcome in a subgroup of ▶breast cancer. ▶REL

References 1. Schwab M (1998) Amplification of oncogenes in human cancer cells. BioEssays 20:473–479 2. Savelyeva L, Schwab M (2001) Amplification of oncogenes revisited: from expression profiling to clinical application. Cancer Lett 167:115–123 3. Schwab M, Westermann F, Hero B et al. (2003) Neuroblastoma: biology, and molecular and chromosomal pathology. Lancet Oncol 4:472–480

Amplified in Breast Cancer 1 J IANMING X U Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

Synonyms Nuclear receptor coactivator 3; NCoA3; Steroid receptor coactivator-3; SRC-3; Receptor-associated coactivator 3; RAC3; Thyroid hormone receptor activator molecule 1; TRAM-1; Coactivator ACTR; p300/CBP-interacting protein; p/CIP; AIB1

Definition AIB1 is a 160 kDa intracellular protein that enhances gene expression though interacting with nuclear hormone receptors and some other transcription factors and serving as a transcriptional coactivator. The AIB1 gene is amplified and overexpressed in some human breast tumors.

Characteristics Molecular Structure and Functional Domains The human AIB1 gene is located in chromosome 20 and it encodes for a 160-kDa intracellular protein with 1402 amino acid residues. AIB1 is a member of the p160 steroid receptor coactivator (SRC) family that also includes SRC-1 and the transcriptional intermediary factor 2 (TIF2). AIB1 contains multiple structural and functional domains (Fig. 1). The N-terminal basic helixloop-helix/Per-Ah receptor nuclear translocator-Sim (bHLH/PAS) domain is the most conserved region in

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the molecule with ~70% sequence similarity to the respective regions of SRC-1 and TIF2. The bHLH/PAS domain contains a nuclear localization signal, which is required for AIB1 to get into the cellular nucleus where AIB1-regulated gene transcription takes place and where AIB1 degrades in a proteasome-dependent manner. The bHLH/PAS domain also can interact with certain transcription factors such as myogenin to mediate their transcriptional functions. The serine/threonine (S/T) rich domain contains many serine and threonine residues and some of these residues are targets of serine/ threonine kinases. The phosphorylation status of AIB1 is related to its interaction specificity and affinity with transcription factors and other coactivators. A sequence in the S/T domain is also found to interact with transcription factor E2F1. Through interaction and function with ▶E2F1, AIB1 can play a role in direct regulation of cell cycle. Following the S/T domain is the second conserved region of AIB1 with ~60% sequence similarity to SRC-1 and TIF2. This region contains three LXXLL (L, leucine, X, any amino acid) α-helix motifs that are responsible for interaction with the ligand-binding domain of nuclear receptors in a hormone binding-dependent manner. The third conserved region located in the C-terminus of AIB1 has ~50% sequence similarity to SRC-1 and TIF2 and contains two poly-glutamine stretches and a weak histone acetyltransferase activity. This domain can steadily interact with CREB (cAMP response elementbinding protein) binding protein (CBP) and p300, which are strong histone acetyltransferases. This domain also can interact with the coactivator-associated arginine methyltransferase (CARM1) and the protein arginine methyltransferase 1 (PRMT1), which are histone methyltransferases. Functional Mechanisms Two transcriptional activation domains of AIB1 have been identified. The first one is located in the region that interacts with CBP or p300 and the second one is located in the region that interacts with CARM1 or PRMT1 (Fig. 1). The transcriptional activation function of AIB1 is mainly executed through these acetyltransferases and methyltransferases, which are chromatin-remodeling enzymes. In the case of steroid hormone-regulated gene expression, hormone binding triggers a series of events for steroid receptors, including the dissociation of heat shock proteins, change of receptor conformation, receptor dimerization and DNA binding. Importantly, the hormone binding also induces the steroid receptors expose their coactivator-binding motifs in their ligand-binding domains and allows coactivators such as AIB1 to be recruited to the enhancer region of the nuclear receptor target genes. Through the further interaction of AIB1 with CBP, p300, the p300 and CBP-associated factor (p/CAF), CARM1 and PRMT1, a steroid receptor-directed

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Amplified in Breast Cancer 1

Amplified in Breast Cancer 1. Figure 1 Schematic presentation of the structure and function of AIB1. CR1, CR2 and CR3, conserved regions 1, 2 and 3 in the p160 SRC family; bHLH/PAS, the basis helix-loop-helix/Per-Ah receptor nuclear translocator-Sim domain; S/T, the serine and threonine-rich domain; L, L and L, the three LXXLL motifs responsible for interaction with nuclear receptors; Q and Q, the two glutamine-rich regions; HAT, the histone acetyltransferase domain; H, hormone; NR, nuclear receptors; CBP, the CREB (cAMP response element-binding protein) binding protein; p300, the 300 kDa protein homologous to CBP; p/CAF, the p300 and CBP-associated factor; CARM1, the coactivator-associated arginine methyltransferase 1; PRMT1, the protein arginine methyltransferase 1; TBP, the TATA binding protein; TAFIIs, TBP-associated general transcription factors (GTFs); Pol II, RNA polymerase II.

transcriptional activation complex is built up on the hormone response elements of their target gene. This protein complex uses its protein acetyltransferase and methyltransferase activities to remodel the chromatin structure, to facilitate the assembly of general transcription factors on the promoter and thereby to promote target gene transcription. In addition to steroid receptors and other nuclear receptors, AIB1 also serves as a coactivator for certain other transcription factors such as E2F1, AP-1 and Ets transcription factors. Physiological Function AIB1 mRNA is expressed in many different human tissues and cell lines when examined by Northern blot analysis. Detail analyses with mouse tissues revealed that AIB1 is mainly expressed in the mammary gland epithelial cells, oocytes, vaginal epithelial layer, hepatocytes, smooth muscle cells, endothelial cells, and the hippocampus and olfactory bulbs of the brain. At this time, our knowledge regarding the in vivo physiological function of AIB1 is mainly learned from the AIB1 knockout mice. AIB1-deficienct mice have a much lower levels of insulin like growth factor-I and 17β-▶estradiol in their circulation. Accordingly, these mice are smaller in size and they exhibit delayed puberty, retarded mammary gland development and reduced female reproductive function. In addition, AIB1 plays a beneficial role in estrogen and ▶estrogen receptor-mediated vascular protection after vessel injury by enhancing estrogen receptor function and contributes to the control of acute inflammatory responses by inhibiting the production of pro-inflammatory cytokines.

Role in ▶Cancer The AIB1 gene is amplified (or increased in the number of gene copies) in about 5–10% human breast tumors. The AIB1 mRNA is overexpressed in about 30–60% breast tumors, depending on the resources of reports. However, some study only found about 10% of breast tumors that have elevated AIB1 protein levels. AIB1 overproduction is observed in breast tumors both positive and negative to the estrogen receptor α. In ▶tamoxifen-treated patients, high levels of AIB1 are associated with the HER2/Neu expression, the tamoxifen resistance and the lower disease-free survival rates. In the cultured ▶breast cancer cells, AIB1, together with the estrogen and estrogen receptor, enhances ▶cyclin D1 expression and cell cycle progression. Down regulation of AIB1 in breast cancer cells inhibits cell proliferation, cell motility and anchorage-independent growth in the culture and tumor formation in the immune-deficient mice. Animal experiments further demonstrate that AIB1-deficient mice are resistant to either transgenic ▶oncogene- or chemical ▶carcinogeninduced mammary gland tumorigenesis. The transgenic v-Ha-ras oncogene can no longer induce mammary gland tumors in the ovariectomized AIB1 knockout mice, suggesting that inhibition of AIB1 function and removal of ovarian ▶hormones may be a potential strategy to control breast tumorigenesis. On the other hand, it has been demonstrated that overexpression of AIB1 in the mouse mammary epithelial cells is sufficient to induce a high frequency of mammary gland tumors, indicating that AIB1 is an oncoprotein. Similar to the role of AIB1 in breast cancer, AIB1 is also found to be overexpressed in certain human prostate

Amrubicin

tumors and to play a detrimental role in prostate epithelial tumorigenesis in mouse models.

References 1. Anzick SL et al. (1997) AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968 2. Xu J, Li Q (2003) Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 17:1681–1692 3. Xu J et al. (2000) The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development. Proc Natl Acad Sci USA 97:6379–6384 4. Kuang SQ et al. (2004) AIB1/SRC-3 deficiency affects insulin-like growth factor I signaling pathway and suppresses v-Ha-ras-induced breast cancer initiation and progression in mice. Cancer Res 64:1875–1885 5. Torres-Arzayus MI et al. (2004) High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene. Cancer Cell 6:263–274

Amrubicin M ICHIKO YAMAMOTO, N ORIYUKI M ASUDA Department of Respiratory Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan

Synonyms (+)-(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-D-erythropentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-naphthacenedione hydrochloride; SM-5887

Definition The anthracyclines tested clinically so far have been limited to those produced by fermentation or semisynthetic processes. In contrast, 9-aminoanthracycline, amrubicin is a fully synthetic drug. Amrubicin differs from daunosamine in that it contains a 9-amino group and a simple sugar moiety (Fig. 1).

Characteristics Amrubicin is converted to its active 13-hydroxy metabolite, amrubicinol, in the liver, kidney, and tumor tissue, through reduction of its C-13 ketone group to a hydroxy group. Despite the similarity of its chemical structure to that of a representative anthracycline, doxorubicin, amrubicin’s mode of action differs from that of doxorubicin. Amrubicin and amrubicinol are

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inhibitors of DNA topoisomerase II, which exert their cytotoxic effects by stabilizing a topoisomerase II-mediated cleavable complex (▶Topoisomerase enzymes as drug targets), and are approximately only onetenth as potent as doxorubicin in producing DNA intercalation. Preclinical Studies In in vitro experiments, amrubicin and its metabolite amrubicinol have been found to be active against a broad spectrum of human cell lines established from cancers of the lung, prostate, urinary bladder, colon, kidney, pancreas, and uterus. Amrubicinol has been shown to exhibit a 20- to 220-fold more potent antitumor activity in vitro than amrubicin itself, being as potent as doxorubicin. In addition, amrubicin and amrubicinol have also been demonstrated to show some degree of noncross resistance with doxorubicin. Amrubicin has been shown to be more effective against five human xenografts (one breast cancer, one ▶lung cancer, and three gastric cancers), equally effective against two gastric cancers, and less effective against two tumor (one lung and one gastric cancer). Amrubicin caused dose-dependent weight loss, ataxia, myelosuppression, and hair loss in mice after a single intravenous (i.v.) injection. The maximum tolerated (MTD) for such administration was estimated to be 25 mg/kg in four mouse strains. Cardiotoxicity is one of the dose-limiting toxicities of anthracyclines. However, amrubicin showed little delayed-type cardiotoxicity in rabbit and dog experimental models. Furthermore, amrubicin did not aggravate the doxorubicin-induced myocardial injury. Clinical Studies Amrubicin Monotherapy Phase I/II Trial of Amrubicin Given Daily for Three Consecutive Days Every 3 Weeks. Based on the finding that amrubicin exhibited enhanced antitumor efficacy against six of eight cell lines when it was given for five consecutive days instead of on a single day at the MTD dose, a phase I/II trial of amrubicin for three consecutive days was carried out in patients with advanced nonsmall cell lung cancer (NSCLC). In the phase I study, four patients were enrolled at dose level 1 (40 mg/m2/day) and four at dose level 2 (45 mg/m2/day). No dose-limiting toxicity (DLT) was observed at these dose levels. At dose level 3 (50 mg/m2/day), three of five patients experienced DLTs (leukopenia, neutropenia, thrombocytopenia, or gastrointestinal toxicities). The MTD and recommended dose (RD) were determined to be 50 and 45 mg/m2/day, respectively, from the results of this trial. Seven partial responses (PR) were observed in a total of 28 patients of the phase I/II study, with an overall response rate of 25%.

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Amrubicin

Amrubicin. Figure 1 Chemical structure of amrubicin and its active metabolite, amrubicinol.

Multicenter Phase II Study of Amrubicin in Patients with Advanced NSCLC. Sixty-one previously untreated patients with stage III or IV NSCLC were entered in this study. Amrubicin was administered by a single i.v. injection daily at the dose of 45 mg/m2/day for three consecutive days every 3 weeks. One complete response (CR) and 16 PR were observed, with an overall response rate of 27.9%. The median survival time (MST) was 9.8 months. The major toxicity was myelosuppression. The incidences of grade 3 or 4 toxicity were 72.1% for neutropenia, 52.5% for leukopenia, 23.0% for anemia, and 14.8% for thrombocytopenia. Phase II Study of Amrubicin in Previously Untreated Patients with Extensive-Stage Small Cell Lung Cancer. Thirty-five previously untreated patients with extensive-disease small cell lung cancer (ED-SCLC) were entered in the study. Amrubicin was given by daily i.v. infusion at the dose of 45 mg/m2/day for three consecutive days every 3 weeks. Of 33 eligible patients, 3 showed CR and 22 showed PR, with an overall response rate of 75.8% (95% confidence intervals (CI), 57.7–88.9%). The MST and 1-year survival were 11.7 months and 48.5%, respectively. The most common toxicity was hematologic toxicity. Phase II Trial of Amrubicin for the Treatment of Refractory or Relapsed Small Cell Lung Cancer: Thoracic Oncology Research Group Study 0301. SCLC patients with measurable disease who had been treated previously with at least one platinum-based chemotherapeutic regimen were entered into the trial.

Amrubicin was administered at a reduced dose of 40 mg/m2 per day × 3 days every 3 weeks, in view of the prior chemo- and radiotherapy. Sixty patients (16 refractory and 44 sensitive) were enrolled. The grade 3 or 4 hematologic toxicities comprised neutropenia (83%), thrombocytopenia (20%), and anemia (33%). Febrile neutropenia was observed in three patients (5%). The nonhematologic toxicities were mild. The overall response rates were 50% (95% CI, 25–75%) in the refractory group and 52% (95% CI, 37–68%) in the sensitive group. The overall survival and 1-year survival in the refractory group and sensitive group were 10.3 and 11.6 months, and 40 and 46%, respectively. Thus, it was concluded that amrubicin shows significant activity against SCLC, with predictable and manageable toxicities. Amrubicin Combination Chemotherapy Phase I-II Study of Amrubicin and Cisplatin for Previously Untreated Patients with Extensive-Stage Small Cell Lung Cancer. This trial was performed to determine the MTDs for combined amrubicin and cisplatin therapy and to assess the efficacy and safety of these drugs at their RD. Patients with histologically or cytologically proven measurable ED-SCLC, no previous chemotherapy, and good prognostic factors were entered into this trial. Amrubicin was administered on days 1–3 and cisplatin on day 1, every 3 weeks. Four patients were enrolled at dose level 1 (amrubicin 40 mg/m2/day and cisplatin 60 mg/m2) and three patients at dose level

Analytic Epidemiological Study

2 (amrubicin 45 mg/m2/day and cisplatin 60 mg/m2). The MTD and RD were found to be at level 2 and level 1, respectively. The response rate at the RD was 87.8% (36/ 41). The MST and 1-year survival rate were encouraging, 13.6 months and 56.1%, respectively. Phase I and Pharmacologic Study of Irinotecan and Amrubicin for Advanced NSCLC. We conducted a phase I trial of irinotecan (CPT-11), a topoisomerase I inhibitor, combined with amrubicin. The aim was to determine the MTD and DLT of amrubicin combined with a fixed dose of CPT-11 in patients with advanced NSCLC. Eleven patients were treated with amrubicin on days 1–3, combined with 60 mg/m2 of CPT-11 on days 1 and 8, every 3 weeks. The starting dose of amrubicin was 25 mg/m2, and the dose was escalated in 5 mg/m2 increments until the MTD was reached. The 30 mg/m2 of amrubicin dose was one dose level above the MTD, since three of the five patients experienced DLT, namely, diarrhea and leukopenia. Amrubicin did not affect the pharmacokinetics of CPT-11, SN-38, or SN-38 glucuronide. There were five PRs among the 11 patients, with an overall response rate of 45%. The RD for phase II studies was determined to be 60 mg/m2 for CPT-11 (days 1 and 8) and 25 mg/m2 for amrubicin (days 1–3), administered every 21 days. Conclusion The preclinical studies described above show that amrubicin has a unique mechanism of action as an anthracycline derivative and a broad spectrum of antitumor activity. The results of clinical studies of amrubicin as a single agent or as one of the drugs in combination regimens for lung cancer have been promising. To determine the exact usefulness of amrubicin in the treatment of SCLC, two randomized trials are now underway in Japan; one of cisplatin + amrubicin versus cisplatin + irinotecan in previously untreated patients of ES-SCLC, and one of amrubicin versus carboplatin + etoposide in elderly patients with ES-SCLC. Although amrubicin has been investigated over the last decade, many of its characteristics remain unclear. Further studies exploring the usefulness of amrubicin as a single agent or as an agent administered in combination with other cytotoxic agents as well as novel molecule-targeted drugs for the treatment of other malignancies are warranted. Lastly, since all the trials with amrubicin have been conducted in Japan, the results of clinical studies to define the benefits and risk associated with amrubicin for cancer therapy conducted overseas are eagerly awaited.

References 1. Sawa T, Yana T, Takada M et al. (2006) Multicenter phase II study of amrubicin, 9-amino-anthracycline, in patients with advanced non-small-cell lung cancer (Study 1): West

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Japan Thoracic Oncology Group (WJTOG) trial. Invest New Drugs 24:151–158 Yana T, Negoro S, Takada M et al. (2007) Phase II study of amrubicin in previously untreated patients with extensivedisease small cell lung cancer: West Japan Thoracic Oncology Group (WJTOG) study. Invest New Drugs 13: 25:253–258 Onoda S, Masuda N, Seto T et al. (2006) Phase II trial of amrubicin for treatment of refractory or relapsed small-cell lung cancer: Thoracic Oncology Research Group Study 0301. J Clin Oncol 24:5448–5453 Oh Y, Negoro S, Matsui K et al. (2005) Phase I-II study of amrubicin and cisplatin in previously untreated patients with extensive-stage small-cell lung cancer. Ann Oncol 16:430–436 Yanaihara T, Yokoba M, Onoda S et al. (2007) Phase I and pharmacologic study of irinotecan and amrubicin in advanced non-small cell lung cancer. Cancer Chemother Pharmacol 59:419–427

Anaerobic ▶Hypoxia

Analgesic Definition The drugs that prevent or reduce pain. ▶Nonsteroidal Anti-inflammatory Drugs

Analytic Epidemiological Study Definition A study in a human population designed to evaluate a specific causal relationship. ▶Allergy ▶Cancer Epidemiology ▶Epidemiology of Cancer

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Anaphase-Promoting Complex

Anaphase-Promoting Complex

Anaplastic Large Cell Lymphoma

Definition

A NGELO R OSOLEN

The anaphase is a phase of mitosis, during which the paired sister-chromatids (▶Sister-chromatids) are separated and drawn to the poles of the cell. This phase is initiated by a ubiquitin-mediated proteolytic pathway (▶Ubiquitination) resulting in the degradation of regulatory proteins. The anaphase-promoting complex (APC/C) functions as a protein ubiquitin ligase.

Department of Pediatrics, Hemato-oncology Unit, University of Padua, Padova, Italy

▶Securin

Anaphylactic Shock Definition A life-threatening allergic reaction characterized by a swelling of body tissues including the throat, difficulty in breathing, and a sudden fall in blood pressure.

Anaplasia Definition Refers o lack of cell differentiation in a tumor.

Anaplastic Astrocytoma Definition Astrocytic tumor characterized by an intermediate degree of histologic and clinical malignancy ▶Brain Tumors

Anaplastic Carcinomas ▶Follicular Thyroid Tumors

Synonyms Ki1 lymphoma

Definition Anaplastic large cell lymphoma (ALCL) was originally described in 1985 as a separate entity among the ▶nonHodgkin lymphomas (NHL); it is characterized by the cohesive proliferation of large cells expressing the CD30/Ki1 antigen on their membrane. The World Health Organization more recently stated that the term ALCL should be applied to tumors with a T-cell or null phenotype, thus further restricting the identification of this specific subtype of ▶lymphoma.

Characteristics

Systemic ALCL accounts for 2–8% of all ▶lymphomas (▶Malignant lymphoma, hallmarks and concept) but represents ~10–15% of NHL of childhood. In addition to the primary systemic disease, a form of ALCL limited to the skin is also recognized. Isolated cutaneous ALCL may spontaneously remit, but can also progress to a more aggressive disease. Systemic ALCL has some clinical features that are less common in other NHL subtypes: patients at diagnosis often have B-symptoms (fever, weight loss, sweats) as in Hodgkin lymphoma, mediastinal (▶Mediastinum) and extranodal involvement, including skin, bone, and soft tissues. Among lymph nodes, the inguinal nodes are often site of disease, particularly in childhood, and rather frequently lymphadenopathy may be painful. Central nervous system and bone marrow are rare sites of disease, although with more sensitive techniques bone marrow may have submicroscopic infiltration of lymphoma cells more often than previously expected. Clinical differences have been reported between ▶ALK (anaplastic lymphoma kinase)-positive and ALK-negative subtypes. Patients with ALK-positive ALCL are significantly younger and have a better prognosis that the negative counterpart, suggesting that ALK-positivity, rather than age, may confer distinct and relevant clinical features to systemic ALCL. Secondary ALCL may arise in the progression of other lymphomas, most commonly during the course of T-cell NHL, mycosis fungoides, Hodgkin lymphoma or lymphomatoid papulosis, and has a poor outcome. Except for the cases of very aggressive disease, ALCL may show multiple recurrences that respond to therapy, although eventually a considerable number of patients die despite intensive treatment.


Diagnosis Diagnosis of ALCL relies on histopathology and immunophenotyping, but for a complete characterization of the tumor, chromosomal analysis and molecular genetic studies are warranted. It is now clear that ALCL includes several variants: classical or common type (corresponding to the original description of the disease) that accounts for ~60–70% of the cases, lymphohistiocytic variant, giant cell-rich, small cell type, and mixed. The large cell-rich type is characterized by multinucleated cells, often with ReedSternberg-like features that make the differential diagnosis with Hodgkin lymphoma (▶Hodgkin disease) sometimes difficult. Irrespective of the histotype, neoplastic ALCL cells are characterized by a distinctive phenotypic profile. They express CD30, a cell membrane glycoprotein, found in activated lymphoid cells, the epithelial membrane antigen (EMA) and T-cell antigens including CD3. Perforin and granzyme B are also expressed in the majority of the cases and, together with absence of CD15 expression, they are useful markers in the differential diagnosis with Hodgkin disease. The development of antibody against the ALCL kinase (ALK) (▶ALK protein) has further refined the immunohistochemical analysis of ALCL. Expression of ALK occurs in tumors carrying ▶chromosomal translocations that involve the corresponding gene. The most common rearrangement between chromosome 2 and 5 causes strong ALK positivity of neoplastic cells in the nucleus and cytoplasm (Figs. 1 and 2), whereas other chromosomal translocations involving ALK produce accumulation of the translocation product at the cytoplasmic level only. ALK reactivity of tumor cells has diagnostic relevance given

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that ALK is not detected in normal lymphocytes or in Hodgkin lymphoma cells and only few cases of rare forms of lymphoma and nonlymphomatous tumors (inflammatory myofibroblastic tumors, rare neuroblastoma, and rhabdomyosarcoma) show ALK reactivity. Ultimately, combination of morphological, immunophenotypic, and genetic analysis allow the diagnosis of ALCL and the differentiation of this lymphoma from other tumors. A peculiar aspect to consider is the differentiation of cutaneous ALCL from other CD30-positive lymphoproliferative disorders including lymphomatoid papulosis. In this case, because the great majority of isolated cutaneous ALCL is ALK-negative, clinical observation and evolution of the disease are of foremost importance. In addition, because skin involvement in the context of a systemic ALCL may bear prognostic implications, suspect skin involvement must be documented through biopsy. Imaging procedures used in the diagnosis and staging of ALCL are similar to other NHL. Special attention should though be given to the examination of soft tissues and skin, given the relatively high frequency of involvement of those sites. Chest x-ray is usually sufficient to detect a mediastinal mass, but a CT scan of the neck, thorax, and abdomen should always be obtained to define the extent of disease. Ultrasound is routinely used for diagnosis and monitoring of lymph nodes, abdominal organ involvement, including liver, spleen, kidney, and soft tissues. This technique is easy to perform and can give accurate information not only for diagnostic purposes, but also to evaluate tumor response to treatment and during follow-up, once treatment is completed. Whole skeletal ▶scintigraphy may be useful, especially in cases with bone pain, and should be complemented with x-ray of positive bones.

Anaplastic Large Cell Lymphoma. Figure 1 This panel depicts the classic variant of anaplastic large cell lymphoma with numerous large tumor cells that often contain horseshoe- or kidney-shaped nuclei, distinct nucleoli and abundant cytoplasm.

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Anaplastic Large Cell Lymphoma. Figure 2 This panel immunohistochemical staining of anaplastic large cell lymphoma containing the chromosomal translocation t(2;5) originating the NPM–ALK fusion protein. Nuclear and cytoplasmic reactivity to an anti-ALK specific monoclonal antibody is detectable in most lymphoma cells (brownish color) Pictures were a courtesy of Dr. E. S. d’Amore, Institute of Pathology, University of Padua, Italy.

Brain MRI or CT scan are usually performed, but central nervous system involvement at diagnosis is infrequent in ALCL. More recently ▶positron emission tomography (▶PET) with 18-fluorodeoxyglucose (FDG) has been introduced in the routine evaluation of ALCL patients, mainly for staging. Lymphoma cells characteristically have a higher uptake of FDG compared with normal cells and this gives high activity features to the vital tumor mass. Because a high reactivity may also be seen in other nonmalignant tissues with high glucose metabolism, including reactive lymph nodes and inflammatory tissues, PET findings have to be interpreted with caution at present, until large prospective clinical studies where this recent technology is routinely applied are completed. To completely define the extent of the disease, as in other NHL, bone marrow biopsy and bone marrow smear should be performed and analyzed for the presence of tumor cells, as well as a lumbar puncture to exclude the presence of lymphoma cells in the central spinal fluid. Genetics The ▶chromosomal translocation t(2;5)(p23;q35) was originally reported in patients with malignant histiocytosis, which indeed represented ALCL cases diagnosed according to old criteria. Break of chromosome 2 and subsequent fusion to chromosome 5 in a reciprocal chromosome rearrangement is the main genetic feature of ALCL. As a result, the ALK gene on chromosome 2 is juxtaposed to the nucleophosmin (▶NPM) gene on chromosome 5. The ▶NPM–ALK fusion gene gives rise to a fusion protein composed of ALK and NPM domains that can be detected by ▶immunohistochemistry using an anti-ALK monoclonal antibody. While

ALK is not usually expressed in normal tissues, except in few neuronal cells, NPM gene encodes a shuttle protein that undergoes dimerization in the cytoplasm and in this conformation can move to the nucleus. In the cytoplasm of NPM–ALK-positive ALCL cells, heterodimers between NPM and NPM–ALK are formed that, while retaining the ability to move to the nucleus, are reactive against the anti-ALK antibody. This explains the cytoplasmic and nuclear reactivity of ALCL cells harboring the t(2;5) translocation. The wide use of antibodies to ALK protein revealed that about 10% of the ALK-positive ALCLs showed an immunohistochemical reactivity confined to the cytoplasm. Molecular genetic studies demonstrated that those cases were associated to a series of different chromosomal translocations, all involving the ALK gene, but with partners other than NPM. The deriving fusion genes all cause hybrid protein overexpression that lack the shuttling properties of NPM-containing fusion proteins, thus preventing them from nuclear localization. From the functional point of view, NPM–ALK can dimerize and as such it possesses constitutive tyrosine kinase activity, mimicking the normal functional activity of the ALK receptor in normal cells upon binding and oligomerization by its specific ligand. A number of experimental data suggest that NPM–ALK has a causative role in tumorigenesis of ALK-positive ALCL, although it may need concomitant events. In fact, NPM–ALK displays transforming activity in both hematopoietic and fibroblastic cell lines in vitro. Overall, ~90% of childhood ALCLs are positive for the ALK-hybrid protein, whereas this percentage is only 50–60% in adult ALCLs. As suggested by clinical studies, ALK-positivity, not only is a relevant tool in the

Anaplastic Lymphoma Kinase

diagnosis of ALCL, but represents also a prognostic marker in that ALK-positive ALCLs fare better than ALK-negative lymphomas in the adult population. Therapy Treatment of ALCL, similarly to other NHL, is almost exclusively based on chemotherapy. The therapeutic approach has witnessed a variety of chemotherapy regimens, ranging from acute lymphoblastic leukemiatype regimens lasting 24 months to shorter chemotherapy more frequently used in aggressive B-cell lymphomas. Differently from most European studies where ALCL is considered as a separate entity, in North America all large cell lymphomas, regardless of the histologic subgroup and ▶immunophenotype, are treated according to the same chemotherapy scheme. Primary systemic ALCL in adults has been treated mostly with CHOP (cyclophosphamide, vincristine, prednisone), CHOP-derived chemotherapy, and MACOP-B (methotrexate, adriamycin, cyclophosphamide, vincristine, prednisone, and bleomycin) regimens, but occasionally patients have been treated with Hodgkin lymphoma-type chemotherapy (e.g., ABVD regimen). Collaborative trials have been conducted in childhood and adolescence ALCLs. Overall results were comparable with very different treatment strategies, ranging from leukemia-like treatment, such as modified LSA2-L2 protocol, to chemotherapy regimes derived from B-cell NHL as the BFM (Berlin-Frankfurt-Munster) and the French and British Pediatric Hemato-Oncology Society protocols. The latter are based on the administration of short (usually 5-days) courses of rather high-intensive chemotherapy (based on the rotational use of corticosteroid, anthracyclines, cyclophosphamide or ifosfamide, cytarabine, methotrexate, epipodophyllotoxin) administered at intervals of ~3 weeks. With these treatments, 60–75% of patients obtain cure of their disease and do not experience disease recurrences. Similar results were obtained with the APO regimen in the Children’s Oncology Group (USA) that includes higher cumulative doses of anthracyclines without ▶alkylating agents (cyclophosphamide/ifosfamide) or epipodophyllotoxins. Treatment intensity and/or duration have been differentiated based on various risk factors. Although stage of disease has some relevance, in the adult population a high International Prognostic Index (IPI) score, high serum lactate dehydrogenase levels, ALKnegativity and expression of the surface antigen CD56 have been associated to a significant lower outcome. In children and young adults, possibly because of the very high frequency of ALK-positivity, ALK expression does not seem to be a relevant prognostic indicator, whereas high stage of disease, elevated lactate dehydrogenase levels, and specific site of disease, including liver, spleen, lung, and skin involvement, seem to be associated to a less favorable prognosis.

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A distinct clinical condition is represented by the isolated cutaneous ALCL. Once other disease localizations have been excluded, given that spontaneous regression of the lesions can occur, a strict monitoring of the patient is often preferred, postponing initiation of chemotherapy when signs of lymph nodes or other organ involvement is demonstrated. A peculiar aspect of ALCL is that most patients who relapse respond well to salvage therapies. Although early disease recurrences can be extremely aggressive, moderate intensity chemotherapy, including single drug treatment with vinblastine, can achieve long-lasting remission. In case of resistant disease or very aggressive relapse, high-intensive chemotherapy with bone marrow transplant has also been used both in children and adults. Further studies are needed to definitively establish the benefit of bone marrow transplant in ALCL and to identify the subpopulations of patients who may benefit from such a treatment approach.

References 1. Falini B (2001) Anaplastic large cell lymphoma: pathological, molecular and clinical features. Br J Haematol 114:741–760 2. Seidemann, K, Tiemann, M, Schrappe M et al. (2001) Short-pulse B-non-Hodgkin lymphoma-type chemotherapy is efficacious treatment for pediatric anaplastic large cell lymphoma: a report of the Berlin-Frankfurt-Munster Group Trial NHL-BFM 90. Blood 97:3699–3706 3. Brugieres, L, Deley, MC, Pacquement H et al. (1998) CD30(+) anaplastic large-cell lymphoma in children: analysis of 82 patients enrolled in two consecutive studies of the French Society of Pediatric Oncology. Blood 92:3591–3598

Anaplastic Lymphoma Kinase Definition Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) having an extracellular, a single transmembrane, and an intracellular domain containing the tyrosine kinase activity. ALK belongs to the insulin receptor subfamily of RTKs, most closely related to leukocyte tyrosine kinase receptor. It localizes mostly in neuronal cells and may play a role in the nervous system development and maintenance. ALK and some of the ALK partners or closely related genes are found implicated both in anaplasic large cell lymphoma and in inflammatory myofibroblastic tumors. ▶Pleiotrophin ▶ALK Protein

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Anatomic Pathology

Anatomic Pathology Definition

phenotype of the cell, such that the cells are now able to grow as colonies in three-dimensional suspension in soft agar. ▶Chemically Induced Cell Transformation

The study of gross and microscopic tissue features in disease diagnosis. ▶Molecular Pathology

Androgen Anchorage-Independent Definition The ability of cells to survive and multiply in the absence of a protein matrix for adhesion.

Definition An agent, usually a hormone (e.g. testosterone) that stimulates the activity of the accessory sex organs of the male. ▶Prostate-Specific Membrane Antigen (PSMA)

▶Syk Tyrosine Kinase ▶Adhesion Molecules

Androgen Insensitivity Syndrome (AIS) Anchorage-Independent Cell Growth

▶Androgen Receptor

Definition In vitro transformed cells and cancer-derived cells are able to survive and grow in the absence of anchorage to the ▶extracellular matrix (ECM) and their neighboring cells, termed anchorage independence of growth, correlates closely with tumorigenicity in animal models. This property of cancer cells presumably reflects the tendency of tumor cells to survive and grow in inappropriate locations in vivo. Such incorrect localization, as occurs in invasion and metastasis, is the characteristic that distinguishes malignant from benign tumors. ▶Sprouty

Androgen Receptor K AUSTUBH DATTA , D ONALD J. T INDALL Department of Urology Research, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA

Synonyms AR; dihydrotestosterone receptor; testicular feminization TFM; spinal and bulber muscular atrophy SBMA; Kennedy disease KD; androgen insensitivity syndrome AIS; NR3C4; SMAX1; HUMARA

Definition

Anchorage-Independent Cell Transformation Definition The process by which carcinogenic chemicals, oncogenic viruses, or radiations change the genotype and

The androgen receptor or AR is a member of the steroid/ thyroid receptor superfamily, in which all members share basic structural and functional homology. The AR is an intracellular ligand (androgen)-dependent transcription factor, which regulates the expression of genes that control cell proliferation, ▶apoptosis, ▶angiogenesis and differentiation in many hormonally regulated tissues including the prostate. It is an important regulator of male sexual differentiation and maturation.

Androgen Receptor

Characteristics The biological function of androgen is mediated through the androgen receptor. Except for the spleen and bone marrow, the androgen receptor is ubiquitously expressed in human organs. The human androgen receptor gene is more than 90-kb long, and is present as a single allele located at chromosome Xq11.2–12. The AR gene has eight exons and possesses a coding region of 2757 bp. This region encodes a 110 KDa protein (919 amino acids) with four distinct functional domains: a conserved Zn-finger DNA-binding domain (exons 2 and 3), a hinge region (exon 4), a COOHterminal ligand-binding domain (exons 4–8), and a less conserved and structurally flexible amino (NH2)terminal transactivation domain (exon 1). Structure–Function Relationships between Different Domains of Androgen Receptor that are Required for its Transcriptional Activity Regulation of Unliganded AR Unbound AR in the cytoplasm remains in an inactive but androgen responsive state as part of a large dynamic heterocomplex composed of heat shock proteins, cochaperones, and tetratricopeptide repeat containing proteins. This large complex helps to modulate the ligandbinding domain (LBD) of AR into a relatively stable, partially unfolded, and inactive intermediate which has a high affinity for the potent biologically active androgen, dihydrotestosterone. The androgen-free LBD and the associated chaperone proteins inactivate the function of

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the transactivation domain of AR. This transactivation domain becomes constitutively active in the mutant AR lacking a ligand-binding domain (LBD). As such, this molecular chaperone complex and the LBD of AR prevent unwanted activation of AR in the absence of androgen Regulation of Ligand-Bound AR Because of their lipophilic nature, androgens cross the cell membrane, both passively, and actively via the transport protein, megalin. Once within the cell the androgen binds to the AR, which is stabilized and translocated into the nucleus. Within the nucleus the AR homo-dimerizes and recruits transcriptional cofactors to the promoters and enhancers of AR-target genes, thus facilitating their transcription (Fig. 1). Androgen binding to the LBD of AR induces an overall change in AR structure leading into an active conformation which is characterized by dissociation of the receptor– chaperone complex. However, molecular chaperones also play important roles in the events downstream of AR activation such as translocation to the nucleus, transcriptional activation, transcription complex disassembly and degradation. LBD relieves its inhibitory function upon androgen binding. AR LBD configuration is highly structured and resembles other steroid receptors’ ligand-binding domains. 1. AR DNA Binding Domain and Hinge Region: Androgen binding to the LBD initiates a conformational change of AR leading to several secondary effects important for AR transcriptional activity.

Androgen Receptor. Figure 1 A schematic diagram of AR structure and its functions.

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Androgen Receptor

One such effect is the unmasking of the bipartite nuclear-localization signal (NLS) that overlaps the DNA-binding domain and hinge regions (amino acid 625–671). Another weak NLS (amino acid 722–805) is present in the LBD of AR, which is also exposed upon androgen binding. The NLS is recognized by an import protein that mediates translocation of the AR through the nuclear-pore complex. AR homodimerization occurs in the nucleus upon treatment with ligand. Both the DNA and ligand-binding domains of AR are involved in subsequent receptor dimerization. The stronger hydrophobic interaction occurs between the ligand-binding domains.The binding of AR dimer to the specific androgen response elements (ARE) of a gene occurs in a co-operative manner. The DNA binding domains of AR, like other nuclear receptor superfamily members, consist of two zinc fingers that provide the structural basis required for ARE recognition in the promoter region of a gene. The consensus ARE is a 6 bp palindromic core sequence (5′-AGEACA-3′) separated by a three nucleotide spacer. Sequences outside the DNA binding domain also play a role in AR-DNA binding. 2. AR AF-2 Domain: A hydrophobic protein-protein interaction surface known as Activation Function-2, or AF2, is present at the carboxy-terminal, and also becomes accessible after androgen binding to AR. AF2 is the potential binding site for AR co-activators such as the p160 family (TIF2, SRC1, and AIB1). These co-activators enhance AR transcriptional activity by modulating AR conformation and by recruiting cofactors to the promoter. Ligand binding also induces interaction of AF2 with a specific motif (FXXLF) (F = phenylalanine, L = leucine and X = any aminoacid) in the NH2-terminal activation domain of AR. The interaction of NTD and LBD or N/C interaction is important for AR transcriptional activity. Although the mechanistic details of how this interaction leads to AR activity are not precisely known, it appears that ligand binding induces intramolecular folding of AR, leading to the interaction between NTD and LBD. This influences receptor dimerization in the nucleus and reduces ligand degradation from LBD, AR protein dissociation, AR chromatin binding ability and receptor transactivation. 3. AR N-Terminal Transactivation Domain: The Nterminal transactivation domain (NTD) of AR is longer and structurally different as compared to other steroid receptors. The highly flexible and disordered domain containing NTD is largely globular in nature. Association with coactivators and transcription factors helps to induce folding in the domains of the NTD to optimize efficient binding. Due to the allosteric nature of binding, the AR NTD is able to bind a broad spectrum of transcriptional coactivators, co-repressors and other factors.

Therefore the AR NTD may be responsible for mediating androgen-regulated expression of genes whose functions consist of protein folding, trafficking and secretion, metabolism, cytoskeletal rearrangement, cell cycle regulation and signal transduction. AF1 and AF5 Domain of NTD: There are two major overlapping activation functions present in the AR NTD, AF1 (amino acids 142–485) and AF5 (amino acids 351–528). These regions contain microsatellite repeats, protein-protein interaction surfaces, and phosphorylation and sumoylation sites. AF1 is considered the major transactivation domain that binds to basal transcription factors, co-regulators, cell cycle regulatory proteins, heat shock proteins etc. The interaction with molecules like TIFIIF increases folding in the AF1 domain and facilitates further protein-protein interactions for the formation of a transcriptionally competent receptor complex. Unlike AF1, activation of AF5 is ligand independent. An inhibitory domain within AF5 inhibits the DNA-binding domain of AR from binding to AREs. Glutamine and Glycine Repeats: AR NTD contains two polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts. The first trinucleotide repeat sequence, CAG, spans amino acids ~58–78, and encodes the amino acid glutamine. The second stretch consists of GGN repeats that encode glycines and span amino acids ~449–472. Shortened trinucleotide repeat stretches result in increased AR activity and are often associated with prostate cancer predisposition. On the other hand, overexpansions of the CAG repeat (more than 40) correlate with a significant decrease in AR activity and are associated with diseases such as X-linked Spinal and Bulbar Muscular Atrophy (SBMA), also known as Kennedy’s disease. Both the polyglutamine and polyglycine repeat size appear to regulate the N/C interaction and thereby influence AR activity. FXXLF and WXXLF Motifs: The AR NTD also contains two short, highly conserved peptide motifs that mediate the liganddependent N/C interaction as discussed previously. These motifs are FXXLF (in humans FQNLF), ranging from amino acids 23–27 and WXXLF (in humans WHTLF), ranging from amino acids 434–438. AR Co-regulators: AR functions as a tripartite receptor system involving AR itself, the androgens and AR coregulators. AR interaction with either co-activators or co-repressors enhances or represses its transcriptional activity, respectively, without affecting the basal levels of transcription. Possible mechanisms of co-regulator function include modulation of chromatin structure, promotion of AR post-translational modifications and control of androgen/AR binding affinity, AR expression, AR stability, AR nuclear translocation, and AR recruitment of transcription machinery.

Androgen Receptor

4. Post-translational Modification of AR: Posttranslational modification of AR is one of the key mechanisms regulating its function. One of the major post-translational modifications of AR is phosphorylation. These phosphorylation sites may be the sites for possible cross-talk between peptide growth factors and the AR signaling axis, which is important for normal prostate epithelial cell growth and function as well as for the progression of cancer. Most of the phosphorylation sites reside in the NTD at different serine residues. However, recent findings suggest that Src kinase mediates phosphorylation at tyrosine 534 of AR, and therefore acts as a potential regulator of AR transcriptional activity. Another post-translational modification is acetylation of AR at the hinge domain (at lysine residues 630, 632 and 633) by Histone Acetyl Transferases such as p300, p/CAF, and TIP60. This modification regulates recruitment of co-regulators as well as the growth properties of the AR. Acetylation is also required for regulation of the AR by the AKT, PKA and JNK signaling pathways. Sumoylation of AR at two specific domains (NRM1 and NRM2 at NTD) also appears to be important for AR activation, localization and degradation. Nongenomic Function of AR Recently, a nongenomic mechanism for AR function independent of its transcriptional activity has been postulated. This nongenomic event is very rapid (2–15 min) and activates signaling events such as the Src/ Raf1/ERK, ▶PI3K-AKT, and IL-6-STAT3 pathways. Interestingly, membrane lipid rafts are considered to be the privileged site for this AR-mediated nongenomic signaling. Upon androgen binding most of the ARs translocate to the nucleus and act as transcription factors. The few remaining cytosolic ARs may then enter into ▶caveolin positive or negative rafts to initiate different signaling pathways. Androgen Receptor in Human Physiology and Pathology Androgen and AR are not only necessary for the initiation of prostate development; they are also important factors for the survival, proliferation, secretary function, morphology, angiogenesis and differentiation of the adult prostate gland. AR is also important for Wolffian duct development and spermatogenesis in males. Various clinical disorders due to functional abnormalities of AR have been reported, suggesting that a wide range of physiological responses and developmental processes are mediated by AR. Androgen Receptor and ▶Prostate Cancer: Approximately 80–90% of prostate tumors are dependent on androgen at the initial diagnosis, suggesting the importance of AR signaling in all stages of ▶prostate carcinogenesis. A recently developed bioinformatics

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approach known as Cancer Outlier Profile Analysis (COPA) together with standard genomic techniques has identified recurrent gene fusions of the 5′ untranslated region of the TMPRSS2 gene to ▶ETS transcription factors (ERG or ETV1) in human prostate cancer tissues. Interestingly, this gene fusion enables expression of the fused product under the control of AR, as the TMPRSS2 promoter contains an ARE. This ▶TMPRSS2-ETS fusion appears to be frequent in prostate cancer and might be an initiating event for prostate cancer, underscoring the importance of ARmediated signaling in prostate cancer. Both the tumor epithelia and adjacent stroma express AR. Retardation of tumor growth occurs in response to androgen ablation therapy. Until now, the primary therapy for advanced (locally extensive or metastatic) prostate cancer consists of androgen ablation by pharmacotherapeutic or surgical means. Eventually, the tumor recurs due to a transition from androgen-dependence to a highly aggressive and androgen-depletion-independent (refractory) phenotype. The detailed molecular mechanism underlying the development of the androgen refractory phenotype of prostate cancer is poorly understood. As such, it has been difficult to develop effective treatments for this stage of the disease. Disruption of androgen receptor function inhibits proliferation of androgen refractory prostate cancer, demonstrating the importance of AR even at subnormal physiological levels of androgen. Mechanisms for ligand-independent AR reactivation include AR mutation, gene amplification, increased stability and nuclear localization, co-regulators and cross talk between different signal transduction pathways. Recent studies also suggest an acquired capacity of recurrent prostate tumors to biosynthesize testicular androgens from adrenal androgens or cholesterol, thereby reactivating the AR. Targeting AR for Therapy: Androgen ablation therapy, which is achieved by pharmaco-therapeutic (steroidal and non-steroidal antiandrogens) or surgical (subcapsular or subepididymal bilateral orchiectomy) means, is the standard initial systemic therapy for locally advanced or ▶metastatic prostate cancer. This therapy is based on inhibiting the synthesis of active androgen or inhibiting the physiological androgen from binding to AR. Novel therapeutic strategies for the androgen-depletion-independent stage of prostate cancer will focus on inhibiting expression of AR, blocking the binding of AR coactivators, and enhancing the binding of AR co-repressors. Using intelligent high-throughput screening and structural and computational chemistry, it may be possible to develop peptide antagonists, ▶small molecules or antisense oligonucleotides that target AR-coactivator binding surfaces. Potential drugs targeting the N-terminal domain may also prevent or delay the progression of both hormonal-dependent and -independent prostate cancer.

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Androgen-Independent Prostate Cancer

References 1. Shen HC, Coetzee GA (2005) The androgen receptor: unlocking the secrets of its unique transactivation domain. Vitam Horm 71:301–319 2. MacLean HE, Warne GL, Zajac JD (1997) Localization of functional domains in the androgen receptor. J Steroid Biochem Mol Biol 62:233–242 3. Dehm SM, Tindall DJ (2006) Molecular regulation of androgen action in prostate cancer. J Cell Biochem 99:333–344 4. Heinlein CA, Chang C (2004) Androgen receptor in prostate cancer. Endocr Rev 25:276–308 5. Debes JD, Tindall DJ (2004) Mechanisms of androgenrefractory prostate cancer. N Engl J Med 351:1488–1490

Anergy Definition A state of unresponsiveness, induced when the T cell antigen receptor is stimulated, which effectively freezes T cell responses pending a “second signal” from the antigen-resenting cell (▶costimulation).

Aneugen Definition

Androgen-Independent Prostate Cancer

Any agent that affects cell division and the mitotic spindle apparatus resulting in the loss or gain of whole chromosomes, thereby inducing ▶aneuploidy. ▶Micronucleus Assay

Definition AIPC. ▶Hormone refractory prostate cancer

Aneuploidy Definition

Androgens Definition Male sexual hormones that are mainly produced in the testicles. Testosterone is the principal androgen hormone. ▶Adjuvant Chemoendocrine Therapy

Anemia Definition Anemia is abnormally low hemoglobin concentration in the blood (females: 100 nm) and differ. For example, the peak emission of renilla luciferase is 480 nm, whereas firefly luciferase is 610 nm. Collectively, these factors ensure that bioluminescent signals from selected pairs of luciferase enzyme can be discerned upon the basis of substrate exclusivity as well as their spectral signature. This is highly useful as it enables the employment of powerful dual-labeled BLI studies, where the light generated by different luciferase enzymes can be detected sequentially to measure multiple parameters within the same cell or individual animal (e.g. viable tumor burden measured by renilla luciferase and the activation of a cellular process by firefly luciferase). The development of increasingly sophisticated ▶spectral unmixing image analysis techniques should soon make it possible to routinely discern the optical spectra from two different luciferases when both substrates are administered simultaneously. How is In Vivo Bioluminescence Detected? The intensity of light generated by luciferase labeled cells in a typical bioluminescence imaging experiment is sufficiently low that a highly sensitive light detector is needed to measure it. Such detectors are commercially available and typically comprise a cryogenically cooled CCD camera (Charge-Coupled Device; cooled to ≤−90°C to reduce thermal noise and increase light sensitivity) that is housed behind a lens within a completely light tight box. The non-visible levels of light associated with bioluminescence imaging can be detected by the pixels of the cold CCD, which results in a fully digitized and quantifiable 2-D map of light intensity across the field of view. An image of this light intensity map is then superimposed over a digital

Bioluminescence Imaging

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Bioluminescence Imaging. Figure 1 The above figure shows a series of bioluminescent images of an individual mouse taken at weekly intervals and shows the development of a spontaneous and bioluminescent prostate tumor. Note that the colors associated with these images and accompanying scale bar correspond to light intensity and do not reflect the color of detected light. This figure is reproduced with modification from Fig. 4A (Lyons SK, Lim E, Clermont AO et al (2006) Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice. Cancer Res 66(9):4701–4707) by copyright permission of the AACR.

photograph of the subject (taken in normal light conditions immediately prior to bioluminescence acquisition) to indicate the regions of the subject where labeled cells reside (Fig. 1). In a manner analogous to conventional photography, the exposure time and aperture settings of the CCD camera can be adjusted to modify the sensitivity of bioluminescence acquisitions. This ensures that CCD pixels do not become saturated when imaging relatively bright subjects and maximizes sensitivity when imaging relatively dim subjects. Computer software can be used to add together (or “bin”) the signals detected by adjacent CCD pixels to further increase imaging sensitivity, but this gain is made at a cost to image resolution. Software tools are also used to create “regions of interest” to quantitatively measure light emission from any area of the image in fully calibrated physical units (i.e. photons/s/cm2/▶steradian). Considerations to Maximize In Vivo Imaging Sensitivity Currently BLI is considered one of the most sensitive non-invasive preclinical imaging modalities when used in conjunction with small animal models of disease. There are, however, several important factors that will affect the sensitivity of any in vivo BLI approach and so influence the minimum number of cells that can be detected or the ability to visualize the activity levels of a cellular process above noise. One obvious issue relates to the extent of luciferase enzyme expression in the target cell; labeled cells that express relatively low levels of luciferase will be harder to detect than an equivalent number of labeled cells that express greater amounts of luciferase.

Another key issue is the depth of signal, as the wavelengths of light produced by the commonly employed luciferases are prone to scatter and absorption as they pass through mammalian tissue. Red wavelengths of light (>600 nm) pass through tissue with greater efficiency than relatively bluer wavelengths (12 Gy/h. VLDR (very low dose rate) radiation is used in permanent radioactive seed implants, at a dose rate of less than 40 cGy/h. Temporary implants are placed into the tumor/adjacent tissues in order to deliver LDR, MDR, or HDR treatments. VLDR implants typically reside permanently in the tissue implanted, but decay over the course of a few months. In the delivery of LDR or MDR radiation, the temporary implant stays in place over several hours, whereas HDR treatments usually last only a few minutes. LDR techniques involve the static placement of radiation isotopes within the applicators for a period of time. 137Cesium (137Cs) for gynecologic brachytherapy or 192Iridium (192Ir) for ▶gynecologic cancers or sarcomas are most commonly used. The radiation is either manually afterloaded by a physician or can be remotely afterloaded if a cesium selectron afterloader (for gynecologic brachytherapy) is available. HDR treatments involve a single 192Ir source fixed to a wire that is guided remotely by a computer. The HDR afterloader attaches to individual applicators by transfer tubes. Computer programming determines the position of the radiation isotope within the applicator, and calculates a radiation ▶isodose curve that may be manipulated by altering the dwell times. ▶Dwell positions are defined along the applicators every

Characteristics of some commonly used radioisotopes in the United States

Isotope

Half-life

Energy (MeV)

137

30 years 74 days 60 days 17 days

0.66 0.29–0.6 0.028 0.023

Cesium 192 Iridium 125 Iodine 103 Paladium

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2.5–10 mm, and the isotope remains at designated dwell positions for a preset time as determined by the optimized plan. LDR radiation may have a ▶radiobiological advantage over HDR radiation, as the normal tissue is more likely to be able to repair sublethal damage. Additionally, the continuous dose may prevent repopulation of the tumor cells, and the longer period of time that the cells are exposed to radiation allows the cell cycle to move through radio-resistant and radiosensitive phases. HDR radiation may lead to an increase in normal tissue toxicity if the total dose delivered compared to LDR is not decreased. It is important to fractionate the HDR radiation sufficiently and deliver as small a fraction size as feasible depending on the tissue treated, the indications for treatments and the amount of normal tissue in proximity to the source. In ▶cervical cancer brachytherapy, packing the vagina can reduce the amount of normal tissue exposed to radiation. Pulsed dose rate (PDR) brachytherapy uses an HDR afterloader and source but attempts to mimic the radiobiologic effect of LDR by giving a large number of very small fractions over a longer period of time than HDR.

Dose Calculations Historically, the dose delivered to a treatment volume was hand-calculated, based on one of three methods of implantation. The Paris and Quimby systems place parallel sources with uniform spacing and source activity, to give a higher central dose compared with the periphery. The Paterson–Parker method utilizes higher peripheral radioactivity compared with the centers resulting in increased ▶dose homogeneity throughout the implant. These methods have been replaced in several radiation oncology clinics by computer programs that utilize information gathered from imaging techniques such as CT scans to define the target volume and identify the implant geometry within that volume to calculate the dose.

Implantation Techniques The placement of the radiation source in relation to the treatment volume is the most important determinant in the effectiveness of brachytherapy. Therefore the techniques used depend on the location of the tissue being targeted. Surface applicators involve sculpting a radiotherapy delivery system on or around the target surface area. A superficial dose may be delivered to lesions of the skin or intraoperatively to exposed tumor beds. Intracavitary radiation utilizes orifices within the human body to introduce applicators in close proximity to the tumor. Common examples of intracavitary

radiation include gynecologic malignancies, in which the vagina, cervical os, and uterine cavity allow for the relatively easy placement of applicators. Other intracavitary treatments include the bronchus, esophagus, and rectum. Interstitial radiation entails passing catheters through normal tissue to reach the target volume or placing tubes within a surgical bed at the time of operation.

Clinical Applications of Brachytherapy Brachytherapy may be administered alone or in combination with external beam radiation, chemotherapy, or surgery to provide either cure or palliation for the patient. The most common uses of brachytherapy are discussed below.

Gynecologic Malignancies The most common gynecologic malignancy treated with brachytherapy in the United States is ▶endometrial cancer. Intracavitary radiation targets the vaginal vault in women thought to be at high risk of local recurrence to the vagina following definitive surgery. This treatment involves the insertion of a cylinder into the vagina (Fig. 1). LDR or HDR radiation may be used. The dose and fractionation of the radiation depend on both the dose rate and the patient’s history of prior external beam radiation therapy. The dose may be prescribed at either the surface of the applicator or at a depth, typically 5 mm, from the applicator. Cervical cancer is treated using a combination of external beam radiation with or without chemotherapy and brachytherapy, commonly referred to as a tandem and ovoid application. A central uterine tandem is placed through the cervical os into the uterine cavity. Vaginal ovoids or a vaginal ring or cylinder are secured to the central tandem (Fig. 2). Historically, cervical cancer brachytherapy was administered using LDR radiation, most commonly using tandem and ovoids placed twice with one week between treatments. In most centers, plain films assess the location of normal tissue structures; however, several radiation oncology clinics have acquired CT imaging capability, allowing for 3D imaging of the normal tissues and more accurate dose calculation. In more recent times, several centers have incorporated HDR radiation into the management of cervical cancer. HDR tandem and ovoid dose is delivered in minutes, and most commonly requires four or five separate insertions, with each treatment lasting several minutes. The HDR isodose curve approximates a standard LDR loading (Fig. 3). Vulvar and vaginal cancers are rare, but their treatment may involve interstitial or intracavitary radiation after external beam radiation.

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Brachytherapy. Figure 1 A high dose rate vaginal cylinder is inserted into the vagina to treat the vaginal surface for patients who have had a hysterectomy for uterine or cervical cancer. The applicator is attached to a brachytherapy board for stabilization.

Brachytherapy. Figure 2 Low dose rate Fletcher–Suit– Delclos tandem and ovoid applicator will be loaded with 137 Cs. The central tandem is inserted into the uterus. The ovoids may have plastic caps placed over them in order to fill the vaginal fornices. A flange rests outside the external os of the cervix. The apparatus is held in place by vaginal packing.

▶Prostate Cancer Brachytherapy in prostate cancer may be the sole treatment for low-risk disease or in combination with external beam radiation as a form of dose escalation. VLDR brachytherapy places permanent radioactive seeds of either 125I or 103 Pd into the prostate through the perineal skin, under image guidance and using catheters. The seeds remain permanently within the prostate and deliver a low dose of radiation continuously until they have decayed. HDR brachytherapy for prostate cancer is currently being investigated in research protocols.

Brachytherapy. Figure 3 High dose rate tandem and ovoid isodose curve demonstrates the 100% isodose line optimized to point A, a point 2 cm above and lateral to the cervical os.

Other Other cancers that can be treated with brachytherapy as part of combined care include head and neck cancers, including nasopharynx and tongue, breast cancers, sarcomas, thoracics and some gastrointestinal cancers.

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References 1. Williamson JF (2006) Brachytherapy technology and physics since 1950: a half century of progress. Phys Med Biol 51(13):R303–R325 2. Hoskin PJ, Bownes P (2006) Innovative technologies in radiation therapy: brachytherapy. Semin Radiat Oncol 16(4):209–217 3. Rivard MJ, Nath R (2006) Interstitial brachytherapy dosimetry update. Radiat Prot Dosimetry 120(1–4):64–69 4. Stewart AJ, Viswanathan AN (2006) Current controversies in high-dose-rate versus low-dose-rate brachytherapy for cervical cancer. Cancer 107(5):908–915 5. Nag S (2004) High dose rate brachytherapy: its clinical applications and treatment guidelines. Technol Cancer Res Treat 31(3):269–287

Bracken Fern Definition A worldwide diffuse plant belonging to Pteridium genus known to cause cancer naturally in animals. Bracken fern eating is also related to human cancer.

Bradykinin Definition Bradykinin is an active peptide of the kinin protein group. It consists of nine amino acid residues and is a potent vasodilator. ▶Kallikreins

B-raf-1 ▶B-Raf Signaling

BRAF1 ▶B-Raf Signaling

B-Raf Signaling T ILMAN B RUMMER Cancer Research Program, Garvan Institute of Medical Research, Sydney, NSW, Australia

Synonyms v-raf murine sarcoma viral oncogene homolog B1; B-raf-1; BRAF1; EC 2.7.11.1; MGC126806; MGC138284; RAFB1; p94; c-Rmil

Definition B-Raf signaling comprises the activation of the protooncogene product B-Raf and its downstream effectors and represents a key regulatory step in the activation of the canonical ▶MAP kinase pathway by various extracellular stimuli and oncogene products such as ▶RAS and activated receptor tyrosine kinases like ▶NTRK and ▶RET. Aberrant B-Raf activity as a result of somatic mutations is observed in 8% of human cancers.

Characteristics Physiological Aspects of B-Raf Signaling B-Raf is a member of the ▶Raf kinase family and represents an important component of the Ras/Raf/ MEK/ERK MAP kinase signal transduction pathway, which plays a pivotal role in growth control and differentiation. Dysregulation of this pathway is observed in about 30% of human tumors and represents an established mechanism for tumorigenesis. In their role as gatekeepers of this pathway, Raf-kinases appear as attractive targets for therapeutic intervention. The Raffamily contains three genes in vertebrates, A-Raf, B-Raf and Raf-1 as well as D-Raf and LIN-45 in Drosophila und Caenorhabditis, respectively. While the RAF1 gene displays a ubiquitous and prominent expression pattern, B-Raf is predominantly expressed in neuro-ectoderm derived tissues, placenta, the hematopoietic system and the testis. However, gene targeting experiments in mice and ▶DT40 B cells revealed that B-Raf represents the major ERK activator, even if it is expressed at barely detectable levels, whereas Raf-1 serves as an accessory ERK activator. Among the three mammalian isoforms, B-Raf displays the highest affinity towards its substrate MEK and has the highest activities in biological and ▶in vitro kinase assays. In many cell types, B-Raf plays a non-redundant role in the maintenance of ERK signaling induced by various extracellular signals and thereby regulates directly, or in concert with other signaling pathways, the expression of important target gene products such as growth factors and cytokines. The importance of B-Raf for the efficient expression of ERK-regulated target gene products is most likely

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explained by the fact that ERK activation is not only required for the induction of ▶immediate early genes transcription, but also for the stabilization of the resulting proteins by phosphorylation through sustained ERK signaling. The correlation between B-Raf expression and sustained ERK signaling has been implicated in various physiological processes such as lymphocyte activation, myelopoiesis, angiogenesis, development of extra-embryonic tissues as well as for the growth-factormediated survival of neurons and their effector functions. The discovery of germ-line mutations with mostly slight to moderate gain-of-function character in the SOS, KRAS, HRAS, SHP2/PTPN11, BRAF and MEK1/ 2 genes in patients suffering from the various ▶neurocardio-facial-cutaneous syndromes illustrates that tight control of this pathway upstream or at the level of the B-Raf/MEK interface is key to the normal development and homeostasis of many organs. B-Raf Signaling and Tumor Development The high biological relevance of B-Raf is also reflected in the discovery that ▶somatic alterations of the BRAF gene occur in about 8% of all human tumors with particular high frequencies in ▶melanoma (70%), ovarian (30%), thyroid (27%), colorectal and biliary tract carcinoma (both 15%). Many of the resulting mutant B-Raf proteins cause chronic ERK activation and transform a variety of cell types in vitro. Furthermore, the B-RafV600E oncoprotein, which is the most frequently found mutant and occurs in 7% of human tumors, induces neoplasms in transgenic mice and zebrafish. Apart from their established role as ERK activators, B-RafV600E and other oncogenic mutants have been shown to activate the ▶NF-κB pathway, although the exact mechanism for this oncologically relevant aspect of B-Raf sigaling remains elusive. Dysregulated B-Raf signaling in the absence of any BRAF mutations has been also implicated in various neoplastic diseases. For example, hyper-activation of wild type B-Raf has been observed in ▶Polycystic Kidney Disease. Similarly, over-expression and deregulation of B-Raf have been implicated in ▶Kaposi Sarcoma. Likewise, amplification and/or overexpression of the BRAF gene were described as alternative events to BRAF mutations in melanoma. Furthermore, B-Raf serves as an important signal transducer of upstream oncogene products such as RAS or activated receptor tyrosine kinases (RTKs) such as ▶RET, ▶NTRK, ▶Epidermal Growth Factor Receptor family members or the ▶Kit/Stem cell factor receptor. In many cell types where the chronic activation of the RAF/ MEK/ERK effector arm by these oncoproteins represents a major mechanism of cellular transformation, a mutual exclusivity is observed between mutations in BRAF or genes encoding its upstream activators. For example, gain-of-function mutations in either the

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▶RET, ▶NTRK, ▶RAS or BRAF proto-oncogenes account for 70% of papillary thyroid carcinoma and provoke similar transformed phenotypes indicating that the activation of B-Raf effectors such as ERK and NF-κB is a major driving force in thyrocyte transformation. Similar constellations have been described for RAS and BRAF in melanoma, colorectal and ovarian carcinoma. However, Ras and B-Raf transformed cells differ in their responsiveness to MEK-inhibitors showing that both oncoproteins, while having a large group of effectors in common, also trigger ▶oncogene addiction through distinct mechanisms. Oncogenic B-Raf not only mimics growth factor signaling, but also induces a variety of auto- and paracrine acting growth factors itself, e.g. ▶Heparin-Binding Epidermal Growth factor (EGF)-Like Growth Factor, chemokines and proinflammatory and angiogenic cytokines like ▶Vascular Endothelial Growth Factor A. Apart from tumor initiation, tissue culture experiments suggest that oncogenic B-Raf also contributes to tumor progression by inducing two additional key events in metastasis: the ▶Epithelial to Mesenchymal Transition of the oncogene-bearing cell and the ▶angiogenic switch in its environment through the aforementioned growth factors and cytokines. Aberrant B-Raf activity does not necessarily result in tumorigenesis unless profound changes in the regulatory network underlying cell cycle control have occurred. Through the ERK and NF-κB pathways, oncogenic B-Raf stimulates not only the production of positive cell cycle regulators such as Cyclin D1, but also induces negative regulators such as cyclin-dependent kinase inhibitors like p16INK4A. Consequently, chronic B-Raf/ ERK signaling ultimately results in cell cycle arrest and cellular ▶senescence. For example, melanocytes with an intact cell cycle control program become growth arrested by chronic B-Raf signaling and develop only into benign nevi. However, if important negative cell cycle regulators and tumor suppressor genes like ▶INK4A or ▶p53 are lost, oncogenic B-Raf signaling will trigger cell cycle progression and drive tumor development. B-Raf Structure and Regulation Like many other protein kinases, B-Raf is part of a large multi-protein complex or ▶signalosome in which the individual components regulate B-Raf conformation and activity through various protein-protein interactions in a dynamic spatio-temporal manner. Key to the understanding of the (dys-)regulation of B-Raf is the knowledge of its modular structure. B-Raf shares three highly conserved regions (CR) with the other members of the Raf-family (Fig. 1): the N-terminal CR1 contains the Ras-GTP binding domain, which initiates the interaction with activated Ras, and the Cystein-rich domain involved in the stabilization of

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B-Raf Signaling. Figure 1 Model of the B-Raf activation cycle. B-Raf contains three conserved regions: CR1 (blue) consisting of the Ras-binding domain (RBD) and the Cystein-rich domain (CRD), CR2 (green) and the kinase domain CR3 (blue). Inactive B-Raf resides in the cytoplasm in a closed, inactive conformation stabilized by 14-3-3. Interaction of B-Raf with a complex consisting of CK2 and the scaffold protein KSR results in phosphorylation of S446 (and perhaps S447) in the N-region thereby transferring B-Raf into a more open conformation. The constitutive basal phosphorylation of B-Raf at S446 suggests that a large fraction of B-Raf resides in this primed state. Interaction with activated Ras (Ras-GTP) leads to phosphorylation of T599 and S602 within the activation loop, which induces a conformational change within the CR3 and renders B-Raf active. B-Raf is supposedly inactivated by phosphatases, re-phosphorylation of the inhibitory residue S365 and transition into the closed conformation.

Ras/Raf interaction. The CR2 contains a negative regulatory serine residue (S365) that serves as a binding site for ▶14-3-3 proteins upon phosphorylation by ▶Akt and other kinases. The catalytic domain (CR3) harbors phosphorylation sites for Raf-regulating enzymes within two segments, the N-region and the ▶activation loop. B-Raf carries a second 14-3-3 binding motif around S729 at the C-terminal end of the CR3 domain, which is essential to couple B-Raf to its downstream effector MEK. Similar to the better-characterized Raf-1 isoform, B-Raf is activated by its interaction with small GTPases of the RAS family. Although no crystal structure for any of the full-length Raf-proteins is available, various experimental approaches imply that Raf activation is accompanied by a transition from a closed, autoinhibited into an open, active conformation in which the N-terminal lobe consisting of the CR1 and CR2 domains is displaced from the C-terminal lobe encompassing the CR3 (Fig. 1). The degree of auto-inhibition of B-Raf is influenced by the inclusion/exclusion of

amino acid sequences within the linker region between N- and C-terminal lobe, which are encoded by alternatively spliced, tissue-specific exons and various phosphorylation events. Among the latter, two phosphorylation sites within the CR3, the N-region and the activation loop, are of particular importance (Fig. 1). The introduction of negative charges into the N-region, which is located at the N-terminal end of the CR3 domain, plays a critical, multi-faceted role in Raf activation. While the N-region of Raf-1 is charged through phosphorylation of its S338SYY341-sequence in a RAS-dependent manner by Ser/Thr- and Tyr-kinases, the equivalent serine residues within the N-region of B-Raf (S446SDD449-motif) are phosphorylated in a constitutive and RAS-independent manner (Fig. 1). Although structural data are still missing, several lines of evidence propose that N-region phosphorylation primes B-Raf for activation at the membrane by reducing the affinity between N-terminal and C-terminal lobe. The significance of the aspartate residues, which are the functional equivalents of the phosphotyrosine residues in the SSYY-sequence of Raf-1, is

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twofold: firstly the negative charge of the aspartate residues are supposed to prime B-Raf for N-region phosphorylation by Casein Kinase 2 (CK2). Secondly, the D448 residue stabilizes the conformation of activated B-Raf through the formation of a salt-bridge with R506 within the αC-helix of the CR3. The important role of the SSDD-sequence is highlighted by the fact that mutation of the serine and/or aspartate residues results in drastic reduction of the basal in vitro kinase and biological activities. Furthermore, it has been suggested that the different mechanisms that supply the N-region of B-Raf and Raf-1 with negative charges, account not only for the aforementioned isoform-specific differences in the enzymatic, biological and transforming activities, but also predispose the BRAF gene for oncogenic hits. However, while tissue culture experiments demonstrated that the rare B-Raf E586K mutant indeed requires an intact SSDDsequence to induce MEK/ERK activation and oncogenic transformation, the biological activity of the most frequently found mutant, B-RafV600E, is not affected by N-region neutralization, at least not in experimental approaches involving the ectopic expression of this oncoprotein. The interaction with Ras recruits B-Raf to the plasma membrane followed by the phosphorylation of the

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activation loop residues T599 and S602 (Fig. 2). This phosphorylation event presumably leads to the dislocation of the activation loop relative to the overall catalytic domain thereby resulting in full B-Raf activity. The importance of the activation segment phosphorylation is established by the fact that mutation of these phosphorylation sites to alanine residues renders B-Raf resistant to extracellular signals and even to strong activators like oncogenic RasG12V. Conversely, mutations that mimic the phosphorylation-induced dislocation of the activation segment, such as BRAFV600E, lock B-Raf in an active conformation and confer high constitutive enzymatic and transforming activities to B-Raf independent of RAS. Consequently, these activation loop mutations are frequently found as somatic alterations of the BRAF gene in human tumors. Intracellular B-Raf activity is also regulated by the phosphorylation-dependent recruitment of ▶14-3-3 proteins in an opposing manner (Fig. 1). Binding of 14-3-3 proteins to phospho-S729 at the C-Terminus of B-Raf is essential to couple B-Raf to the MEK/ERK pathway. In contrast, phosphorylation of S365 within the CR2 by Protein kinases A, Akt or Serum-andGlucocorticoid-induced kinase (SGK) generates a second binding site for 14-3-3 proteins, which negatively regulates B-Raf activity, most likely through the

B-Raf Signaling. Figure 2 Modulation of B-Raf signaling. Extracellular signals received by various receptor classes trigger the activation of Ras-GTPases by stimulating their loading with GTP. Activated Ras not only recruits B-Raf and promotes its phosphorylation by unknown activation loop kinases, but also stimulates its homo- and hetero-dimerisation. The activity of B-Raf (and Raf-1) is fine tuned by a multitude of positive and negative modulators. The longevity of B-Raf/Raf-1 heterodimers is determined by a rapid negative feedback loop from ERK. In a delayed negative feedback loop, sustained B-Raf/ERK signaling also induces the transcription of Sprouty-2, a negative regulator of B-Raf.

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stabilization of the auto-inhibited conformation through the simultaneous binding of the 14-3-3 dimer to S365 and S729 (Figs. 1 and 2). 14-3-3 proteins are also involved in the RAS-stimulated formation of homodimers of B-Raf and its hetero-dimerisation with Raf-1 (Fig. 2). Indeed, B-Raf/Raf-1 hetero-dimers represent the most potent form of Raf-activity within the cell. Activated ERK limits the longevity of these dimers by targeting an evolutionary conserved phosphorylation motif at the C-terminus of B-Raf (Fig. 2). In addition, B-Raf activity is modulated by other components of the signalosome such as the ▶HSP90/Cdc37 chaperone complex and ▶scaffold proteins like Kinasesuppressor-of-Ras (KSR) and Connector-and-enhancerof-KSR (CNK). Membrane phospholipids such as phosphatidylserine (PS) and phosphatidic acid (PA) are also discussed as important regulators of Raf activation. B-Raf is also negatively regulated by Sprouty-2 and Rafkinase-inhibitory protein (RKIP), two proteins, which are both often down-regulated in human cancer raising the possibility that their epigenetic silencing represents an alternative mechanism to gain-of-function mutations in genes linked to the Ras/Raf/MEK/ERK pathway in human cancer. Similarly, B-RafV600E and other activation loop mutants are incapable of interacting with Sprouty demonstrating that the V600E mutation not only uncouples B-Raf from positive (interaction with Ras, N-region and activation loop phosphorylation) but also negative regulatory mechanisms. B-Raf as a Therapeutic Target The growing importance of B-Raf in tumor biology has fostered the development of therapeutic strategies aiming at either reducing the expression or activity of B-Raf or its downstream effector MEK. Various MEK inhibitors are currently in clinical trials and experiments in tissue culture and xenograft models indicate that tumor cells harboring the BRAFV600E mutation, but not those with RAS mutations, are highly “addicted” to ERK activity and are consequently particularly sensitive towards MEK inhibition. Similar results have been obtained in experiments in which the expression of B-RafV600E but not of wild type B-Raf was specifically abolished by allele-specific ▶RNA interference illustrating the importance of this oncoprotein for the maintenance of the tumor phenotype. Recent strategies also target B-Raf directly. The orally available multikinase inhibitor BAY 43-9006 (also known as Sorafenib or Nexavar), which was originally designed to block Raf-1, inhibits B-Raf as well as several receptor tyrosine kinases (RTKs) involved in neo-angiogenesis and tumor progression. However, it is assumed that the inhibition of the latter kinase class or the simultaneous inhibition of several kinases, rather than the inhibition of Raf itself, is responsible for the anti-tumor activity of BAY 43-9006, in particular in renal cell

carcinoma. A third approach employs the requirement of the HSP90/Cdc37 chaperone complex for the stability of B-Raf. In this regard, the HSP90 inhibitor ▶Geldanamycin was shown to trigger the degradation of B-Raf by disrupting its association with the HSP90/ Cdc37 chaperone complex. The stability of most activated B-Raf mutants, including B-RafV600E, appears to be more reliant on the chaperone complex than those of wild type B-Raf suggesting that tumor cells driven by BRAF mutations will be particularly sensitive to Geldanamycin.

References 1. Brummer T, Martin P, Herzog S et al. (2006) Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein. Oncogene 25:6262–6276 2. Galabova-Kovacs G, Kolbus A, Matzen D et al. (2006) ERK and beyond: insights from B-Raf and Raf-1 conditional knockouts. Cell Cycle 5:1514–1518 3. Ritt DA, Zhou M, Conrads TP et al. (2007) CK2 is a component of the KSR1 scaffold complex that contributes to Raf kinase activation. Curr Biol 17:179–184 4. Schreck R, Rapp UR (2006) Raf kinases: oncogenesis and drug discovery. Int J Cancer 119:2261–2271 5. Wellbrock C, Karasarides M, Marais R (2004) The RAF proteins take centre stage. Nat Rev Mol Cell Biol 5:875–885

B-Raf Somatic Alterations T ILMAN B RUMMER Cancer Research Program, Garvan Institute of Medical Research, Sydney, NSW, Australia

Definition Somatic alterations of the BRAF gene in cancer, either caused by point mutation or genomic rearrangement of the BRAF proto-oncogene.

Characteristics The Ser/Thr-kinase B-Raf, a product of the human BRAF proto-oncogene, plays a pivotal role in the activation of the classical ▶ERK/MAP kinase pathway that is involved in the control of proliferation and differentiation of various tissues. Consequently, alterations of the expression level or the activity of B-Raf are associated with malignancies like ▶polycystic kidney disease and various cancers. Proto-oncogenes can be converted into oncogenes by point mutations, amplifications, genomic rearrangement, e.g. translocation or inversion, or by retroviral transduction. Interestingly, all four mechanisms of oncogene activation have


been documented for the BRAF genes in human and/or animal tumors. History of the BRAF Proto-Oncogene The discovery of the raf-oncogenes originates back to the isolation of the chicken Mill Hill 2 (MH2) retrovirus by Begg in 1927. Genetic studies in the 1980s demonstrated that MH2 contains two unrelated retroviral oncogenes that were designated as v-myc and v-mil. Subsequent analysis of v-mil revealed a high sequence homology to the v-raf oncogene of the murine sarcoma retrovirus 3611. Further analyses showed that both v-mil and v-raf arose independently by retroviral transduction from the chicken c-mil and mammalian raf-1 genes, respectively. In 1988, a v-mil related oncogene was discovered in ▶transforming retroviruses that were generated by passaging the nononcogenic Rous-associated virus type 1 (RAV-1) on embryonic chicken neuroretina cells. Due to its origin in retinal cultures, this relative of v-mil was designated as v-Rmil. Subsequent studies showed that v-Rmil was generated by retroviral transduction from the

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proto-oncogene c-Rmil, which is related but distinct to the c-mil/raf-1 proto-oncogenes and represents the avian ▶orthologue of BRAF. Similar to the avian c-Rmil/B-raf gene, the BRAF genes of other vertebrates display a conserved exon/intron structure with 18–20 coding exons, in which the first eight exons encode the N-terminal autoinhibitory region (Fig. 1a). At the same time as v-Rmil was discovered, the human BRAF oncogene was identified in a ▶NIH3T3 transformation assay using ▶Ewing sarcoma DNA. Importantly and in striking analogy to v-Mil and v-Raf, both the v-Rmil and the B-Raf oncoprotein from the Ewing sarcoma isolate represent N-terminally truncated B-Raf proteins (Fig. 1b), which have lost the N-terminal regulatory lobe and consequently the ability for autoinhibition (▶B-Raf signaling). Therefore, all these Rafoncoproteins display constitutive activity and induce chronic activation of the ERK pathway. Thus, loss of exons encoding for the auto-inhibitory N-terminal moiety is a common mechanism of oncogenic activation of raf proto-oncogenes. This notion is further supported by recent experiments showing that the

B-Raf Somatic Alterations. Figure 1 B-Raf oncoproteins. (a) Situation for wildtype B-Raf. In its inactive state, the BRAF proto-oncogene product resides in a closed conformation stabilized by 14-3-3 proteins. Activation of B-Raf by activated RAS results in a displacement of the N-terminal auto-inhibitory region (Conserved region (CR) 1 in blue, CR2 in green) from the CR3 or kinase domain (red) allowing access of the activation loop kinase to the the TVKS-motif. Phosphorylation of T599 and S602 within this motif renders B-Raf active. (▶B-Raf signaling). (b) Schematic representation of v-Rmil. Due to the retroviral transduction event, the genome of RAV encodes for a fusion protein flanking the B-Raf (CR3) kinase domain with an N-terminal portion encoded by the env gene and a C-terminal moiety encoded by a portion of the gag gene. Both the env and gag genes are integral components of retroviral genomes. (c) Schematic representation of AKAP9-B-Raf. (d) Schematic representation of B-Raf proteins with point mutations as exemplified for the activation loop mutant B-RafV600E.

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murine Braf gene represents a frequent integration point for the Sleeping Beauty transposon. All transposon integrations were observed between exons 9 and 10 resulting in a disruption of the coding sequence of full-length B-Raf and expression of an N-terminally truncated B-Raf protein with an intact kinase domain and structural similarity to v-Rmil and v-Raf. However, it should be mentioned that neither retroviral B-Raf oncogenes nor transposon-mediated oncogenic activation of the BRAF gene have been observed in human beings. Likewise N-terminal truncations of B-Raf like those found in the original publication on the human B-Raf gene, which most likely represents a transfection artifact, have not been found in human tumors until recently. Nevertheless, the human BRAF protooncogene is affected by somatic alteration in about 7% of human tumors. The following alterations are observed in human tumors: Chromosomal Aberrations A recent study has identified an oncogenic BRAF allele in about 11% of papillary thyroid carcinomas (PTC) in children and adolescents that had been exposed to radiation following the Chernobyl nuclear power plant station accident in 1986. This oncogene was generated via a paracentric inversion of the BRAF locus on chromosome 7q34 resulting in an in-frame fusion with exons 1–8 of the A-kinase anchor protein 9 (AKAP9) gene on 7q21-22. The resulting AKAP9-B-Raf fusion protein is made up by exons 1–8 of AKAP9 and exons 9–18 of BRAF. Thus, this AKAP9-B-Raf protein contains an intact kinase domain, but the auto-inhibitory N-terminal regulatory domain of B-Raf is replaced by the AKAP9 moiety, which cannot confer autoinhibition (Fig. 1c). Consequently, the activity of this fusion protein is, similar to the situation in v-Rmil, unrestrained and able to transform NIH3T3 cells. Interestingly these mutations were only found in tumors that had developed within a short latency period suggesting that this chromosomal aberration is a driver of radiation induced PTC rather than being a secondary event. Another recent study has reported the occurrence of chromosomal translocations involving the human BRAF gene in two cases of large congenital melanocytic nevi, which can progress into malignant melanoma. In both cases and similar to the situation of the AKAP9B-Raf fusion protein, these translocations give rise to fusion proteins, which lack the exons encoding the auto-inhibitory N-terminal regulatory domain, but again contain an intact B-Raf kinase domain. Somatic and Germ-Line Point Mutations Although Raf proteins were implicated early on as important effectors of human oncoproteins, e.g. Ras, they were not considered as frequent mutational targets

in cancer. In 2002, however, the ▶cancer genome project (CGP) reported a high frequency of somatic point mutations in the human BRAF gene in malignant melanoma (27–70%). Subsequent studies also revealed high point mutation frequencies in thyroid (36–53%), ovarian (30%), biliary (14%) and colorectal cancer (522%) and lower frequencies in a wide range of other human tumors. It is estimated that the human BRAF gene bears somatic mutations in about 7% of all human cancers. In contrast to the aforementioned alterations of the BRAF gene, these point mutations do not affect the overall primary structure of B-Raf (Fig. 1d), but mostly bypass critical regulatory events required for the activation of wildtype B-Raf (▶B-Raf signaling). While mutations in the human CRAF gene are still considered as a very rare event, over 40 different somatic mutations, involving 24 different codons, have been identified in BRAF since 2002. Most alterations represent point mutations, however, codon deletions or in-frame insertions have been occasionally identified as well. A detailed overview on these mutations can be found on the CGP homepage (http://www.sanger.ac.uk/perl/genetics/ CGP/cgp_viewer?action =gene&ln=BRAF). Most mutations cluster within the activation loop codons and, to a lesser extent, within the nucleotide sequence encoding the glycine-rich loop (also known as P-loop; Fig. 1d). Among the activation segment mutations, the thymidine to adenine transversion at nucleotide 1799, which results in the substitution of valine 600 within the T599V600KS602-motif in the activation segment by glutamate, represents the most common mutation and is found in 6% of human cancers. Structural analysis of the B-Raf kinase domain suggests that the inactive conformation of B-Raf is stabilized by a hydrophobic interaction between the activation loop residues with the glycine rich loop, with V600 and F467 playing key roles in this process. Upon activation of wildtype B-Raf by activated Ras, T599 and S602 in the activation loop become phosphorylated by an unknown kinase resulting in the disruption of the inhibitory hydrophobic interaction between the activation and glycine rich loop and consequently full activation of B-Raf (Fig. 1a). In a similar way, any mutation in either the activation or glycine-rich loop mutation that disrupts this hydrophobic interaction, e.g. replacement of V600 by bulky and/or charged amino acids like glutamate, mimics the activated state and confers constitutive activity to B-Raf. As described in ▶B-Raf signaling, the current model of B-Raf activation proposes a sequence of positive regulatory events leading to a relief of auto-inhibition by the N-terminal lobe followed by activation loop phosphorylation and full B-Raf activation. According to this sequential model of B-Raf activation, the V600E mutation not only bypasses these events, but is also

Brain Microvascular Endothelial Cells

able to counteract auto-inhibition, which would explain why this mutation is so frequently found in tumors driven by chronic ▶B-Raf signaling. However, why the V600E mutation occurs more frequently than any other activation loop or glycine-rich loop mutations that would also disrupt the inactive conformation, remains controversial. The V600E codon might represent a mutational “hotspot” or, due to still unknown details of B-Raf activation, might be an extremely efficient oncogene that subjects B-RafV600E expressing cells to a particularly strong positive selection. It should be also mentioned that the occurrence of the BRAFV600E allele in colorectal cancer is correlated with ▶microsatellite instability (MSI) and widespread methylation of CpG islands in a highly statistically significant manner. However, it remains to be clarified as to whether the MSI phenotype that is caused by absence or hypo-activity of DNA mismatch repair genes and is characterized by a widespread methylation of CpG islands, reflects a cause or a consequence of dysregulated B-Raf signaling. In 2006, germ-line mutations in the human BRAF gene were found in patients suffering from the ▶cardiofacial-cutaneous (CFC) syndrome. Some of these mostly gain-of-function mutations in CFC patients are also found in cancer, however, mutations conferring high activity to B-Raf such as BRAFV600E have not been found. Indeed, knock-in experiments in mice have shown that ubiquitous expression of B-RafV600E confers early embryonic lethality suggesting that high levels of chronic B-Raf activity would not be tolerated during human development as well. However, it should be noted that not all of the point mutations found in cancer or CFC patients represent obvious gain-of-function mutations as some of them actually display impaired in vitro kinase activity. Nevertheless, these impaired activity mutants still appear to activate the ERK pathway within the cell, either through stimulating the activity of Raf-1 in Raf-1/B-Raf heterodimers or, potentially, by acting as a buffer against negative regulators, e.g. RKIP, or negative feedback loops controlling ▶B-Raf signaling. Amplification Amplification of the BRAF locus is another mechanism contributing to elevated B-Raf protein expression and activity. Studies in malignant melanoma have described the amplification of BRAF alleles with point mutations such as V600E at the expense of the wildtype BRAF allele. Genetic experiments have identified B-Raf as an important factor for ERK activation under basal and steady state conditions (▶B-Raf signaling). Experiments in various cell types have shown that increasing levels of wildtype B-Raf enhanced basal and steady state ERK signaling suggesting that over-expression of endogenous

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wildtype B-Raf might contribute to tumorigenesis. Indeed, amplification of the BRAF locus in the absence of any mutations in exon11 (Gly-rich loop) and exon 15 (activation loop) was described as an important contributor to the proliferation of malignant melanoma cell lines.

References 1. Ciampi R, Knauf JA, Kerler R et al. (2005) Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. J Clin Invest 115:94–101 2. Collier LS, Carlson CM, Ravimohan S et al. (2005) Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436:272–276 3. Dessars B, De Raeve LE, Housni HE et al. (2007) Chromosomal translocations as a mechanism of BRAF activation in two cases of large congenital melanocytic nevi. J Invest Dermatol 127:1468–1470 4. Dhomen N, Marais R (2007) New insight into BRAF mutations in cancer. Curr Opin Genet Dev 17:31–39 5. Tanami H, Imoto I, Hirasawa A et al. (2004) Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines. Oncogene 23:8796–8804

Bragg (Curve) Peak Definition A characteristic dose distribution of a single-energy charged particle beam (e.g. protons) with a sharp peak close to the end of the range. The range is a distance that particles travel inside the medium. ▶Radiation Oncology

Brain Capillaries ▶Blood–Brain Barrier

Brain Microvascular Endothelial Cells ▶Blood–Brain Barrier

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Brain Tumors

Brain Tumors YASUYUKI H ITOSHI , PAULA M. K UZONTKOSKI , M ARK A. I SRAEL Departments of Pediatrics and of Genetics, Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH, USA

Definition

Primary ▶brain tumors present most commonly as ▶meningioma or various grades of ▶astrocytoma. ▶Gliomas constitute 78% of all malignant brain and central nervous system tumors. It is estimated that 20,500 individuals will be diagnosed with cancer of the brain and nervous system in 2007, or about 1.4% of all newly occurring malignancies. Of those diagnosed, there will be 10% more men than women. Primary brain and nervous system cancers will account for 2.3% of the estimated 560,000 cancer deaths in 2007. Based on the most recent report of the Central Brain Tumor Registry of the United States, benign tumors of the CNS arise in numbers comparable to malignant brain tumors. In children and young adults, brain tumors are responsible for 25% of all cancer-related deaths, second only to leukemia in this age group. The estimated 5-year relative survival rate for malignant brain tumors is 29%, but there is much variation in survival, depending on tumor histology. The 5-year survival rate exceeds 91% for pilocytic astrocytomas, but is less than 4% for glioblastomas. Generally, survival decreases with increasing age at diagnosis.

Characteristics Classification and Pathology The cell of origin of commonly occurring brain tumors is not known, although recent evidence suggests that these tumors arise either from ▶neural stem cells or from other cells that take on many characteristics of neural stem cells as a result of malignant transformation caused by the

Brain Tumors. Table 1 Tumor type

activation of oncogenes and the inactivation of ▶tumor suppressor genes within the cells. Pathologically, these tumors are classified according to the World Health Organization (WHO) nomenclature and grading criteria. Tumors that share cytologic and histologic evidence of astrocytic differentiation are known as ▶astrocytoma and are the most frequent primary intracranial neoplasms. Their neuropathological appearance is highly variable. Tumors with evidence of oligodendroglial differentiation are known as ▶oligodendroglioma. Some tumors that have cells reminiscent of both lineages are known as ▶mixed oligo-astrocytomas. Each of these tumor types can be graded histologically according to a four-tiered system of increasing malignancy from Grades I through IV. Grade I, for example, has an excellent prognosis following surgical excision, and Grade IV, ▶glioblastoma multiforme, has multiple features of clinical aggressiveness and is typically incurable. Hypercellularity with evidence of high mitotic activity, nuclear and cytoplasmic atypia, endothelial proliferation, and necrosis correspond closely to tumor virulence and are most characteristically present in Grade IV tumors. The overwhelming majority of gliomas arising in adults are high-grade and arise in a supratentorial location. Highgrade tumors do not have a clear margin separating neoplastic and normal tissue. This finding is consistent with the observation that tumor cells usually have infiltrated adjacent normal brain by the time of diagnosis, when complete resection is oftentimes not possible. Tumor cells capable of initiating new tumor foci can now be recognized as tumor stem cells. Cytogenetic examination of chromosomes within the cells of a ▶brain tumor has revealed characteristic regions that tend to be altered in specific tumor types (Table 1). Frequent sites for chromosomal DNA loss in astrocytic tumors include chromosomes 17p, 13q, and 9. In oligodendroglioma, DNA from 1p and 19q is frequently lost, and in ▶meningiomas, 22q is often lost. Molecular genetic analysis can also reveal evidence of tumor-specific genetic alterations at sites where chromosomes appear normal upon cytogenetic

Cytogenetic and genetic alterations in brain tumors Chromosomal alteration

Genetic changes Oncogene

1p–, 7+, 9p–, de110, 11p–, 12q–, 12q+, 13q–, 13q+, 18p+, 17p–, 17q–, 19p–, 19q–, 22q–, DMsa Oligodendroglioma 1p–, 19q–, 7+, 10– Astrocytoma

Medulloblastoma a

EGFR, PDGFRA, KIT, CROS, MET, CDK4, NEU, RAS, MDM2, GLI, CMYC EGFR

5p+, 5p–, 5q–, del6, 8p–, 8q+, 9q–,10q–, GLI, CMYC, CTNNB1 17p–, 17q–, 17q+, 21q+

DMs, double minute chromosomes.

Tumor suppressor gene P53, RB1, NF1, PTEN, DMBT, CDKN2A, CDKN2D, RASSF1A TP53, PTEN, CDKN2A, CDKN2D, PIK3CA TP53, PTCH, SUFU, APC, RASSF1A

Brain Tumors

analysis. Using a variety of molecular technologies, it has been possible to document the alteration of many different genes in brain tumors, particularly astrocytic tumors (Table 1). While the particular constellation of genetic alterations that activate oncogenes and inactivate tumor suppressor genes varies among individual brain tumors that appear to be histologically indistinguishable, an accumulation of mutations is typically associated with increasingly aggressive malignant behavior. Glioblastoma multiforme (▶GBM) typically presents without evidence of a precursor lesion, referred to as de novo or primary GBM. These tumors typically have evidence for chromosome 10 deletions in the region where the tumor suppressor ▶PTEN is known to be located, and activation of the ▶epidermal growth factor receptor (▶EGFR) gene either by amplification or deletion of 275 amino acids from the extracellular domain of the receptor. This and closely related mutations occur in 60% of GBM. EGFR is the gene most frequently activated in malignant astrocytomas. EGFR amplification and activation by mutation occurs in 5% of low-grade astrocytomas and about 30% of GBM, indicating that this molecular change is principally associated with the progression from lowor intermediate-grade neoplasia to high-grade astrocytic neoplasia. In fewer than 20% of cases, GBM arises in association with progressive genetic alterations after the diagnosis of a lower-grade astrocytoma. These tumors are referred to as secondary GBMs. The most widely described alterations are amplification or overexpression of the ▶PDGF receptor, mutations of ▶p53 or ▶MDM2 and ▶INK4a, and loss of PTEN (Table 1). Deletion of the ▶CDKN2 gene, which encodes the cyclin-dependent kinase inhibitor p16, has been reported to occur in 40–70% of glioblastoma. The ▶RB1 tumor suppressor gene is homozygously deleted or mutated in about 30% of high-grade gliomas. The protein products of tumor suppressor genes are proteins that act to regulate or suppress cell growth or promote cell death. These genes are inactivated during tumorigenesis, and several such genes have been implicated in the development of astrocytoma. Occasionally, inactivation of one of these alleles in the germline can occur without disturbing development, and patients who carry germline mutations of some tumor suppressor genes can be predisposed to the development of cancer. Several inherited cancer-predisposition syndromes are known to be associated with the development of different brain tumors. Patients with ▶Li-Fraumeni syndrome, caused by an inherited constitutional p53 mutation, have a predisposition for the development of brain tumors. The p53 gene, located on chromosome 17p, has been found to influence multiple cellular functions thought to be important in tumorigenesis. p53 mutations have been reported in sporadically arising astrocytic tumors of all grades, occurring in 40% of astrocytomas,

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in 30% of ▶anaplastic astrocytomas, and in a slightly smaller fraction of GBM. Other brain tumor predisposition syndromes associated with the inactivation of one copy of a particular gene in the germline include ▶neurofibromatosis type 1 (NF1 gene), which is associated with meningioma and optic glioma; ▶neurofibromatosis type 2 (NF2 gene), which is associated with acoustic neuroma and glioma; familial ▶retinoblastoma (▶Rb gene), which is associated with retinoblastoma and pinealoblastoma; ▶von Hippel-Lindau syndrome (VHL gene), which is associated with cerebellar hemangioblastoma; ▶tuberous sclerosis (TSC1 and TSC2 genes), which is associated with subependymal giant cell astrocytoma; ▶Turcot syndrome (▶APC gene), which is associated with astrocytoma and ▶medulloblastoma; and Gorlin’s syndrome (PTCH gene), which is associated with desmoplastic medulloblastoma. The second most common primary brain tumor is oligodendroglioma, which has a more benign course than astrocytoma. Many ▶gliomas have mixtures of cells with astrocytic and oligodendroglial features. If this mixed histology is prominent, the tumor is termed a mixed glioma or an ▶oligoastrocytoma. Many investigators believe that the greater the oligodendroglial component, the more benign the clinical course. The presence of such histologic characteristics as mitosis, necrosis, and nuclear atypia generally is associated with a more aggressive clinical course. If these features are prominent, the tumor is termed a malignant oligodendroglioma. The highest grade oligodendroglioma are indistinguishable from glioblastoma multiforme. Other malignant primary brain tumors include ▶primitive neuroectodermal tumors (▶PNET) such as ▶medulloblastoma, ▶ependymoma, and ▶atypical teratoid/▶rhabdoid tumors; ▶germinomas; and CNS ▶lymphoma. Cerebral PNETs and medulloblastoma, a PNET that arises in the posterior fossa, are highly cellular malignant tumors thought to arise in neural precursor cells. These tumors are difficult to distinguish from one another and typically appear histologically as sheets of small round malignant cells. Germline mutation of PTCH and SUFU in rare patients has called attention to the importance of sonic hedgehog signaling in medulloblastoma. Similarly, APC germline mutations in rare patients implicate WNT signaling as well. These tumors most commonly occur in children. Ependymomas are rare tumors, and when these occur in children, they typically are within the fourth ventricle, where they are thought to arise from cells lining the fourth ventricle. In adults, they arise more frequently in the spinal cord. Patients with neurofibromatosis type 2 are at increased risk of developing ependymoma, and 30% of sporadically occurring tumors exhibit deletion of Ch22q where the NF2 gene is located. Histologically, these tumors exhibit diagnostic ependymal rosettes.

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Brain Tumors

Atypical teratoid/rhabdoid tumors histologically appear as fields of undifferentiated malignant neuroectodermal cells that are indistinguishable from PNET, except for infrequent cells that exhibit evidence of rhabdoid differentiation and the presence of mesenchymal and epithelial elements. Germinomas arise most commonly during the second decade of life at midline locations. Both malignant and benign variants occur frequently. These tumors present with hypothalamic–pituitary dysfunction and visual field deficits. Primary CNS lymphomas are most commonly seen in immunocompromised patients, and have a clinical presentation similar to other primary brain tumors with signs and symptoms referable to cerebral and cranial nerve involvement. Imaging studies typically demonstrate a uniformly enhancing mass lesion. Secondary CNS lymphoma almost always occurs in association with the progression of systemic disease. Several kinds of tumors that are most often benign also occur in the nervous system. ▶Meningiomas are derived from cells of the arachnoid membranes. They are more frequent in women than in men, with a peak incidence in middle age. Meningiomas rarely have histological evidence of malignancy. Other tumors that have a benign clinical course include giant cell astrocytomas, pleomorphic xanthroastrocytomas, neurocytomas, and gangliogliomas. Colloid cysts, dermoid cysts, and epidermoid cysts also occur in the brain. Clinical Presentation of Brain Tumor Patients The most common symptoms that bring patients with a tumor arising in the brain to their physician include a slow progressive focal neurological disability, or a nonfocal neurological syndrome such as headache, dementia, gait disorder, or seizure. Other systemic symptoms suggest a tumor from some other location that may have metastasized to the brain, since patients with primary brain tumors typically do not exhibit systemic symptoms. Patients with primary brain tumors rarely have any biochemical abnormalities; thus CT (Computerized Tomography) and MR (Magnetic Resonance) imaging are key diagnostic modalities for the identification of brain tumors. The characteristic imaging features of brain tumors are mass effect, edema, and contrast media enhancement. Positron emission tomography (PET) scanning and single photon emission computed tomography (SPECT) have ancillary roles in the imaging of brain tumors. Meningiomas and other slow-growing tumors may be found incidentally on a CT or MRI scan or they may present with a focal seizure, a slow progressive focal deficit, or symptoms of increased intracranial pressure. As described above, brain tumors are also recognizable in many inherited syndromes including von Recklinghausen syndrome (neurofibromatosis type 1), neurofibromatosis type 2, Li-Fraumeni syndrome,

▶Multiple endocrine neoplasia type 1, tuberous sclerosis, Turcot syndrome, and Gorlin syndrome. Clinical Management of Brain Tumor Patients and Prognosis Stereotaxic needle biopsy may establish the histological diagnosis of primary brain tumor, although open biopsy is also often utilized to establish the diagnosis. The primary modality of treatment for most primary brain tumors is surgery. The goals of surgery are to obtain tissue for pathological examination, to remove tumor, and to control mass effect. In the case of low-grade and benign tumors, the removal of tumor tissue can be curative or contribute substantially to extending the time to symptomatic progression. In higher-grade tumors, the role of surgery in contributing to curative therapy is less clearly defined, but in younger patients most surgeons aggressively pursue the removal of as much tumor as possible. Following total excision of an ependymoma, the prognosis is excellent. However, many ependymomas cannot be totally excised. Following surgery, ▶radiation therapy has been shown to prolong survival and improve the quality of life of patients with high-grade glioma, PNET, ependymoma, or meningioma when malignant histologic elements can be pathologically identified within the tumor. The medical management of most brain tumors is symptomatic, although a role for chemotherapy is clearly defined in oligodendroglioma and medulloblastoma. In patients with oligodendroglioma, a combination of procarbazine, lomustine, and vincristine has been shown to be most effective in patients with a deletion of Ch1p. Various combination therapies have been shown to contribute to the treatment of medulloblastoma, which has a propensity to spread throughout the neuroaxis. If medulloblastoma is limited to the posterior fossa and completely resected, this tumor has a good prognosis. Temozolomide given during radiation therapy for glioblastoma has been shown to contribute to longer overall survival time. Chemotherapy and radiation typically play a central role in the treatment of germinomas, although there is a role for surgery as well. Patients whose brain tumors are associated with surrounding edema benefit symptomatically from the administration of high doses of glucocorticoids. Anticonvulsants are useful in the control of seizures. Some glioma patients receive anticoagulation therapy to avoid complications of venous thrombosis that occurs in these patients. The prognosis for patients with primary brain tumors varies greatly as a function of the histology and location of the tumor. Benign tumors are often cured by surgery alone. ▶Germinomas and medulloblastomas are more sensitive to cytotoxic therapies than are other brain tumors, and the prognosis for patients with these tumors is generally better than for patients with high-grade

BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk

glioma. In modern studies, the median survival of patients with high-grade glioma is 1–2 years. Complications of Therapy Neurological damage associated with surgical intervention presents a key challenge in the management of brain tumors. Furthermore, the nervous system is vulnerable to injury by therapeutic radiation, and this is frequently manifested by neuropsychological compromise and disability, particularly in very young children who have been treated with high doses of radiation. Pathologically, there is demyelination, hyaline degeneration of small arterioles, and eventually brain infarction and necrosis. Endocrine dysfunction is also commonly seen when the hypothalamus or pituitary gland has been exposed to therapeutic radiation. Depending on the radiated field, secondary tumors such as glioma, meningioma, sarcoma, and thyroid cancer occur following radiation therapy. Toxicities associated with chemotherapy can be significant, but they are not usually different from the toxicities associated with comparable treatments for tumors arising elsewhere in the body. ▶Neuro-Oncology: Primary CNS Tumors

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the growth and differentiation of new neurons and synapses. In the brain, it is active in the hippocampus, cortex, and basal forebrain—areas vital to learning, memory, and higher thinking. BDNF was the second neurotrophic factor to be characterized after NGF.

Branching Morphogenesis Definition Branching morphogenesis refers to the formation of tree-like networks of epithelial tubes through reiterated cycles of branch initiation, branch outgrowth and branch arrest. This process relies on the precise spatio-temporal control of gene expression, cell proliferation and migration, and is essential for the physiological function of many organs including the lung, the vascular system and the kidney. ▶Sprouty

References 1. Central Brain Tumor Registry of the United States (2005) Statistical report: primary brain tumors in the United States, 1998–2002. Central Brain Tumor Registry of the United States, Illinois 2. Jemal A, Siegel R, Ward E et al. (2007) Cancer statistics. CA Cancer J Clin 57(1):43–66 3. Beger M, Prados M (2007) Berger M, Prados M (eds) Textbook of neuro-oncology. Elsevier Saunders, Philadelphia 4. World Health Organization Classification of Tumours (2000) Tumours of the nervous system. In: Kleihues P, Cavenee WK (eds) Pathology and genetics. International Agency for Cancer, Lyon 5. Bigner DD, McLendon RE, Bruner JM (1998) Russell OS, Rubinstein LJ (eds) Pathology of tumors of the nervous system. 6th edn. University Press, New York

BRCA1-associated Ring Domain (Gene/Protein) 1 ▶BARD1

BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk P ETER D EVILEE

Brain-Derived Neurotrophic Factor

Leiden University Medical Center, Leiden, The Netherlands

Definition Definition BDNF; Is a neurotrophin in the central nervous system (CNS), predominantly in the brain and the periphery. This is in contrast to ▶NGF acting predominantly in the peripheral nervous system. It acts on certain neurons of the CNS and the peripheral nervous system that helps to support the survival of existing neurons and encourage

Mutations in the ▶breast cancer genes BRCA1 and BRCA2 cause elevated risks to breast and ovarian cancer. BRCA1 maps to chromosome 17 (band q21), BRCA2 maps to chromosome 13 (band q12). At the genetic level there are interesting analogies between the two genes, even though they are not detectably related by sequence. Both genes are large (coding regions of 5.6 and 10.2 kb, respectively),

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complex (22 and 26 coding exons, respectively), and span about 80 kb of genomic DNA. Both have extremely large central exons encoding >50% of the protein. The majority of the mutations in both genes detected to date lead to premature termination of protein translation, presumably resulting in an inactive truncated protein. Gene changes are distributed nearly ubiquitously over the coding exons and immediate flanking introns. Even though more than half of all mutations are found only once, many mutations have been detected repeatedly in certain populations. For most of these, this has been shown to be the result of a founder effect: these mutations arose a long time ago, and have since spread in the population. Typical founder mutations are the 1185delAG and 15382insC in BRCA1 and 26174delT in BRCA2 that have a joint frequency of about 2.5% among individuals of Ashkenazi Jewish descent.

Characteristics Clinical Characteristics Female carriers of a deleterious BRCA1 mutation were estimated by the Breast Cancer Linkage Consortium (BCLC) to have an 87% cumulative risk to develop breast cancer before the age of 70, and 40–63% risk to develop ovarian cancer before that age (Fig. 1). The gene frequency of BRCA1 was estimated at 1 in 833 women, implying that 1.7% of all breast cancer patients diagnosed between the ages of 20 and 70 are carrier of such a mutation. The estimated cumulative risk of breast cancer conferred by BRCA2 reached 84% by age 70 years. The corresponding ovarian cancer risk was 27% (Fig. 1). These estimates imply that BRCA2

mutations are about as prevalent as BRCA1 mutations. It has been suggested that the ovarian cancer risks are dependent on the position of the mutation in the gene, for BRCA1 as well as BRCA2 mutations. There is also some evidence that cancer risks can be modified by other factors. For example, a strong variability in phenotype can be seen among families segregating the same mutation. This can range from early-onset breast cancer and ovarian cancer, to late-onset breast cancer without ovarian cancer. Even within a single pedigree, ages of onset of cancer can vary substantially. It seems likely that environmental and hormonally related factors (smoking, oral contraceptives) importantly co-determine disease outcome in carriers. Molecular and Cellular Characteristics Tumor Suppressor Genes The first clues to the roles of BRCA1 and BRCA2 in tumorigenesis were genetic. The fact that most germline mutations are predicted to inactivate the protein, and the observed loss of the wild type allele in almost all breast and ovarian cancers arising in mutation carriers, are strong indicators that BRCA1 and BRCA2 proteins act as tumor suppressors. This is supported by the finding that induced overexpression of wild type but not mutant BRCA1 in MCF-7 breast cancer cells leads to growth inhibition and inhibited tumor growth in nude mice. Expression of BRCA1 and BRCA2 In normal cells, BRCA1 and BRCA2 encode nuclear proteins, preferentially expressed during the late-G1/ early-S phase of the cell cycle, but down-regulated in quiescent cells. While apparently at odds with the

BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk. Figure 1 Overall penetrances of BRCA1 and BRCA2 for breast and ovarian cancer. Estimates were obtained by maximizing the LOD score with respect to all the different penetrance functions in those families with strong evidence of the breast and ovarian cancers being caused by the gene (done by linkage analysis). This is equivalent to maximizing the likelihood of the marker data, which is determined only by disease phenotype data. This will give an unbiased estimation of the penetrance irrespective of ascertainment of families on the basis of multiple affected individuals. Data were compiled from Ford et al. (1994) Lancet 343:692–695 and Ford et al (1998) Am J Hum Genet 62:676–689. The graphs can be read in such a way that, for example, an unaffected carrier of a BRCA1 mutation has a 50% risk to develop breast cancer before age 50.


above-mentioned observations that BRCA1 expression inhibits cellular proliferation, the proliferation-induced expression could represent a negative feedback loop tending to decrease breast cancer risk. However, BRCA1 expression can also be up-regulated in a proliferation-independent way in mammary epithelial cells induced to differentiate into lactating cells by glucocorticoids. Hence, BRCA1 might also play a role in controlling mammary gland development. In mice, expression of BRCA1 and BRCA2 is coordinately up-regulated with proliferation of breast epithelial cells during puberty, pregnancy and lactation. Intriguingly, BRCA1 might suppress estrogen-dependent mammary epithelial proliferation by inhibiting ER-α mediated transcriptional pathways related to cell proliferation. Whatever the cellular function of BRCA1, it appears to be regulated by phosphorylation: it becomes hyperphosphorylated at G1/S with dephosphorylation occurring at M phase. BRCA1 might regulate the G1/S checkpoint by binding hypophosphorylated retinoblastoma protein. BRCA1 and BRCA2 have also been suggested to regulate the G2/M checkpoint by controlling the assembly of mitotic spindles and the appropriate segregation of chromosomes to daughter cells. BRCA1- and BRCA2-Related Breast Cancer A close examination of the pathology of BRCA1- and BRCA2-related breast cancers has defined a typical pathology for each category, differing from that in sporadic cases. In general, cancers in carriers are of higher grade than age-matched controls (Fig. 2), and the

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BRCA1-cancers more frequently display a “medullary”-like appearance. This is due to a higher mitotic count and lymphocytic infiltrate. BRCA2-related breast cancers generally show fewer mitoses and less tubule formation. For both BRCA1- and BRCA2-related cancers, greater proportions of the tumor show continuous pushing margins. Although a role for BRCA1 and BRCA2 in non-inherited sporadic breast cancer is unclear, protein expression of BRCA1 was found to be reduced in most sporadic advanced (grade III) ductal breast carcinomas. BRCA1 and BRCA2 as Caretakers of the Genome To date, several biological roles for BRCA1 and BRCA2 have been demonstrated, and a number of observations indicate that they function in a similar pathway. Both maintain genomic stability through their involvement in homologous recombination, transcription-coupled repair of oxidative DNA damage and double-strand break repair. These roles are suggested by interactions of the Brca1 and/or Brca2 proteins with proteins known to be involved in DNA damage repair, most notably RAD50 and RAD51. Murine embryonic stem cells and mice in which both copies of BRCA1 or BRCA2 have been mutated show a repair deficiency and defects in cell-cycle checkpoints. BRCA1 and BRCA2 play a role in transcriptional regulation, through interactions or complex formation with RNA polymerase II and various transcriptional regulators, although this is presently more firmly established for BRCA1 than for BRCA2. A transcriptional response to DNA damage is well-documented,

BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk. Figure 2 BRCA1- and BRCA2-related breast cancers are generally of higher grade than age-matched controls. Histological sections from 118 breast tumors attributable to BRCA1, and 78 attributable to BRCA2, were evaluated by five histopathologists, all experts in breast disease. Every slide was seen by two pathologists. An age-matched group of 547 apparently sporadic female breast cancer cases served as control. The overall grade of both BRCA1 and BRCA2 breast cancers was significantly higher than that of controls (p < 0.0001 and p < 0.04, respectively). For BRCA1 breast cancers this was due to higher scores for all three grade indices, whereas for BRCA2 breast cancers the grade was only significantly higher for tubule formation. Data taken from The Breast Cancer Linkage Consortium (1997) Lancet 349:1505–1510.

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and identification of downstream targets of BRCA1/2mediated transcription regulation might help to further understand how BRCA1 and BRCA2 suppress tumor formation. Microarray-based screening of genes regulated by BRCA1 were recently found to fall into two categories, cell-cycle control genes and DNA damage response genes. Clinical Relevance When to Take the DNA-test? Diagnosis of gene defects became possible after the identification of BRCA1 and BRCA2 in 1994 and 1995, respectively. In many countries, testing for mutations is being offered to women with a high priori familial risk in Clinical Genetic Centres or multidisciplinary Cancer Family Clinics. A few studies have presented models to determine the prior probability that the counselee is a BRCA mutation carrier, by combining breast and ovarian cancer family history data with results from comprehensive mutation-testing. These models enable the genetic counselor to decide when a DNA-test is indicated. Why Take the DNA-test? A clear positive result of the DNA-test, i.e. the presence of a deleterious mutation, is being used to enter these women into early-detection cancer screening programs or in the decision for or against prophylactic surgery. A woman in which breast cancer has just been diagnosed can benefit from knowledge about gene carrier status, since the risks to the contralateral breast and ovaria must

be considered. The treatment of such cancer by lumpectomy will not reduce recurrence risks dramatically, as opposed to complete mastectomy. Healthy women who test positive can take action to prevent cancer developing, although the efficacy of the preventive options currently offered to a woman remains without formal supporting evidence. Chemoprevention is still controversial, and good prospective data on BRCA carriers will probably never become available, given the ethical and clinical difficulties surrounding randomization. Prophylactic surgery, intuitively the most secure way to reduce breast cancer risk to below population levels, is socially ill-accepted in many parts of the world, and formal proof of its preventive effect in BRCA carriers is also lacking. Clearly, this area is fraught with clinical dilemmas. Interpreting a Negative Test Result Paradoxically, a negative test result (the absence of a deleterious mutation) presently still has limited power in excluding the presence of a strong susceptibility allele. A negative test result is presently being found in 70–80% of all probands tested in most non-Ashkenazi Jewish populations. Among probands with a family history for ovarian cancer, a negative test result is found less frequently (although still in 40–60% of the cases). There are several levels of uncertainty. . The first is technical: no single mutation-detection method is 100% sensitive, and therefore only exhaustive testing, using a range of different methodologies sensitive to various types of mutation-mechanisms,

BRCA1/BRCA2 Germline Mutations and Breast Cancer Risk. Table 1 Mutation types in BRCA1 and BRCA2 and their predicted effects BRCA1 % of Total Mutation type Frameshifting Nonsense Splice-site In-frame del/ins Missense Neutral Intronic change Mutation effect Protein truncating Missense Neutral polymorphism Unclassified variant

% of Distinct

BRCA2 % of Total

% of Distinct

47.1 11.3 4.4 0.6 28.4 3.5 4.7

38.7 11.1 7.9 1.8 28.4 3.9 8.3

33.7 11.5 2.2 0.4 44.3 3.1 4.9

36.5 10.2 3.6 1.0 35.4 5.5 7.8

62.6 2.2 11.0 24.2

56.9 1.5 7.2 34.4

41.4 0.7 14.4 43.4

47.9 1.9 13.7 36.4

The entire Breast Cancer Information Core (BIC) database was down-loaded on March 1, 2000 from http://www.nhgri.nih.gov/ Intramural_research/Lab_transfer/Bic. There were 3,086 BRCA1 mutations and 1,892 BRCA2 mutations. The total numbers of distinct changes were 724 and 670, respectively.

Breakpoint Cluster Region

and investigating the entire coding regions and regulatory domains, can detect any changes. This is obviously very cost- and labor-intensive. . The second level of uncertainty relates to the interpretation of sequence changes that do not predict a truncated protein. Of the almost 5,000 BRCA1 and BRCA2 mutations submitted to the Breast Cancer Information Core (BIC) database, about one-third are either missense, in-frame deletions or insertions, base-substitutions not leading to an amino acid change (neutral changes) or intronic changes with unknown effect on mRNA-processing (Table 1). Only a small proportion of these have been unmasked as polymorphisms unrelated to disease outcome. They include missense changes and intronic variants, but, intriguingly, also a nonsense mutation in BRCA2. The K3326X mutation was found in 2.2% of over 400 controls tested. Only a few missense changes (e.g., BRCA1C61G) have been called a deleterious disease-related mutation, mainly because they reside in a validated functional domain of the protein or affect an evolutionary conserved residue. As a result, about 35% of all the distinct gene changes detected to date are lumped into the “unclassified variant” category, meaning that their relevance to disease outcome is uncertain. Almost certainly, a substantial proportion of these represent rare polymorphisms but equally certainly, a number of them will turn out to be true deleterious mutations. . A third reason for a negative test result is that the familial clustering of breast cancer in a family is due to an unknown gene or in fact is a non-genetic chance event. The proportion of truly missed, deleterious mutations is therefore difficult to gauge. A study by the BCLC has suggested that a combination of incomplete testing and missed or misinterpreted gene changes, causes false-negative test results in over 30% of all family types with some evidence of being linked to BRCA1. This proportion was independent of the mutation-screening methodology used.

4. Ponder B (1997) Genetic testing for cancer risk. Science 278:1050–1054 5. Welcsh PL, Owens KN, King MC (2000) Insights into the functions of BRCA1 and BRCA2. Trends Genet 16:69–74

BRCT Domain Definition Named after BRCA1 c-terminal repeat. The domain consists of a 90–100 amino acid unit that occurs as a single element or as multiple repeats in several proteins involved in the DNA-damage response. Heterodimerization between BRCT repeats promotes proteinprotein interactions. A subset of tandem BRCT repeats adopt a conserved head-to-tail structure. Such tandem repeats can function as a phospho-peptide binding module that binds proteins with specific phosphorylation motifs. ▶Fanconi Anemia ▶BRIT1 Gene ▶BRCA1/BRCA2 Germline orientation and Breast Cancer Risk

Breakpoint Definition Point of separation on a chromosome involved in translocation or other structural rearrangement. ▶E2A-PBX1 ▶Chromosomal Translocations

References 1. Devilee P (1999) BRCA1 and BRCA2 testing: weighing the demand against the benefits. Am J Hum Genet 64:943–948 2. Ford D, Easton DF, Stratton M et al. (1998) Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. Am J Hum Genet 62:676–689 3. Lakhani SR, Jacquemier J, Sloane JP et al. (1998) Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations. J Natl Cancer Inst 90:1138–1145

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Breakpoint Cluster Region Definition A localized site of recurrent DNA breakage. ▶ALU Elements ▶BCR-ABL1

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Breast Cancer

Breast Cancer B ILL G ULLICK Department of Biosciences, University of Kent at Canterbury, Canterbury, Kent, UK

Definition Breast cancer may originate from more than one cell type in the breast as a result of different subsets of molecular changes. It is therefore a collection of diseases with different characteristics, different risks and different treatments. It occurs predominantly in women but it may also occur in men (0.5% of cases). In addition to invasive disease, several benign pre-malignant and noninvasive forms exist. Although the broad pathological categories are generally accepted there are several alternative systems of sub-categorization. . Benign conditions: include sclerosing conditions and obliterative mastitis; mild moderate and severe hyperplasias and atypical hyperplasias; fibrocystic conditions, fibroadenomas and related conditions (note that the Oxford Textbook of Pathology presents a “simplified nomenclature” of 50 subtypes of benign breast disease). . Non-invasive carcinoma: generally divided into Lobular Carcinoma in situ (LCIS) and Ductal Carcinoma in situ (DCIS). DCIS was originally sub-divided into comedo, cribriform, papillary, solid and clinging but has more recently been categorized as well, moderately and poorly differentiated. . Invasive carcinoma: broadly defined as of “special type” (30% of cases) and “no special type” (70% of cases).

Characteristics Epidemiology Breast cancer is the most common fatal malignancy in women in the Western world representing about 10% of all cancer deaths. It is however much less common in other countries probably as a result of environmental rather than genetic factors. Behavioral risk factors have been identified. Early pregnancy to term and multiple pregnancy are protective for breast cancer incidence probably due to a reduced exposure to estrogens. There are several reports of weak associations with diet and use of oral contraceptives. Screening In some countries mammographic screening is available to women to detect early disease, as it is likely that earlier treatment is beneficial for survival. Educational programs are also active, for instance in promoting selfexamination as a method of early detection. The value

of these approaches for improving patient survival is not yet fully established.

Genetics About 5–10% of breast cancer cases are associated with a genetic predisposition to the disease. Recently three genes have been identified where the inheritance of variants is associated with a very high incidence (penetrance) of the disease. The ▶BRCA1 gene was the first to be identified, and gene carriers are thought to have as much as a 70–80% chance of developing the disease, generally at an earlier age than women with “sporadic” (not genetically predisposed) disease. Subsequently a second gene called ▶BRCA2 was also found in other families that predisposes to breast and ovarian cancers. The particular risk in an individual gene carrier may be determined by the nature of the particular gene defect and by environmental and hormonal factors and by their background genetic makeup (▶breast cancer genes BRCA1 and BRCA2). A further inherited condition, called ▶Li-Fraumeni syndrome is associated with increased risk to breast and many other types of cancers. This is due to the inheritance of a rare mutant copy of the ▶p53 gene. Molecular Biology Breast cancer is the most studied form of human cancer, due to its common occurrence and to the availability of many immortal breast cancer derived cell lines that can be grown in tissue culture (in contrast to prostate cancer for instance). About 60% of breast cancers at diagnosis express the estrogen receptor. Several genes have been found to be altered by mutation or amplification in invasive cancer and in DCIS. The ▶HER-2 or ERBB2 gene (also known as c-erbB-2 and neu) is amplified in about 25% of invasive breast cancers leading to overexpression of the growth factor receptor which it encodes. The c-myc gene is similarly found to be amplified in about 20% of breast cancer also resulting in overexpression of the c-myc protein. The ▶cyclin D gene, which specifies a protein important in regulating the cell cycle, is also amplified in a proportion of breast cancers. The p53 gene has been found to be point mutated in 20% of invasive breast cancers and to be overexpressed in about 50% of cases. Other point mutations have been found in E-cadherin gene, which encodes a cell adhesion molecule. In this case the mutations are most common in lobular cancers. More subtle changes occur in the expression of apparently normal proteins including growth factors such as those in the epidermal growth factor (EGF) family and the FGF family of proteins, the receptor tyrosine kinase c-erbB-3 and the Src tyrosine kinase. Some of these altered proteins represent targets for new forms of treatments.

Breast Cancer Genes BRCA1 and BRCA2

Treatment Three methods of treatment are available, surgery, radiotherapy and chemotherapy/hormonal therapy. Benign disease and lobular carcinoma in situ are rarely treated but are observed, as they are associated with an increased risk of developing breast cancer. DCIS of the breast has often been treated by mastectomy as it is frequently quite widespread within the breast but may also be treated by local surgery. It is possible that the different pathologically defined forms of DCIS may be associated with different relative risks of recurrence and recurrence as invasive disease. Invasive disease is generally first treated by surgery, and lymph nodes are sampled to determine, by pathological diagnosis, if there is evidence of tumor spread. This procedure may be limited to a single node (called the sentinel node) or may involve a greater degree of surgery. Patients with invasive breast cancer are often treated with radiotherapy to the breast to reduce the chances of local recurrence. Even if the cancer has not apparently spread, patients are sometimes offered preventative or “adjuvant” therapy using drugs, as this helps to prevent recurrence of the disease. Chemotherapy or hormonal therapy are generally offered to patients where metastatic spread of the disease has occurred. Hormonal therapy is frequently offered to women whose tumors express the estrogen receptor. Specific methods of treatment still vary depending on the patient and the institution where it is given, although more generally agreed protocols are becoming accepted. New Treatments Breast cancer is frequently a hormonally dependent disease. Thus treatments with drugs such as tamoxifen, which binds to the estrogen receptor and reduces its activity, or other drugs that suppress the production of estrogen, such as aromatase inhibitors, are frequently employed. However, new drugs directed to known molecular changes in the cancer cells are under development. These include signal transduction inhibitors directed to molecules such as the epidermal growth factor receptor, monoclonal antibodies to the c-erbB2 receptor and drugs which inhibit proteolytic enzymes thought to be involved in the process of metastasis. Several of these are now in clinical trials that will determine their usefulness for the treatment of the disease.

References 1. Fisher B (1999) From Halsted to prevention and beyond: advances in the management of breast cancer during the twentieth century. Eur J Cancer 35:1963–1973 2. Gayther SA, Pharoah PD, Ponder BA (1998) The genetics of inherited breast cancer. J Mammary Gland Biol Neoplasia 3:365–376

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3. Gullick WJ, Srinivasan R (1998) The type 1 growth factor receptor family: new ligands and receptors and their role in breast cancer. Breast Cancer Res Treat 52:43–53 4. Hortobagyi G (2000) Adjuvant therapy for breast cancer. Annu Rev Med 51:377–392 5. O’D McGee, Isaacson PG, Wright NA (1992) The breast. In: Oxford textbook of pathology, Oxford University Press, Oxford, pp 1643–170

Breast Cancer Genes BRCA1 and BRCA2 A SHOK R. V ENKITARAMAN Hutchison/MRC Research Centre, Cambridge, UK

Definition

▶BRCA1 and ▶BRCA2 are cancer-predisposition genes, germline mutations in which are associated with a high risk of developing breast, ovarian and other cancers. Much information has been accumulated on the function of their large, nuclear-localized protein products, which implicates them in the cellular response to DNA damage, the control of mitotic cell division, and the regulation of gene transcription. BRCA1 and BRCA2 are very distinct genes, despite the similarity in their acronyms.

Characteristics

Roughly one-tenth of all ▶breast cancer cases exhibit a familial pattern of inheritance. Of these familial cases, germline mutations in either one of two genes, BRCA1 or BRCA2, occur in 20–60% (that is, in 2–6% of all cases). Somatic mutations in BRCA1 or BRCA2 do not appear to be a feature of non-familial (that is, sporadic) breast cancer, but there is evidence that epigenetic suppression of BRCA gene expression, or genetic alterations affecting the biological pathways in which they participate, can occur in sporadic breast cancer. BRCA1 and BRCA2 were first identified in 1994– 1995 through the analysis of families exhibiting a predisposition to early-onset breast cancer. Founder mutations affecting these genes occur in Iceland and amongst the Ashkenazim, where they confer a highly penetrant risk of breast, ovarian and other cancers (including cancers of the male breast, pancreas and prostate). In other populations, germline BRCA1 or BRCA2 mutations are found in the great majority (up to 80%) of families that suffer from multiple occurrences of breast plus ovarian cancer. Germline BRCA2

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mutations affecting both alleles also occur in the rare D1 complementation group of Fanconi anaemia. The BRCA1 and BRCA2 genes have been assigned to human chromosomes 17q and 13q, respectively. In both genes, exon 11 (3.4 kb in BRCA1, or 5 kb in BRCA2) encodes a large portion of the protein. Overall, the murine and human genes are no more than 60% identical at the amino acid level, although small regions exhibit a much higher degree of conservation. Proteins encoded in alternative splice products, such as the IRIS protein encoded by the BRCA1 gene, also exist. Protein BRCA1 and BRCA2 encode large proteins (human BRCA1 is 1,863 amino acids long; and human BRCA2 is 3,418 amino acids) that localise to the nucleus in mitotic and meiotic cells (Fig. 1). They bear little resemblance to proteins of known function. At its N-terminus BRCA1 protein contains a RING-domain known to mediate hetero-dimerization with the RING domain of BARD1, forming an active E3 ubiquitin ligase. At its C-terminus, BRCA1 includes two copies of a 95 amino acid motif (the BRCT domain, for BRCA1 C-terminal) later detected in a number of different proteins implicated in DNA repair and cell cycle checkpoint control. This domain, whose atomic structure has been elucidated, mediates a number of protein-protein interactions with phosphorylated targets by serving as a phosphopeptide-binding module. Eight repeated sequences (the BRC repeats), each of about 30 amino acids, are encoded in BRCA2 exon 11. The BRC repeats, but not their intervening sequences, are conserved between several mammalian species suggestive of a conserved function. Indeed, the interaction of BRCA2 protein with RAD51, a mammalian homologue of bacterial RecA essential for genetic recombination, occurs through the BRC repeats. There is good evidence from genetic, structural and biochemical studies that the BRC repeats regulate the activity of RAD51 in reactions that lead to DNA repair by recombination. Two other regions of BRCA2 have been implicated in the control of recombination. A domain carboxyl-terminal to the BRC repeats interacts with

the small protein Dss1 to form a structure capable of binding junctions between single-stranded and doublestranded DNA, which can displace the ssDNA-binding protein RPA from recombination substrates, whereas an additional RAD51-binding region of uncertain function is located near the extreme C-terminus of BRCA2. Cellular and Molecular Regulation The transcripts and protein products encoded by BRCA1 and BRCA2 are expressed in dividing cells of many types. Expression is also high in meiotic cells. These expression patterns speak to the possible functions of BRCA1 and BRCA2 proteins. Role in the Cellular Response to DNA Damage Both BRCA1 and BRCA2 proteins localize to the nucleus. In meiotic cells, co-localisation has been reported to the synaptonemal complexes of developing axial elements. This is suggestive of a role in meiotic recombination, a process that is initiated by DNA double-strand DNA breakage. Similarly, there is good evidence that BRCA1 and BRCA2 are essential in mitotic cells for the repair of DNA double-strand breaks by homologous recombination. Several lines of evidence are indicative of such a role: . Cells in which BRCA1 or BRCA2 or their homologues in other species have been inactivated exhibit genotoxin hypersensitivity and chromosomal instability suggestive of defects in DNA double-strand break repair. . Second, homology-directed repair of double-strand DNA breaks introduced into chromosomal substrates is impaired by the disruption of BRCA1 or BRCA2, although pathways for non-homologous end joining remain largely unaffected. . Finally, BRCA1 and BRCA2 localize after DNA damage to nuclear foci where they interact with molecules implicated in DNA recombination, including RAD51, and the Fanconi anemia proteins. The precise mechanisms that may underlie such a function remain to be determined. BRCA2 interacts directly, and at a relatively high stoichiometry, with

Breast Cancer Genes BRCA1 and BRCA2. Figure 1 Structural features of the BRCA1 and BRCA2 proteins (not drawn to scale).


RAD51, a protein essential for DNA repair by recombination, to modulate RAD51 activity or availability. The interaction of BRCA1 with RAD51 is less well defined, although both proteins co-localize – along with BRCA2 – to discrete nuclear foci following DNA damage. BRCA1 may participate in the cellular mechanisms that sense and signal DNA damage, culminating in the activation of cell-cycle checkpoints and the machinery for DNA repair. The protein kinases ATM (encoded by the gene mutated in Ataxia telangiectasia), ATR, chk1 and chk2 (mutated in ▶Li-Fraumeni syndrome) are proximal components of these sensing/ signaling mechanisms. ATM, chk1 and probably the other checkpoint kinases, phosphorylate BRCA1 following DNA damage, a modification essential for its proper function. These observations are important because they place BRCA1 – and by extension, possibly BRCA2 – in the same pathway as genes such as ATM (▶ATM protein), germline mutations in which are also associated with an increased risk of breast and other cancers. Thus, a common DNA damage response pathway may be defective in a significant fraction of breast cancers. BRCA1 and BRCA2 have also been implicated in the enforcement of cell cycle checkpoints during the G2 and M phases, and in the regulation of centrosome number. Additional functions have recently been described in the control of mitosis. BRCA1 regulates proteins such as MAD2 that act in the mitotic spindle assembly checkpoint, and has an essential function in directing the correct formation and function of the mitotic spindle, whereas BRCA2-deficient cells exhibit defects in the completion of cell division by cytokinesis. Thus, BRCA1 and BRCA2 appear to work in multiple processes responsible for maintaining the integrity of chromosome number as well as structure in dividing cells, which may help to explain why they are potent tumor suppressors. Other Functions It is difficult to reconcile the disparate nature and severity of the cellular and developmental defects induced by the disruption of murine homologues of BRCA1 and BRCA2, with functions exclusively in the response to DNA damage. Indeed, evidence is accumulating that BRCA1, in particular, can control gene transcription. Several proteins that interact with BRCA1 are known to regulate transcription or mRNA processing. Moreover, at least a fraction of the total intracellular pool of BRCA1 is linked to the general transcription machinery – the RNA polymerase II holoenzyme – through its RNA helicase subunit. Intriguingly, BRCA1 has been implicated in the control of X-inactivation in female cells, a process whose dysregulation is associated with breast cancer predisposition. How this function may be exerted is not clear, but it may work through the control of localization of the Xist product.

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In addition, roles for BRCA1 and possibly BRCA2 have been reported in the control of oestrogen receptor expression and signaling. Tumor Suppression by BRCA1 and BRCA2 Inheritance of a single defective copy of BRCA1 or BRCA2 confers cancer predisposition in humans. However, the second allele is almost invariably lost in the cancers that arise in predisposed individuals, indicating that BRCA1 and BRCA2 behave in some respects as ▶tumor suppressor genes. Abnormalities in growth or in DNA repair have not yet been reliably detected in murine or human cells heterozygous for BRCA1 or BRCA2 mutations. Thus, there is currently little to suggest that cancer predisposition arises solely from haplo-insufficiency, or a transdominant deleterious effect induced by a single mutant BRCA1 or BRCA2 allele. Rather, as has been proposed for other tumor suppressor genes, germline mutations in one allele may simply increase the likelihood that the gene is wholly inactivated by loss of the second allele through somatic mutation. However, aneuploidy as a consequence of abnormal cytokinesis has been reported in cells and tissues heterozygous for BRCA2 mutations, and the possibility that this effect of haplo-insufficiency contributes to carcinogenesis remains to be determined. Whatever the events that lead to loss-ofheterozygosity, inactivation of BRCA1 or BRCA2 would initiate genetic instability by destabilizing chromosome structure and number, allowing the rapid evolution of tumors due to increased somatic alterations in genes that control cell division, death or lifespan. Thus, BRCA genes are proposed to work as “caretakers” of genetic stability. This “caretaker” role is most likely to arise through the function of the BRCA proteins in DNA repair and mitosis. Cells that harbor disruptions in BRCA1 or BRCA2 accumulate aberrations in chromosome structure, reminiscent of diseases like Bloom syndrome or ▶Fanconi anemia, where chromosomal instability is associated with cancer predisposition. They also exhibit aneuploidy and defects in cell division. These defects could together elevate the rate of genomic instability, leading to somatic mutations or alterations in gene copy number that promote carcinogenesis. It is unclear why carcinogenesis accompanied by loss of the second BRCA gene allele in individuals who inherit one mutant allele should occur preferentially in tissues such as the breast or ovaries. Both BRCA1 and BRCA2 are widely expressed and appear to perform functions essential to all tissues. Currently there is little evidence to help distinguish between the several possible explanations that can be advanced. The chronology of the molecular events during carcinogenesis in BRCA gene mutation carriers is not known. Loss of the second allele is clearly very frequent, but it is unclear at what stage in tumor

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Breast Cancer Resistance Protein

evolution this may occur. However, the catastrophic cellular consequences of homozygous inactivation of BRCA1 or BRCA2, which quickly lead to cell death, does emphasize that other genetic alterations will be necessary. Current evidence favors the notion that the inactivation of cell-cycle checkpoint genes, particularly those that enforce mitotic checkpoints, is an important additional step during carcinogenesis in BRCA gene mutation carriers. Viewed in this way, it is conceivable that the tissue specificity of carcinogenesis represents differences in the ability of cells which have lost both alleles of BRCA1 or BRCA2 to survive for long enough to acquire these additional genetic alterations. For instance, BRCAdeficient cells in epithelial tissues such as the breast and ovary may take advantage of hormonal or local inter-cellular interactions to support survival until the accumulation of additional genetic alterations allows outgrowth. By contrast, BRCA-deficient cells in nontarget tissues may quickly be eliminated.

Clinical Relevance Germline mutations in BRCA1 or BRCA2 are frequently associated with familial, early-onset, breast and ovarian cancer, particularly in those families that suffer from multiple cases of cancer in both sites. This has obvious important implications for genetic testing and counselling in the clinic. The mutations have been estimated to carry a cumulative life-time cancer risk of between 40–70%. There is some evidence that the pathological features of breast and ovarian cancers associated with BRCA1 or BRCA2 mutations differ from those of sporadic tumors. So far, these differences seem to be insufficiently wellmarked to be of diagnostic significance. One notable association is that BRCA1-deficient cancers often exhibit a “basal-like” phenotype usually characterized by the expression of specific markers, and negativity for oestrogen receptor expression. It is also unclear if the prognosis of breast and ovarian cancers associated with BRCA1 or BRCA2 mutations will differ significantly from that of sporadic cases. Conflicting results have been reported in the literature, their interpretation made difficult by the varied study designs and by the relatively small numbers of cases that have been compared. Similarly, the value of prophylactic interventions, whether surgical or drug-based, in BRCA gene mutation carriers awaits evaluation. Emerging evidence suggests that the DNA repair defect inherent in BRCA-mutant tumors can be exploited in cancer therapy. Thus, both BRCA1 and BRCA2deficient cancers appear to be hypersensitive to the effect of DNA cross-linking agents such as carboplatin, and also to novel chemical inhibitors of the PARP1 enzyme.

Breast Cancer Resistance Protein Definition The breast cancer resistance protein is a plasma membrane transporter and member 2 of the subfamily G of ATP-binding cassette transporters. It is predominantly localized in the epithelium of small and large intestine, in hepatocyte canalicular membranes, in ducts and lobules of the breast, on the placental syncytiotrophoblast, and in the plasma membrane of stem cells. Physiological substrates include sulfated steroids. Transported xenobiotics are e.g., several dietary carcinogens, the chlorophyll breakdown product pheophorbide, and the receptor tyrosine kinase inhibitor imatinib. The breast cancer resistance protein is involved in conferring multidrug resistance. ▶Membrane Transporters

Breast Conservation Definition Surgical removal of cancer from the breast while preserving the remainder of the breast tissue. ▶Oncoplastic Surgery

Breast Imaging Reporting and Data System Definition BIRADS; http://www.birads.at/index.html

Breast Implant Definition Device placed into the body to enlarge or reconstruct the breast after removal. ▶Oncoplastic Surgery

BRIP1

Breast Reconstruction Definition

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Breast Transillumination ▶Optical Mammography

Surgical procedure to simulate the appearance of the breast after it has been removed for the treatment of cancer. ▶Oncoplastic Surgery

Breslow Depth Definition

Breast Reduction Definition Surgery to reduce the size of the breast. ▶Oncoplastic Surgery

Breast Regressing Protein 39 Kd

Referring to ▶cutaneous desmoplastic melanoma tumor thickness (according to Breslow). The single most important factor in predicting survival for patients with stage I/II melanoma. Is measured from the top of the granular layer (for non-ulcerated lesion) or from the ulcer base overlying the deepest point of invasion (for ulcerated lesions) to the deepest extension of the tumor using an ocular micrometer. According to the AJCCcriteria for malignant melanoma, tumor thickness and the presence or absence of ulceration are the primary criteria for the tumor classification. ▶Desmoplasia ▶Desmoplastic Melanoma

▶Serum Biomarkers

Breast Stem Cells Definition

BRG- and BRM-associated Factor, 47 kDa ▶hSNF5/INI1/SMARCB1 Tumor Suppressor Gene

Self-renewing cells in the breast that are capable of producing all of the breast epithelial cell types and likely play an important role in breast cancer. ▶Basal-like Breast Cancer

Brilliant Yellow S ▶Curcumin

Breast Surgery Definition Surgical procedures on the breast used to treat congenital deformities or to remove cancerous tissue. ▶Oncoplastic Surgery

BRIP1 ▶BACH1 Helicase

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BRIT1 Gene

BRIT1 Gene D EBORAH J ACKSON -B ERNITSAS 1 , K AIYI L I 2 , S HIAW-Y IH L IN 1 1

Department of Systems Biology, The University of Texas M.D., Anderson Cancer Center, Houston, TX, USA 2 Department of Surgery, Baylor College of Medicine, Houston, TX, USA

Synonyms Microcephalin; MCPH1

Definition BRCT-repeat inhibitor of hTERT expression.

Characteristics Significance of BRIT1 in the Development of Cancer ▶BRIT1 was first described through a genetic screen for transcriptional repressors of the catalytic subunit of ▶human telomerase, (hTERT). This catalytic subunit, hTERT, is the rate-limiting determinant of and is necessary for telomerase activity and thus is highly significant for cellular immortalization by preventing natural cellular senescence. Therefore, molecules that negatively control hTERT activity, such as BRIT1, directly influence the development of pre-cancerous and cancerous cells. The BRIT1 amino acid sequence matched a previously identified gene, ▶Microcephalin (▶MCPH1) that is implicated as one of the contributing factors in the autosomal recessive neuro-develomental disorder, primary microcephaly. Additional biological roles for BRIT1 in the DNA damage response pathway were suggested by the protein structure. The presence of three ▶BRCA1 carboxy-terminal (BRCT) domains within its structure connected BRIT1 with a group of proteins involved in DNA damage repair and checkpoint control such as ▶53BP1, ▶MDC1 and BRCA1. Proper cellular response to DNA damage is essential for the maintenance of genomic stability and is consequently crucial in prevention of neoplastic transformation. Depletion of BRIT1 by experimental manipulation abolishes normal DNA damage response and introduces chromosomal and centrosomal abnormalities. The reduction of BRIT1 expression in normal human mammary epithelial cells by experimental RNA interference generated chromosomal breaks, dicentric chromosomes and telomeric fusions. Additional chromosomal aberrations were introduced when BRIT-deficient cells were submitted to genotoxic insult. The resultant genomic instability generated from the loss of

an appropriate BRIT1-mediated checkpoint and DNA repair mechanism may contribute to tumor formation. Functional impairment or loss of proteins, such as BRIT1, may significantly contribute to tumorigenic development by allowing the perpetuation of damaged or mutated genes within a cell, resulting in the inappropriate expression and control of the affected genes. In addition to the influence on hTERT expression, BRIT1 appears to control the expression of two vital checkpoint-regulating proteins; BRCA1 and Chk1. BRCA1 and Chk1 protein levels are dramatically reduced in cells where BRIT1 has been experimentally reduced by RNA interference. A concurrent reduction in mRNA levels of BRCA1 and Chk1 were observed in BRIT1 knockdown cells suggesting that BRIT1 exercises an influence on the transcription of these genes. The significance of BRCA1 in breast cancer has been previously established. Whether BRIT1 functions directly as a specific transcription factor or as a chromatin modifying factor is unclear at this time, however, as a controller of these key players in the DNA damage checkpoint control network, BRIT1 is extremely important to the maintenance of normal cell function and thus in the prevention of tumorigenesis. Aberrations of BRIT1 have been identified in various different human cancers. BRIT1 mRNA and protein expression is aberrantly reduced in several breast cancer cell lines and is reduced in some human ovarian and prostate epithelial tumors as compared to the corresponding normal tissue. Reduction of BRIT1 gene copy number significantly correlated with genomic instability found in the specimens. Additionally, reduced BRIT1 expression correlated with the duration of the relapse-free intervals and with the occurrence of metastases in some breast cancer patients suggesting that BRIT1 deficiency may contribute to the aggressive nature of breast tumors. A mutant form of BRIT1, isolated from one human breast tumor specimen, lacked two C-terminal BRCT-domains of the protein. This shorter form of BRIT1 resulted in a loss-of-function with respect to DNA damage response when tested experimentally. Therefore, significant evidence exists to directly link defective or reduced BRIT1 protein expression to several forms of cancer and to implicate BRIT1 as a novel tumor suppressor gene.

BRIT1 Function in DNA Damage Response In the normal course of events, cellular DNA is subjected to a variety of endogenous and environmental factors that induce damage within its structure. In response to these insults, normal cells activate complex mechanisms to detect, signal the presence of and subsequently repair DNA damage when possible. Propagation of the DNA damage alarm progresses

BRIT1 Gene

through a complex signal transduction network that includes the BRIT1 protein. Initially, sensor proteins recognize the location of damaged or altered DNA structure and transmit a signal through mediator molecules to transducer proteins. The transducer proteins transmit the signal to numerous downstream effectors involved in specific pathways (Fig. 1). Two distinct DNA damage repair networks, both requiring BRIT1 activity, have been described. The ▶ATM (ataxia telangiectasia mutated) pathway is activated by doublestranded breaks in the DNA observed after exposure to ionizing radiation while the ▶ATR (ATM and Rad3related) pathway is activated by prolonged presence of single-stranded DNA induced by either ultraviolet radiation or stalled DNA replication. ATM and ATR are essential kinases that are responsible for phosphorylating numerous transducer and effector proteins in the DNA damage network. BRIT1 co-localizes with numerous molecules associated with these two signaling networks including ▶γH2AX, MDC1, 53BP, ▶NBS1, p-ATM, ATR, p-RAD17 and p-RPA34 after DNA damage is induced. In the absence of functional BRIT1, all of these molecules with the exception of γH2AX, fail to localize to sites of DNA damage strongly suggesting that the BRIT1 molecule is an essential mediator for the subsequent repair processes. However, BRIT1 expression does not influence the chromatin binding of proteins unrelated to DNA damage such as Orc-2 indicating that the BRIT1 function is highly specific. The depletion of BRIT1 inhibits the recruitment of phosphorylated ATM to double-stranded DNA broken ends and subsequently blocks the phosphorylation of multiple down-stream members of the ATM repair pathway including Chk2

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and NBS1. Depletion of BRIT1 also abolishes the UVinduced phosphorylation of RPA34 and reduces the levels of phosphorylated RAD17 indicating the importance of BRIT1 in the ATR signaling pathway. Based on these observations, it has been determined that BRIT1 is a pivotal protein in both of the DNA damage signaling networks and for this reason has great significance in the prevention neoplastic transformations of cells. Figure 1 illustrates a simple model for the function of BRIT1 based on current experimental evidence. After exposure to ionizing radiation, double-stranded breaks in DNA occur resulting in the recruitment the MRE11/RAD50/NBS1 (MRN) complex and MDC1 to the damaged site thus facilitating the recruitment and kinase activity of ATM. Activated p-ATM phosphorylates NBS1, H2AX and BRCA1 which localize to sites of DNA damage. Increased single-stranded DNA by exposure to ultraviolet radiation induces the coating of RPA on DNA leading to the recruitment of ATRIP and ATR to the sites of DNA damage. Activated ATR then phosphorylates critical downstream molecules such as Rad17 and Chk1 further propagating the DNA damage signal in the cell. BRIT1 appears to regulate the recruitment of NBS1, MDC1 and thus the MRN complex in the ATM pathway. BRIT1 also regulates the recruitment of RPA, which in turn recruits ATRIP/ ATR complex initiating the ATR signaling cascade. BRIT1 Function in Cell Cycle Control Normally, the progression of a cell through the cell division cycle is stalled to allow for its DNA repair and if the damage cannot be repaired, the cell enters programmed cell death (apoptosis). This retardation or cell cycle checkpoint is essential to maintain the

BRIT1 Gene. Figure 1 BRIT1 Function in DNA Damage and Cell Cycle Control.

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BRM

integrity of cellular DNA that insures normalcy in consecutive descendant cells. Key molecules involved in cell cycle arrest, (p53, chk1 and chk2), are all activated when phosphorylated by ATM or ATR. BRIT1 clearly impacts the activity of both ATM and ATR by affecting the association of these molecules to damaged DNA. Activated p53 induces cell cycle arrest at G1 while p-Chk1 and p-Chk2 negatively regulate Cdc25 phosphatases that promote transition through the cell cycle thereby inducing the execution of G1/S, G2/M and intra-S checkpoints of the cell cycle. BRIT1 is required for the activation of intra-S and G2/M cell cycle checkpoints after cellular exposure to ionizing radiation. The influence of BRIT1 on control of these cell cycle checkpoints may result from BRIT1’s regulation of three checkpoint regulator proteins, Chk1, BRCA1 and NBS1. In the absence of BRIT1, BRCA1 and Chk1 expression is significantly reduced and NBS1 fails to be phosphorylated. BRCA1 plays a significant role in homologous recombination DNA repair and possibly serves as a scaffold for ATM and ATR thus affecting phosphorylation of many downstream effectors proteins. Therefore the regulation of BRCA1 by BRIT1 dramatically affects multiple aspects of cell cycle control and DNA damage repair. The normal cellular response to ionizing radiation is to arrest the cell cycle also at G2, allowing for the initiation of DNA repair, however, BRIT1-depleted cells continue to progress through G2 indicating that BRIT1 is essential in the activation of this important cell cycle checkpoint. Additionally, BRIT1-depleted cells continue to synthesize DNA and proceed through mitosis unlike normal cells exposed to ionizing radiation. Replication of DNA damaged by ionizing radiation could easily result in the propagation of mutated or disrupted genes and contribute to tumorigenesis. BRIT1 also controls the cell’s entry into mitosis by affecting the stabilization of the cdc25A, a key phosphatase in cell cycle control. Cells derived from a microcephaly patient (BRIT1 defective) maintained a persistent level of the phosphatase, cdc25A following UV treatment. Cdc25A is targeted for degradation when phosphorylated by Chk1 kinase during normal cell division and its degradation is usually amplified by UV exposure. Degradation of cdc25A abolishes the activation of the Cdk2-cyclin complex inhibiting DNA synthesis. Conversely, inappropriate persistence of cdc25A allows for the continued DNA synthesis despite aberrations or damage in the structure. These BRIT1 mutant cells also harbor reduced levels of phosphorylated Cdk1-cyclin B complex that is essential for mitotic entry. It was proposed that the regulation of mitosis by BRIT1 is both ATR dependent through regulation of cdc25A stability and ATR independent through regulation of Cdk1-cyclin B phosphorylation.

The affect of BRIT1 on cell cycle control is therefore multi-faceted (Fig. 1). Complete loss of the BRIT protein results in reduced protein levels of BRCA1 and Chk1 and impairs the activity of a multitude of vital proteins through their diminished phosphorylation. Presence of a mutated BRIT1 protein allows for expression of BRCA1 and Chk1 but still blocks proper signal transduction by inhibiting the activities of both ATM and ATR kinases.

References 1. Xu X, Lee J, Stern DF (2004) is a DNA damage response protein involved in regulation of Chk1 and BRCA1. J Biol Chem 279:34091–34094 2. Lin S, Rai R, Li K et al. (2005) BRIT1/MCPH1 is a DNA damage responsive protein that regulates the BRCA1Chk1 pathway, implicating checkpoint dysfunction in microcephaly. PNAS 102:15105–15109 3. Rai R, Dai H, Multani AS et al. (2006) BRIT1 regulates early DNA damage response, chromosomal integrity and cancer. Cancer Cell 10:1–13 4. Chaplet M, Rai R, Jackson-Bernitsas D et al. (2006) BRIT1/MCPH1: A Guardian of the genome and an enemy of tumors. Cell Cycle 5(22):2579–2583 5. Alderton G, Galbiati L, Griffith E et al. (2006) Regulation of mitotic entry by Microcephalin and its overlap with ATR signaling. Nat Cell Biol 8:725–733

BRM Definition Biological response modifier.

BRMS1 A LEXANDRA C. S ILVEIRA , DANNY R. W ELCH Department of Pathology and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA

Definition BRMS1 (Breast Metastasis Suppressor 1) is a human ▶metastasis suppressor gene that, when re-expressed, suppresses metastasis of human breast carcinoma, ovarian, and melanoma cell lines in immunocompromised mouse models.

BRN-5547136

Characteristics The BRMS1 gene is located on chromosome 11q13.1-q13.2. It spans over 8.5 kb and is comprised of ten exons, the first exon being untranslated. BRMS1 cDNA is 1485 base pairs and encodes a 246 amino acid protein (MW 28.5 kD, although it runs more slowly (35 kDa) by SDS-PAGE). It is highly conserved with the mouse ortholog (Brms1) having 95% homology at the amino acid level. Like human BRMS1, the mouse ortholog has suppresses metastasis in murine models of breast cancer. BRMS1 protein contains two nuclear localization sequences, two coiled-coil motifs and imperfect leucine zippers. It also contains a glutamic acid rich N-terminus and a potential endoplasmic retention signal. BRMS1 shows almost ubiquitous expression in human tissues; highest expression is in kidney, placenta, peripheral blood lymphocytes and testis and lowest expression in brain and lung. Subcellular fractionation and immunofluorescence studies have determined that BRMS1 protein is predominantly (>90%) nuclear. BRMS1 protein is stabilized by interaction with the chaperone protein, Hsp90, and is further regulated by proteasomal degradation. Cellular and Functional Characteristics BRMS1 re-establishes gap junctional cell-cell communication in human breast cancer cell lines. Studies using MDA-MB-435, MDA-MB-231, and the ovarian cancer cell line HO-8910PM show an inverse effect of BRMS1 expression on cell motility. Further, over-expression of BRMS1 in H1299 human lung carcinoma cells and MDAMB-435 cells results in suppressed growth in soft agar. Additionally, BRMS1 transfection increases apoptosis in suspended non-small cell lung carcinoma. The exact mechanism by which BRMS1 affects these phenotypes is as yet unknown. However, it is known that BRMS1 interacts with several different proteins and large (megadalton) protein complexes, most notably with class I and class II histone deacetylases (HDACs) and the transcription factor NFκB. BRMS1 is specifically a core member of mSin3-HDAC chromatin remodeling/transcriptional repression complexes but its involvement is implicated in other HDAC complexes. Additionally, BRMS1 and HDAC1 function as NFκB co-repressors; chromatin bound BRMS1 facilitates HDAC-1-mediated deacetylation and inactivation of NFκB. Studies also suggest that direct interaction of BRMS1 and the RelA/p65 subunit of NFκB represses the transactivation potential of NFκB. Further, BRMS1 leads to a reduction in NFκB translocation by inhibiting the phosphorylation and degradation of the NFκB inhibitor, IκB. Studies show reduced phosphoinositide signaling in BRMS1 transfected cells. Decreased phosphoinositide

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signaling results in decreased mobilization of intracellular calcium, a known regulator of metastasis. BRMS1 expression also downregulates fascin, an actin-bundling protein associated with cell motility. Further, repression of NFκB results in decreased expression of anti-apoptotic genes and two tumor-metastasis activators, osteopontin and urokinase-type plasminogen activator. Clinical Relevance BRMS1 is regulated at both the RNA and protein levels. To date, only a single study has examined levels of BRMS1 protein in patient samples to find a loss of BRMS1 in nearly 25% of 238 breast cancer cases. Further, the study showed loss of BRMS1 correlated with disease-free survival when stratified by loss of estrogen receptor, progesterone receptor, or Her2 overexpression. Other clinical studies have studied BRMS1 mRNA expression in human cancers compared to adjacent non-cancerous tissues or regional lymph nodes. Since BRMS1 is regulated at the protein level, looking exclusively at mRNA may be misleading. Nonetheless, the majority of studies show high levels of BRMS1 correlates with increased disease-free survival and diminished progression.

References 1. Samant RS, Clark DW, Fillmore RA et al. (2007) Breast cancer metastasis suppressor 1 (BRMS1) inhibits osteopontin transcription by abrogating NF-kappaB activation. Mol Cancer 6:6 2. Hicks DG, Yoder BJ, Short S et al. (2006) Loss of breast cancer metastasis suppressor 1 protein expression predicts reduced disease-free survival in subsets of breast cancer patients. Clin Cancer Res 12:6702–6708 3. Liu Y, Smith PW, Jones DR (2006) Breast cancer metastasis suppressor 1 functions as a corepressor by enhancing histone deacetylase 1-mediated deacetylation of RelA/p65 and promoting apoptosis. Mol Cell Biol 26:8683–8696 4. Meehan WJ, Samant RS, Hopper JE et al. (2004) Interaction of the BRMS1 metastasis suppressor with RBP1 and the mSin3 histone deacetylase complex. J Biol Chem 279:1562–1569 5. Hurst DR, Mehta A, Moore BP et al. (2006) Breast cancer metastasis suppressor 1 (BRMS1) is stabilized by the Hsp90 chaperone. Biochem Biophys Res Commun 348:1429–1435

BRN-5547136 ▶Temozolomide

B

432

Broder Histological Classification

Broder Histological Classification

Brown Adipose Tissue

Definition

Definition

Refers to histological classification of differentiation in ▶squamous cell carcinoma. Devised by Broder. Grades 1, 2 and 3 denoted ratios of differentiated to undifferentiated cells of 3:1, 1:1 and 1:3, respectively. Grade 4 denoted tumor cells having no tendency towards differentiation.

BAT is present in many newborn or hibernating mammals, and its primary function is to generate heat. ▶Cachexia

brp-39 Bromodeoxyuridine (BrdU)

▶Serum Biomarkers

Definition A compound that, due to its chemical structure, can substitute for thymidine in DNA. ▶Fragile Sites

Brush Border Definition

Bromodomain Definition Conserved domain that specifically recognizes and binds to acetylated lysine residues that occur within a protein. ▶Histone Modifications

Bronchogenic Carcinoma ▶Lung Cancer

Is formed by the densely packed microvilli of the surface of columnar epithelial cells, e.g., in the intestine and in the proximal tubules of the kidney. Microvilli are small projections of the plasma membrane which greatly enlarge the surface area of the cell. Individual microvilli can only be distinguished using an electron microscope; in a light microscope, the microvilli are observed collectively as a fuzzy fringe at the surface of the epithelium which has, therefore, been termed brush border. ▶Membrane Transporters

Bryostatin-1 B ASSEL E L -R AYES Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

Brother of the Regulator of Imprinted Sites ▶BORIS

Definition Bryostatins are a class of macrocyclic lactones. Bryostatins are potent modulators of ▶Protein Kinase C (PKC). Bryostatin-1 was isolated from the marine invertebrate Bugula neritina. Bryostatin-1 is currently not available for commercial use.

Bryostatin-1

Characteristics Rationale for Targeting the PKC PKC is a family of homologous serine/threonine protein ▶kinases that transduce signals linked to diverse cellular processes including proliferation, differentiation, angiogenesis, and ▶apoptosis. The PKC family includes 12 isoforms subdivided into three major classes based on their co-factor requirements for activation. Aberrant regulation of the PKC enzymes activity has been demonstrated in a number of malignancies including: breast, colorectal, pancreatic and non-small cell lung cancer. Preclinical Activity of Bryostatin-1 Treatment of cancer cell lines with bryostatin-1 results in the activation of PKC. However, prolonged exposure to bryostatin-1 induces PKC inhibition most probably through ubiquitin-mediated degradation. Inhibition of PKC activity results in cell cycle arrest, apoptosis, cell differentiation and modulation of chemoresistance. Bryostatin-1 has been shown to potentiate the effects of several classes of cytotoxic agents including: vincristine in diffuse large cell lymphoma, melphalan in Waldenstrom’s macroglobulinemia, gemcitabine in pancreatic and breast cancer, paclitaxel and mitomycin C in gastric cancer cell lines. The synergism between bryostatin and cytotoxic agents is sequence dependent. Single Agent Activity of Bryostatin-1 Phase I trials of bryostatin-1 used two different schedules. The maximal tolerated doses were 25 μg/ m2 when infused over 24 h and 120 μg/m2 when infused over 72 h. The most common side effects included ▶myalgia. Other observed toxicities included headache, phlebitis, and transient ▶thrombocytopenia. Single agent bryostatin-1 has been studied in phase II trials for lymphoma, renal, colorectal, head and neck, sarcoma, and melanoma. Bryostatin-1 did not demonstrate single agent activity in any of these diseases. Bryostatin-Based Combinations Since PKC activation contributes to chemoresistance the combinations of bryostatin-1 and cytotoxic agents were tested. In Chronic Lymphocytic Leukemia (CLL) and indolent Non-Hodgkin Lymphoma, bryostatin-1 was evaluated in combination with fludarabine. Patients received fludarabine daily for 5 days and bryostatin-1 over 24 h infusion either before or after fludarabine. The combination was well tolerated. Partial and complete responses were observed in 6 and 2 patients (total number 27), respectively. Bryostatin-1 and vincristine was evaluated in patients with refractory B-cell lymphoma. Twenty four patients were enrolled on the study. The bryostatin-1 was well tolerated at a dose of 50 μg/m2 over 24 h infusion. The regimen had activity with five patients having objective response and five having stable disease.

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Bryostatin-1 was also evaluated in combination with cisplatin in two phase I trials. In the first trial, bryostatin-1 (30 μg/m2 over 24 h infusion), had no significant activity. In the second trial bryostatin-1 was administered at a dose of 15–55 μg/m2 over 72 h. In this study, three responses were reported. A phase I trial of gemcitabine and bryostatin-1 (25–35 μg/m2 over 24 h) revealed that the regimen was well tolerated and resulted in stable disease in 8 out of 36 patients. Bryostatin-1 (15–50 μg/m2 infused over 24 h) was evaluated in combination with paclitaxel. Partial responses were observed in patients with pancreatic and gastroesophageal cancer. The common finding in these trials is that bryostatin-1 can be combined safely with cytotoxic chemotherapy agents. Phase II trials evaluating the activity of bryostatin-1 with cisplatin in cervical cancer had disappointing results. Fourteen patients were enrolled on the trial and there were no treatment responses. Ajani et al. reported on thirty seven patients with gastroesophageal and gastric cancer treated with bryostatin-1 (40 μg/m2 infused over 24 h) and weekly paclitaxel (80 mg/m2). The response rate was 29% which is higher than the previously reported response rates with paclitaxel. In a phase II trial, bryostatin-1 (50 μg/m2 infused over 24 h) and weekly paclitaxel (90 mg/m2) were evaluated in patients with non-small cell lung cancer. Of eleven response evaluable patients, stable disease was seen in five patients. Therefore, the bryostatin-1 and paclitaxel combination did not demonstrate significant activity in lung cancer. Future Directions Mixed results have been observed in the trials evaluating bryostatin-1 and cytotoxic chemotherapy agents. The future challenges in the development of bryostatin-1 include, the identification of biomarkers that can predict activity and the development of combination therapy with other targeted agents. Another approach for the development of bryostatins is through the modification of the chemical structure in order to identify analogues with better safety or efficacy profiles than bryostatin-1.

References 1. Choi SH, Hyman T, Blumberg PM (2006) Differential effect of bryostatin 1 and phorbol 12-myristate 13-acetate on HOP-92 cell proliferation is mediated by downregulation of protein kinase Cdelta. Cancer research 66: 7261–7269 2. Deacon EM, Pongracz J, Griffiths G et al. (1997) Isoenzymes of protein kinase C: differential involvement in apoptosis and pathogenesis. Mol Pathol 50: 124–131 3. Kortmansky J, Schwartz GK (2003) Bryostatin-1: a novel PKC inhibitor in clinical development. Cancer investigation 21: 924–936

B

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BSA

BSA

BUB1

Definition

Definition

Body surface area; The current practice of using bodysurface area (calculated using a formula derived from height and weight) in dosing anticancer agents has been implemented in clinical oncology about half a century ago. By correcting for BSA, it was generally assumed that the interindividual variation in the pharmacokinetics of the drug administered would be reduced which would lower the risk of serious adverse effects without reducing the agent’s therapeutic effect. Recently, doubt has arisen to this hypothesis, and for many anticancer drugs the rationale for individualization of dosage based on BSA is lacking.

budding uninhibited by benzimidazoles 1 is a protein that is required for the spindle assembly checkpoint. BUB1 is a protein kinase; it phosphorylates CDC20 and inhibits ubiquitin ligase activity of APC/C. In mammals, BUB1 depletion causes embryonic lethality in mice.

▶Irinotecan ▶Pharmacokinetics/Pharmacodynamics

▶Mitotic Arrest-Deficient Protein 1 (MAD1) ▶Synucleius

Bulk Minerals Definition

B-Scan Ultrasonography Definition Ophthalmologic ultrasound the provides a two-dimensional ultrasound image of the echogencity of the ocular structures providing a cross-sectional view allowing the for the diagnosis and characterization of multiple disorders including retinal and choroidal detachments, vitreous hemorrhages, vitritis, and intraocular tumors. ▶Uveal Melanoma

Are mineral nutrients that are typically required to be ingested by humans in amounts of hundreds of milligrams to a few grams per day. This category includes calcium, phosphorus, and magnesium and, along with electrolytes, are sometimes referred to as macrominerals. ▶Mineral Nutrients

Bulky Definition A lymphoma is bulky if a nodal lymphoma mass with largest dimension of 10 cm or greater is present.

BSF-2

▶Diffuse Large B-Cell Lymphoma

▶Interleukin-6

Burkitt Lymphoma BTAK ▶Aurora Kinases

Definition

Burkitt lymphoma is caused by ▶Epstein-Barr virus (EBV) and occurs mainly in sub-Saharan Africa.

Bystander Effect

Burkitt Lymphoma Cell Lines

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Bystander Effect B

Definition

Definition

Burkitt lymphoma cell lines are EBV-infected B cell lines established from Burkitt lymphoma biopsies; these cells are tumorigenic in nude mice.

When cells are killed indirectly by virtue of neighboring cells that transfer toxic products to them.

▶BCL6 Translocations in B-cell Tumors ▶Epstein-Barr Virus

▶HSV-TK/Ganciclovir Mediated Toxicity

C

C33 ▶Metastasis Suppressor KAI1/CD82

C75 Definition Derivative of cerulenin inhibits fatty acid synthase activity by interfering with the binding of malonyl-CoA to the β-ketoacyl synthase domain of fatty acid synthase. ▶Fatty Acid Synthase

CA19-9 Definition Carbohydrate antigen 19-9 is used as a tumor-marker when measured in serum. It is thought to be a sialylated Lewis blood group antigen. CA19-9 levels are elevated in many gastrointestinal malignancies including cholangiocarcinoma and pancreatic cancer, as well as some non-malignant conditions such as cholangitis and peritoneal ▶inflammation/infection. Patients who have a genetic deficiency in a fucosyltransferase specified by the Le gene are Lewisa-b- and are unable to make this antigen; thus CA19-9 testing in Lewisa-b- patients can be falsely negative. ▶Cholangiocarcinoma ▶Fucosylation

CA125 c-Abl Definition A protein tyrosine kinase which activation leads to cell cycle arrest and apoptosis. A chromosomal translocation of c-Abl to the Bcr locus results in a fusion gene. Constitutively active tyrosine kinase Bcr/Abl expression associates with chronic myelogenic leukemia. ▶Protein Kinase C Family ▶BCR-ABL1

C.elegans cell death 4 homolog ▶APAF-1 Signaling

Definition CA125 (cancer antigen 125) is a mucin-like protein of high molecular mass estimated at 200–20,000 kDA. CA125 cell surface expression is upregulated when cells undergo metaplastic differentiation into a Mülleriantype epithelium. CA125 is the most extensively studied biomarker for possible use in ovarian carcinoma early detection. It is elevated in some cases of ▶endometriosis. ▶Mesothelin ▶Ovarian Cancer ▶Serum Biomarkers

Ca2+-activated Phospholipid Dependent Protein Kinase ▶Protein Kinase C Family

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Ca2+ ATPase

Ca2+ ATPase Definition ATPases are a class of enzymes that catalyze the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP). The Ca2+ ATPase uses this process to transport Ca2+ against a concentration gradient (e.g., Ca2+ transported from the cytoplasm into the endoplasmic reticulum). ▶Celecoxib

Ca2+-Release Channels Definition Membrane receptors localized in the sarcoplasmic/ endoplasmic reticulum that once activated mediates the release of Ca2+ from the ER to the cytosol. ▶Endoplasmic Reticulum Stress

CaBP3 ▶Calreticulin

Cachexia C HEN B ING Division of Cellular and Metabolic Medicine, School of Clinical Sciences, University of Liverpool, Liverpool, UK

Definition

Cachexia came from the Greek “kakos” and “hexis” meaning “bad conditions.” Cachexia is a complex metabolic syndrome characterized by progressive weight loss with extensive loss of skeletal muscle and adipose tissue, which is secondary to the growing malignancy.

Characteristics Most cancer patients develop cachexia at some point during the course of their disease, and nearly one-half of all cancer patients have weight loss at diagnosis. Cachexia prevents effective treatments for cancer and predicts a poor prognosis because the severity of wasting inversely correlates with survival. The consequences of cachexia are detrimental and cachexia is considered to be the direct cause of about 20% of cancer deaths. The pathogenesis of cancer cachexia remains to be fully understood, but it is evidently multifactorial. Weight Loss Clinically, cachexia should be suspected if involuntary weight loss of more than five percent of premorbid weight occurs within a six-month period. Weight loss is not simply caused by competition for nutrients between tumor and host as the tumor burden may be only 1–2% of total body weight. The frequency of weight loss varies with the type of malignancy, being more common and severe in patients with cancers of gastrointestinal tract (▶gastrointestinal tumors) and lung (▶lung cancer). Gastric and pancreatic cancer patients may lose large amounts of weight, up to 25% of initial body weight. Over 15% of weight loss in patients is likely to cause significant impairment of respiratory muscle function, which probably contributes to premature death. Weight loss can arise from several metabolic changes that take place during malignancy, for example, reduced food intake, increased energy expenditure, and tissue breakdown. Poor Appetite Loss of the desire to eat or lack of hunger is common in cancer patients. It can be related to the mechanical effect of the tumor such as obstructions (especially of the upper gastrointestinal tract), side-effects of chemotherapy or radiotherapy (▶chemoradiotherapy), and emotional distress. Some tumors may secrete products which act on the brain to inhibit appetite. Regulation of food intake involves the integration of the peripheral and neural signals in the hypothalamus and other brain regions. In the hypothalamus, the orexigenic signals such as ▶neuropeptide Y (NPY), the most potent appetite stimulant, increase food intake, and the anorexigenic signals including the pro-opiomelanocortin/cocaine and amphetamine regulated transcript (POMC/CART) inhibit appetite. Dysregulation of NPY in the hypothalamic pathway can lead to decreased energy intake but higher metabolic demand for nutrients. It has been demonstrated that NPY-immunoreactive neurons in the hypothalamus are decreased in experimental model of cancer anorexia. In contrast, reduced food consumption can be restored to normal levels by blocking the POMC/CART pathway in tumor-bearing animals.

Cachexia

High level of leptin, a hormone primarily secreted by adipocytes, inhibits the release of hypothalamic NPY. In cancer cachexia the leptin feedback loop appears to be deranged, altering the signaling pathway of NPY. Cytokines such as interleukin 1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) are implicated to be involved in cancer anorexia, possibly by stimulating corticotrophin-releasing factor, a neurotransmitter which suppresses food intake at least in rodents, and/or by inhibiting neurons that produce NPY in the hypothalamus.

Increased Metabolism and Energy Expenditure Maintaining normal body weight requires energy intake to equal energy expenditure. In some patients with cancer cachexia, energy balance becomes negative as reduced food intake is not accompanied by a parallel decrease in energy expenditure. For example, patients with lung and pancreatic cancers generally have higher ▶resting energy expenditure (REE) compared with normal control subjects; however, REE is usually normal in patients with colorectal cancer. The mechanisms of increased energy expenditure are not clear although studies suggest that it might be through the upregulation of uncoupling proteins, a family of mitochondrial membrane proteins, which are proposed to be involved in the control of energy metabolism. ▶Uncoupling protein-1 (UCP-1), which decreases the coupling of respiration to ADP phosphorylation thereby generating heat instead of ATP, is only expressed in ▶brown adipose tissue (BAT). UCP-1 mRNA levels in BAT are increased in mice bearing the MAC16 colon adenocarcinoma. Although BAT is uncommon in adults, the prevalence of BAT has been found to be higher in cancer cachectic patients than the age-matched control subjects. mRNA levels of UCP2 (expressed ubiquitously) and UCP-3 (expressed in skeletal muscle and BAT) in skeletal muscle are upregulated in rodent models of cancer cachexia. In humans, skeletal muscle UCP-3 mRNA levels are over fivefold higher in cachectic cancer patients compared with patients without weight loss and health controls. Elevated expression of UCP-2 and -3 has been suggested to contribute to lipid utilization rather than whole-body energy expenditure. Cytokines such as TNFα and/or other tumor products may be responsible for the changes in UCP expression at least in rodents. Additional energy consumption could arise from the metabolism of tumor-derived lactate via “futile cycles” between the tumor and the host. The main energy source for many solid tumors is glucose, which is converted to lactate and transferred to the liver to convert back into glucose. This “futile cycle” requires large amount of ATP, resulting in an extra loss of energy in cancer patients.

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Loss of Adipose Tissue Fat constitutes 90% of normal adult fuel reserves and depletion of adipose tissue together with ▶hyperlipidemia becomes a hallmark of cancer cachexia. Computed tomography (CT) scanning has revealed that cachectic cancer patients with gastrointestinal carcinoma had significantly smaller visceral adipose tissue area than control subjects. Increased lipolysis is implicated in cancer-associated adipose atrophy. The activity of hormone-sensitive lipase, a rate-limiting enzyme of the lipolytic pathway, is increased in cancer cachectic patients, which causes elevated plasma levels of free fatty acids and triglycerides. Meanwhile, there is a fall in lipoprotein lipase (LPL) activity in white adipose tissue, thus inhibiting cleavage of triglycerides from plasma lipoproteins into glycerol and free fatty acids for storage, causing a net flux of lipid into the circulation. Finally, glucose transport and de novo ▶lipogenesis in the tissue are reduced in tumor-bearing state, leading to a decrease in lipid deposition. There is also evidence that loss of adipose tissue in cancer cachexia could be the result of impairment in the formation and development of adipose tissue. The expression of several key adipogenic transcription factors including CCAAT/enhancer binding protein alpha, CCAAT/enhancer binding protein beta, peroxisome proliferator-activated receptor gamma, and sterol regulatory element binding protein-1c are markedly reduced in adipose tissue of cancer cachectic mice. Various factors produced by tumors or the host’s immune cells, responding to the tumor can disturb lipid metabolism. TNFα has been shown to affect adipose tissue formation by inhibiting the differentiation of new adipocytes, causing dedifferentiation of mature fat cells and suppressing the expression of genes encoding key lipogenic enzymes. TNFα has also been associated with increased lipolysis probably through suppression of LPL activity in adipocytes. In addition, both TNFα and IL-1 are able to inhibit glucose transport in adipocytes and consequently decrease the availability of substrates for lipogenesis. Certain prostate, gut and pancreatic tumors secrete a lipid-mobilizing factor (LMF), also produced by a mouse adenocarcinoma model. LMF has been shown to be identical to the plasma protein zinc-α2-glycoprotein (ZAG). It is recently found to be secreted by human adipocytes and upregulated in adipose tissue of mice with cancer cachexia. ZAG causes rapid lipolysis in vitro and in vivo, possibly through activation of intracellular cyclic AMP. ZAG also stimulates expression of UCPs in brown fat of mice, which may contribute to increased energy expenditure as well as lipid catabolism during cachexia. Moreover, ZAG has been shown to reduce glucose metabolic rate in adipose tissue, consistent with a decrease in glucose transportor-4 transcript in white fat of mice bearing a ZAG-producing tumor.

C

440

Cachexia-inducing Agent

Loss of Muscle Protein Weakness, commonly seen in cancer cachectic patients, is directly related to wasting of muscle that accounts for almost half the body’s total protein and bears the brunt of enhanced protein destruction. Reduced protein synthesis together with enhanced proteolysis have been observed in experimental animal models and in muscle biopsies from cancer patients with cachexia, and whole-body protein turnover can be markedly increased in cachectic cancer patients. Some mediators and pathways of excessive protein breakdown have been incriminated in cancer cachexia. TNFα appears to be involved, as treatment with recombinant TNFα enhances proteolysis in rat skeletal muscle and activates the ubiquitin–proteasome system. Ubiquitin, an 8.6 kD peptide, is crucially involved in targeting of proteins undergoing cytosolic ATP-dependent proteolysis. There is an increase in ubiquitin gene expression in rat skeletal muscle after incubation with TNFα in vitro. Tumors also produce cachectic factors such as proteolysis-inducing factor (PIF), a 24 kD glycoprotein initially isolated from a cachexia-inducing tumor (MAC16) and the urine of cachectic cancer patients. PIF induces muscle protein breakdown by stimulation of the ubiquitin–proteasome proteolytic pathway. There is increasing evidence that both cytokines and PIF cause protein degradation by activation of ▶nuclear factor kappa B (NFκB), a transcription factor that regulates the expression of a number of proinflammatory cytokines. TNFα and PIF can upregulate components of the ubiquitin–proteasome pathway in an NFκB-dependent manner. Activation of NFκB by TNFα in murine muscle cells suppresses mRNA of the transcription factor ▶MyoD, inhibiting skeletal muscle cell differentiation as well as preventing the repair of damaged skeletal muscle fibers. Treatment Current treatment designed to ameliorate cancer cachexia has limited benefit. Nutritional supplementation (oral or parenteral) alone has little effect and, critically, does not restore muscle mass, improve quality of life or prognosis in cancer patients. Appetite stimulants such as megestrol acetate and medroxyprogesterone acetate are commonly used at present in the treatment of anorexia and cachexia. These agents are believed to stimulate orexigenic peptide NPY in the hypothalamus, and inhibit the synthesis and release of proinflammatory cytokines. Their effects on appetite and well being are short-termed and they do not influence lean body mass and survival. Cannabinoids (▶cannabinoids and cancer) have also been studied as potential appetite stimulants. However, dronabinol has failed to prevent progressive weight loss in patients with advanced cancer. Therapeutic interventions include anticytokines such as thalidomide (▶thalidomide and its analogs) with

multiple immunomodulatory properties. It suppresses the productionofTNFα, IL-1β, IL-12, and cyclooxygenase-2, which is probably through inhibiting NFκB activity. Thalidomide has been shown to attenuate total weight loss and loss of lean body mass in cachectic patients with advanced pancreatic cancer. ▶Eicosapentaenoic acid (EPA), a polyunsaturated fatty acid from fish oil, has attracted attention as a potential anticachectic agent. EPA has been shown to attenuate the increased expression of the components of the ubiquitin–proteasome proteolytic pathway in skeletal muscle of mice with cancer cachexia, and EPA can block PIF-induced protein degradation in vitro. In randomized clinical trials, cachectic patients with unresectable pancreatic cancer receiving EPA have shown a stabilization in the rate of weight loss, fat and muscle mass as well as the REE. Recent data from animal studies suggest that EPA combined with the leucine metabolite beta-hydroxy-betamethylbutyrate seems to be more effective in the reverse of muscleprotein wasting.

References 1. Tisdale MJ (2002) Cachexia in cancer patients. Nat Rev Cancer 2:862–871 2. Fearon KC, Moses AG (2002) Cancer cachexia. Int J Cardiol 85:73–81 3. Bing C, Brown M, King P et al. (2000) Increased gene expression of brown fat uncoupling protein (UCP)1 and skeletal muscle UCP2 and UCP3 in MAC16-induced cancer cachexia. Cancer Res 60:2405–2410 4. Bing C, Russell S, Becket E et al. (2006) Adipose atrophy in cancer cachexia: morphologic and molecular analysis of adipose tissue in tumour-bearing mice. Br J Cancer 95:1028–1037 5. Argiles JM, Busquets S, Lopez-Soriano FJ (2006) Cytokines as mediators and targets for cancer cachexia. Cancer Treat Res 130:199–217

Cachexia-inducing Agent ▶Leukemia Inhibitory Factor

Caco-2 Definition Caco-2 is an immortalized cell line originally derived from a human ▶colon cancer. It can be grown in-vitro in

Cajal Bodies

such a way as to mimic the gastrointestinal tract wall and is used in cell culture models to measure drug intestinal permeability. ▶ADMET Screen

Cadherin 1 Definition CDH1; synonyms Epithelial cadherin, Uvomorulin. ▶E-Cadherin

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Cajal Bodies V INCENZO

DE

L AURENZI

Department of Experimental Medicine and Biochemical Sciences, University of Tor Vergata, Rome, Italy

Synonyms Coiled bodies

Definition

Small nuclear organelles (0.1–2.0 μM in diameter), present in all eukaryotic cells, involved in a number of different nuclear functions.

Characteristics

Cadherins Definition Cadherins are a family of cell adhesion receptor glycoproteins that are involved in the calcium-dependent cell-to-cell adhesion. E-, V-, and N-cadherins are distinct in immunological specificity and tissue distribution. They promote cell adhesion via their homophilic binding interactions. Class of type-1 transmembrane proteins important in cell adhesion. They are dependent on calcium (Ca2+) ions to function, hence their name. Cadherins play an important role in regulation of morphogenesis. Cadherins inhibit invasiveness of tumor cells. ▶Doublecortin ▶E-Cadherin ▶EpCAM ▶Tight Junction ▶Adhesion ▶Cell Adhesion Molecules

Cafe Au Lait Spots Definition Synonym Cafe-au-lait Macule; Coffee-with-milk-colored spots on the skin that are seen characteristically in the neurofibromatosis type 1 (NF1) syndrome.

The nucleus of eukaryotic cells contains a number of different highly specialized organelles. Unlike cytoplasmic organelles these nuclear structures are not delimited by a membrane but are by all means compartments that contain a number of specific proteins. Most of the organelles can be clearly identified trough immuno-staining using antibodies directed against specific marker proteins; however, it should be kept in mind that these organelles are highly dynamic structures that often exchange components and therefore many proteins can be found in more than one organelle. Among these organelles are Cajal Bodies (CBs), described over a century ago by Ramon y Cajal. CBs were originally described in neuronal cells but have since been described in a variety of cell types, both in animals and in plants, suggesting that they are involved in some fundamental cellular process. Due to their characteristic ultra-structural appearance as a tangle of coiled fibrillar strands they have also been called coiled bodies. They usually vary in size from 0.2 μM to 2 μM, but can be occasionally larger. The number of CBs is usually between 0 and4 in normal diploid cells; however, many more can be found in some cancer cells. The number of CBs per cell is regulated during the cell cycle. Indeed, CBs disappear in prophase nuclei, to reappear in G1 at the same time of the nucleolus. Their number is then doubled, usually reaching the number of four, in the S phase. It has been suggested that in these cells the number of CBs depends on the ploidy of the cells or more specifically on the number of chromosomes 1 and 6. CBs can be found associated with specific gene loci such as snoRNA, snRNA and histone gene clusters. In addition, CBs can also be found in association with other nuclear bodies such as cleavage bodies and ▶PML bodies, suggesting that there is an exchange of components between the different nuclear organelles.

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CBs have a heterogeneous composition, containing different Small Nuclear Ribonucleoproteins (snRNPs), Small Nucleolar Ribonucleoproteins (snoRNPs), cell cycle regulating proteins, and transcription factors, as well as other proteins, whose function still needs to be determined. The generally recognized marker of CBs is p80 coilin. The function of this protein is still unknown, its deletion in mice results in reduced coilin –/– animal litters, suggesting a developmental defect; however surviving animals appear normal. Deletion of coilin results in residual bodies that still contain some components such as fibrillarin, Nopp140 and FLASH, but not others like splicing snRNPs. While their function is still in part elusive, recent work suggests that they are involved in several nuclear functions. CBs are supposed to be the site of assembly of the three eukaryotic RNA polymerases (pol I, pol II and pol III) with their respective transcription and processing factors that are then transported as multiprotein complexes to the sites of transcription.They are also involved in the modification of Small Nuclear RNAs (snRNAs) and Small Nuclear Ribonucleoproteins (snRNPs), which are important for spliceosome formation. Indeed CBs contain newly assembled snRNPs and snoRNPs that later accumulate in speckles and nucleoli and it has recently been suggested that CBs are sites of modification for snRNPs and particularly sites where 2′-O-methylation and pseudouridine formation occur. This process requires a novel class of CB specific small RNAs (scaRNAs) that pair with the snRNAs and function as guides for 2′-Omethylation. The reaction is probably mediated by the fibrillarin, a CB and nucleolar associated protein with methyl transferase activity. CBs have also been implicated in ▶replication dependent histone gene transcription, and a subset of CBs is physically associated with histone gene clusters on chromosomes 1 and 6. Phosphorylation of a CB component p220/NPAT by cyclinE/Cdk2 is required for activation of histone transcription, exit from G1 and progression through S phase of the cell cycle. Moreover it has been shown that another CBs component FLASH is essential for this function. Downregulation of FLASH results in structural alteration of CBs, reduction of replication dependent histone gene transcription, and block of cells in the S phase (▶S-phase damage-sensing checkpoints) of the cell cycle. In addition, CBs are involved in U7 snRNA dependent cleavage of the 3′ end of histone pre-mRNA before the mature mRNA can be exported to the cytoplasm. Finally, a role for CBs in regulating ▶telomerase function has been proposed. Based on the presence of the RNA component of telomerase (hRT) in CBs of cancer cells, it has been suggested that CBs play a role in the maturation of hRT or in the assembly of the telomerase complex. However, CBs might represent

only a site of accumulation of hRT; alternatively, this could be an altered localization only present in cancer cells; therefore further studies are required to clarify this potential CB function. Alteration of CB structure, as well as other nuclear structure alterations, has been observed in various diseases; however, in most cases it is not clear if these defects are a consequence of altered nuclear functions or play a role in the disease pathogenesis. CBs have been found associated with the aggregates formed in CAG triplet expansion diseases and ataxin-1; mutated in ▶Spinocerebellar ataxia type 1 (SCA1) it has been shown to interact with coilin. The role of these findings in the disease pathogenesis is yet to be established. Spinal Muscular Atrophy (SMA) is an autosomal recessive disease characterized by motor neuron degeneration associated with muscular atrophy and paralysis; it is usually caused by mutations of the surviving motor neurons 1 gene (SMN1). SMN is a 294 amino acid protein, ubiquitously expressed; it bears no homology to other known proteins and its function is still unknown. It is localized both in the cytoplasm and in the nucleus where it is found in two different nuclear organelles: Cajal bodies (CBs) and Gems (for Gemini of CBs). Pathogenesis of SMA is not clearly understood but reduction of SMN levels results in an alteration of CB structure. Alteration of CBs in cancer has not been thoroughly studied yet and a role for these organelles in cancer has not been clearly established. However cancer cell lines often show an increased number of CBs and some alteration of CBs can be found in specific cancers. In ▶MLL-ELL leukemia (▶Acute myeloid leukemia) the presence of the MLL-ELL fusion protein results in alteration of CBs structure and altered localization of coilin. The TLS/CHOP fusion protein generated by the t(12;16) translocation (▶Chromosome Translocations), found in liposarcomas shows high transforming capacity and is in part localized in CBs. In conclusion, while recent studies have started to shed light on the function of CBs and on the interrelationship between these organelles and other nuclear structures, more work is required to clearly understand the molecular mechanisms involved in their formation and clarify their different roles in nuclear function. This in turn will provide information on their potential role in the pathogenesis of a range of human diseases.

References 1. Gall JG (2000) Cajal bodies: the first 100 years. Annu Rev Cell Dev Biol 16:273–300 2. Ogg SC, Lamond AI (2002) Cajal bodies and coilin – moving towards function. J Cell Biol 159(1):17–21 3. Cioce M, Lamond AI (2005) Cajal bodies: a long history of discovery. Annu Rev Cell Dev Biol 21:105–131

Calcitonin

CAK1 Antigen ▶Mesothelin

Cal Definition

Is a member of the ▶zyxin family of proteins. Also known as migfilin. ▶Lipoma Preferred Partner

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Characteristics Almost all cells of human body synthesize and secrete procalcitonin (proCT), a precursor of the CT peptide, in response to infection/▶inflammation. Only cells of the thyroid and ▶neuroendocrine organs can process proCT to produce mature CT molecule. CT sequence among various species shows remarkable divergence. However, all sequences contain 32 amino acids, a carboxy-terminal proline amide, and a disulfide bridge between cysteine residues at positions 1 and 7. In addition to CT, other biologically and chemically diverse molecules such as CT gene-related peptide (CGRP), ▶adrenomedullin (▶ADM), and ▶amylin (AMY) are considered as CT family of peptides because of their ability to interact with CT receptor (CTR) and induce biological response. Each of these peptides displays selective tissue distribution and distinct physiological effects. For example, CGRP is predominantly present in central and peripheral nervous system, and is important for neurotransmission and neuromodulation. ADM is relatively abundant in vascular space, and plays an important role in the regulation of cardiovascular and respiratory functions, and CT is essential for calcium balance. However, CT does not regulate calcium in extrathyroidal tissues but is implicated to play an important role in cell growth, cell differentiation, and other regulatory functions.

Definition

Is a highly conserved, Ca2+/calmodulin-dependent serine/threonine phosphatase, also called protein phosphatase 2B (PP2B). Calcineurin is best known for its role in the Ca2+-dependent regulation of the nuclear factor of activated T-cells (NF-AT) pathway which is involved in T-cell activation. ▶Calreticulin

Calcitonin G IRISH V. S HAH Department of Pharmacology, University of Louisiana College of Pharmacy, Monroe, LA, USA

Definition Calcitonin (CT) is a 32-amino acid peptide synthesized in mammals by the C cells of the thyroid gland. Several extrathyroidal sites including the prostate gland, gastrointestinal tract, thymus, bladder, lung, pituitary gland, and central nervous system (CNS) also produce this peptide molecule.

Biosynthesis Four CT genes, CALC-I, CALC-II, CALC-III, and CALC-IV with significant nucleotide homologies have been identified. However, CT is encoded by only CALC-I gene. CALC-I and CALC-II encode two different forms of CGRP, CGRP-I and CGRP-2. CALC-III is thought to be a pseudo gene, and CALC-IV produces AMY. Human (h) CT (CALC-I) gene is located in the p14qter region of chromosome 11. CT gene encodes two distinct peptides CT and CGRP, which arise by tissue-specific alternative splicing of the same primary mRNA transcript. The primary mRNA transcript is spliced almost exclusively to CT mRNA in thyroid, and to CGRP in the nervous system. CT is synthesized as part of a larger precursor protein of 136 amino acids. The DNA sequence of the hCT gene predicts that the hormone is flanked in the precursor by N- and C-terminal peptides. Both N-terminal and C-terminal flanking peptides are detected in the plasma and thyroidal tissues of both normal and ▶medullary thyroid carcinoma (MTC) patients. However, no biological function for either of these two peptides has been conclusively determined. Cyclic adenosine monophosphate (cAMP), pentagastrin, and progesterone are potent stimulators of CT gene expression. In contrast, testosterone and estrogens have inhibitory effect.

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There is evidence for ▶polymorphisms in CALC-I gene that leads to increased risk for ovarian ▶cancer in carrier women (T-C 624 bp upstream of translation initiation codon); and 16 bp microdeletion polymorphism has been reported in a family multiple cases of unipolar and bipolar depressive disorders. Biological Actions Actions on Bone CT is the only hormone that inhibits bone resorption by direct action on osteoclasts in the bone. It is characterized by the rapid loss of osteoclast ruffled borders, reduced cytoplasmic spreading, decreased release of lysosomal enzymes, and inhibition of collagen breakdown. This role is physiologically more relevant at times of stress on skeletal calcium conservation such as pregnancy, lactation, and growth, when bone remodeling by osteoclasts and the consequent release of calcium stores in the bone need to be tightly regulated to prevent unnecessary bone loss. In normal adult humans, even large dose of CT has little effect on serum calcium. However, in pathologies created by increased bone turnover such as thyrotoxicosis, ▶metastatic bone disease, or Paget’s disease, CT treatment effectively inhibits bone resorption and lowers serum calcium. Renal Actions CT increases urinary excretion rate of sodium, potassium, phosphorus, and magnesium. CT also enhances 1-hydroxylation of 25-hydroxy vitamin D in the proximal straight tubule by stimulating the expression 25-hydroxy vitamin D 1-hydroxylase. Central Actions Central administration of CT produces analgesia, affects sleep cycles producing insomnia, major reduction in slow wave sleep and long period of alteration of rapid eye movement (REM) sleep and wakening. The centrally mediated actions of CT correlate well with the location of CT binding sites. CT also demonstrates multiple hypothalamic actions such as modulation of hormone release, decreased appetite, gastric acid secretion, and intestinal motility. Administration of CT in clinical situations of bone pain is very effective in ameliorating the pain symptoms. Other Actions CT and its receptors have been identified in a large number of other cell types and tissue sites suggesting multiple roles for CT–CTR axis. CTR binding sites have been identified in the kidneys, brain, pituitary, testis, prostate, spermatozoa, lung, and lymphocytes. There is evidence to suggest the involvement of CT in cell growth and differentiation, tissue development and tissue remodeling. CT appears to be important for

blastocyst implantation and development of the early blastocyst. CT in Cancer Overexpression of CT has been reported in cancerderived cells from ▶thyroid, ▶lung, ▶breast, ▶prostate, ▶pancreas, ▶pituitary, ▶bone (osteoclastoma, osteogenic sarcoma), and embryonal carcinoma, suggesting the deregulation of CT expression is an important event in several malignancies. The results from our laboratory have shown that CT and CTR are present in undifferentiated basal cells, but absent in differentiated secretory cells of normal human prostate gland. However, CT and CTR become detectable in malignant secretory epithelium suggesting malignancyassociated deregulation of CT/CTR expression. CT and CTR transcripts in malignant human prostate become detectable as early as high-grade ▶PIN, and progressively increase with increase in tumor grade. In human pancreas, CTR is present in benign as well as malignant regions but CT is exclusively detected in malignant sections of multiple pancreatic carcinomas, including ductal adenocarcinomas. Mechanism of CT Action Receptors CT acts by binding to receptors on the plasma membrane of responding cells. CTR cDNA has been cloned in multiple mammalian species. Analysis of the protein translated from CTR cDNA sequence reveals the size of approximately 500 amino acids, and the receptor belongs to the class B family of G protein-coupled receptors (▶GPCRs), which also includes numerous potentially important drug targets. The human CTR gene is located on chromosome 7 at 7q21.3. The CTR gene exceeds 70 Kb in length, comprises of at least 14 exons, separated by introns ranging in size from 70 nucleotides to >20,000 nucleotides. Multiple polymorphic sites in CTR gene have been identified, and several of them lead to lower bone mineral density in postmenopausal women. Receptor Isoforms Human CTR (hCTR) is known to exist in mutiple isoforms that arise from alternative splicing of the same primary transcript. The two most common hCTR variants arise by alternative splicing of intracellular domain 1. The most common variant (Type 1 hCTR) leads to the addition of a 16 amino acid insert in the first intracellular loop. Alternative splicing of this small exon leads to the expression of type 2 hCTR, which differs from type 1 hCTR (abundant in the brain and the kidneys) by the absence of a 16-amino acid insert in the first intracellular loop. Type 2 CTR is predominantly expressed in malignant prostate and pancreatic cells. It has been shown that the lack of 16-amino acid insert in

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first intracellular loop enables type 2 hCTR to coactivate both adenylyl cyclase and phospholipase C. In addition, another receptor referred as calcitonin receptor-like receptor (▶CRLR) has been reported.

CT coactivates protein kinases A and C, and pathways activated by these enzymes play an important role in CT-stimulated growth, invasiveness and tumorigenicity of prostate cancer cells.

Modulation of CTR Specificity CTR displays high affinity for CT, but low affinities for other CT family peptides. However, the ligand specificity of CTR is significantly altered when it binds to a ▶RAMP protein. Several ▶RAMPs have been identified but three (RAMP1, RAMP2, RAMP3) are investigated. hCTR displays low affinity for AMY, but association with RAMPs enables hCTR to bind AMY with high affinity. Similarly, ligand specificity of CRLR depends on the complexed RAMP. For example, CRLR-RAMP1 serves as CGRP receptor whereas CRLR-RAMP2 acts as ADM receptor. CRLR-RAMP3 displays high affinity for ADM as well as CT. This phenomenon opens up a possibility that ligand specificity of CTRs can be regulated by modulation of RAMPs expression.

G Protein-Independent Signaling Recent evidence suggests that GPCRs also activate G protein-independent signaling by interacting with proteins referred as GPCR interacting proteins (GIPs). GPCRs activate this signaling by binding to GIPs through one or more of structural interacting domains such as ▶Src homology 2 (SH2) and SH3, plackstrin homology, ▶PDZ and Eva/WASP homology (EVH) domains. Examination of CTR sequence reveals that the last four amino acids at the extreme C-terminus of the ▶C-tail form E-S-S-A tetramer (amino acids 447–50), which conforms to the canonical type I ▶PDZ ligand. A single serine-to-alanine substitution in the PDZ ligand of prostate CTR almost abolished CT-elicited increase in invasiveness and tumorigenicity of PC-3 prostate cancer cell line, raising a strong possibility that metastasizing ability of CTR is dependent upon its ability to interact with intracellular proteins containing ▶PDZ domain(s). CTR seems to induce ▶metastasis by disassembly of tight junctions on prostate cancer cells, which leads to the loss of cell polarity, activation of proteases such as urokinase type plasminogen activator, matrix metalloproteinases 2 and 9. These results raise a possibility that the prevention of interaction between CTR and its intracellular partner through the PDZ ligand can be an effective strategy to prevent CT-mediated metastasis. With current advances in medicinal chemistry and peptide mimetics, it should be possible to design a small peptide of 4–6 amino acids or a small molecule to prevent this interaction. CT also activates ▶phosphoinositol-3-kinase (PI3K)– Akt–Survivin pathway and induces chemoresistance and ▶apoptosis in multiple prostate cancer cell lines through as yet uncharacterized mechanism. CT activated protein kinase A plays a key role in multiple actions of CT on prostate cancer cell lines, suggesting that both, G proteindependent and G protein-independent actions of CTR may act in concert to increase oncogenicity of prostate cancer cell lines.

CTR Signaling G protein-Mediated Signaling The intracellular mechanisms by which CTR produces biological effects are still being elucidated. However, the signaling pathways appear to vary with cell type as well as animal species. As with most other GPCRs, the CTRs show coupling with multiple G proteins, which also depends on the isoform of CTR. For example, type I CTR preferentially couples to ▶Gαs, leading to the activation of adenylyl cyclase and elevation in the intracellular levels of cAMP. The inhibitory action of CT on osteoclasts is accompanied by increase in cAMP levels. Forskolin, a direct activator of adenylyl cyclase, as well as dibutyryl cAMP, which elevates intracellular cAMP levels independent of adenylyl cyclase, mimic CT actions on bone resorption. Similarly, CTR is known to activate adenylyl cyclase in kidney as well as in cancers of lung, breast, and bone. Unlike type 1 CTR, type 2 CTR simultaneously couples with Gαs and ▶Gαq, leading to the coactivation of adenylyl cyclase and phospholipase C. This results in the elevation of intracellular levels of cAMP, as well as inositol triphosphates, and thence increased cytosolic calcium levels. This, together with coliberated diacyl glycerols, activates protein kinase C. In brain tissue, CT couples to G proteins other than Gαs as indicated by limited activation of adenylyl cyclase in neural tissues. In hepatocytes, CT increases cytosolic calcium levels without activating adenylyl cyclase. In LLC-PK1 kidney cells, CT increases either intracellular cAMP levels or cytosolic calcium levels in a cell cycledependent manner; and in pituitary lactotrophs, CT inhibits TRH-induced increases in cytosolic levels and activation of protein kinase C. In ▶prostate cancer cells,

Significance of CT–CTR axis in Cancer: Clinical Aspects CT is “Oncogene” for Prostate Cancer but “Tumor Suppressor” for Breast Cancer Although growing body of evidence suggests elevated expression of CT and CTR in multiple cancers, extensive studies on CT actions have been conducted only in ▶prostate cancer and ▶breast cancer (▶tumor suppressor) cell lines. Interestingly, CT displays sexual dimorphism in these two cancers, raising a possibility

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of the modulatory role of sex hormones on CT actions in these two organs. For example, CT is a potent ▶oncogene for prostate cancer as indicated by the progressive increase in CT and CTR expression in primary prostate cancers with tumor progression, and potent stimulatory actions of CT on tumorigenicity of prostate cancer cell lines. In contrast, CT and CTR is constantly expressed in normal mammary ductal epithelium, the loss of CTR expression is associated with the progression of breast cancer to ▶metastatic phenotype, and CT inhibits growth of some breast cancer cell lines. Although opposing actions of CT on prostate and breast cancer cell lines remain to be thoroughly investigated, initial studies in the author’s laboratory suggest that CT protects junctional complexes in breast cancer cell lines, and estradiol attenuates the actions of CT in estradiol receptorpositive breast cancer cell lines. These results emphasize the importance of CTR actions on junctional complexes in cancer, and its significance in cancer cell growth and metastasis. CT is an Angiogenic Factor ▶Angiogenesis, the process of new vessel formation or neovascularization, has aroused increasing interest over last 25 years. Expansion of the tumor cell mass is dependent on both the degree of tumor vascularization and the rate of angiogenesis. Our recent results have demonstrated the presence of CTR in ▶HMEC-1 cell line, and that CT stimulates in vivo angiogenesis in nude mice, and directly stimulates all major phases of in vitro angiogenesis including endothelial cell migration, invasion, proliferation, and tube morphogenesis. The stimulatory actions of CT on in vitro angiogenesis are comparable to the actions of ▶vascular endothelial growth factor (VEGF). Importantly, silencing of CTR in HMEC-1 cells completely abolishes CT-induced tube morphogenesis. Furthermore, ▶prostate and thyroid cancer cell lines expressing high levels of CT form large, highly vascular tumors. In contrast, the silencing of CT expression in these cell lines markedly reduces tumor growth and vascularity. These results may also explain the findings that malignancies displaying high levels of CT expression (such as MTCs and ▶multiple endocrine neoplasias) also produce highly vascular tumors. Considering that therapeutic use of CT for pain relief is fairly widespread in cancers as well as other diseases, it will be important to consider oncogenic and angiogenic effects while determining CT therapy in these patients. In summary, CT and CTR expression has been wellinvestigated in ▶breast and prostate carcinomas. CT is a potent stimulator of tumor growth, angiogenesis, and metastasis in prostate cancer cell lines. In contrast, CTR expression is lost with breast cancer progression, and CTR attenuates growth of breast cancer cell lines.

Significant expression of CT and CTR has also been reported in MTCs, multiple endocrine neoplasias, and ▶carcinomas of lung, ▶pancreas, ▶gastrointestinal tract, ▶thymus, and ▶bladder. However, the significance of CT–CTR axis in these carcinomas remains to be investigated.

References 1. Findlay DM, Saxton PM (2003) Calcitonin. Henry HL, Normon AW (eds) Encyclopedia of hormones and related cell regulators. Academic Press, New York, NY, pp 220–230 2. Thomas S, Chigurupati SA Muralidharan, Girish S (2006) Calcitonin increases tumorigenicity of prostate cancer cells: evidence for the role of protein kinase A and urokinase-type plasminogen receptor. Mol Endocrinol 20(8):1894–1911 3. Han B, Nakamura M, Zhou G et al. (2006) Calcitonin inhibits invasion of breast cancer cells: involvement of urokinase-type plasminogen actor and uPA receptor. Int J Oncol 28(4):807–814 4. Ball DW (2007) Medullary thyroid cancer: therapeutic targets and molecular markers. Curr Opin Oncol 19 (1):18–23 5. Seck T, Pellegrini M, Florea AM et al. (2005) The delta e13 isoform of the calcitonin receptor forms six transmembrane domain receptor with dominant negative effects on receptor surface expression and signaling. Mol Endocrinol 19(8):2132–2133

Calcitriol DAVID F ELDMAN , A RUNA K RISHNAN , J ACQUELINE M ORENO, S RILATHA S WAMI Division of Endocrinology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

Synonyms CALCITRIOL; Activated vitamin D; 1α,25-Dihydroxyvitamin D3

Definition Calcitriol, the hormonally active form of vitamin D, is the major regulator of calcium homeostasis in the body and is critically important for normal mineralization of bone. Calcitriol is produced by sequential hydroxylations of vitamin D in the liver (25-hydroxylation) and the kidney (1α-hydroxylation) to produce the active hormone. Like other steroid hormones, calcitriol, working through the vitamin D receptor (VDR), functions by a genomic mechanism similar to

Calcitriol

the classical steroid hormones to regulate target gene transcription. In other words, vitamin D is converted into a hormone that acts similarly to other hormones (steroids, thyroid hormone, retinoids, etc.) whose mechanism of action is via nuclear receptors. The traditional actions of calcitriol are to enhance calcium and phosphate absorption from the intestine in order to maintain normal concentrations in the circulation and to provide adequate amounts of these minerals to the bone-forming site to allow mineralization of bone to proceed normally. This action is critical to prevent rickets in children and osteomalacia in adults. However, in the past two decades, it has become increasing clear that calcitriol has many additional functions that implicate the hormone in a wide array of actions relating to bone formation as well as to other areas unrelated to bone or mineral metabolism including antiproliferative, prodifferentiating and immunosuppressive activities. Pharmaceutical companies and academic centers have actively studied analogs of calcitriol in an attempt to design a drug with increased potency to treat osteoporosis, cancer or autoimmune diseases while being less likely to cause hypercalemia and renal stones, the predictable sideeffects of high doses of calcitriol, Several recent reviews of the mechanism of action and function of calcitriol have been published as well as a comprehensive book addressing all areas of vitamin D.

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northern latitudes or other life-styles that limit vitamin D production. Calcitriol and Cancer A number of epidemiologic studies have found a protective relationship between vitamin D status and decreased risk of cancer. Higher rates of cancer mortality have been observed in regions with less sunlight UV-B radiation, among African-Americans and among overweight people, each associated with lower levels of circulating 25(OH)D, the precursor of calcitriol. These data suggest that there is a beneficial effect of vitamin D on cancer development and mortality. The preponderance of observational studies of vitamin D status in relation to the risk of colon, breast, prostate and ovarian cancers has found that vitamin D sufficiency lowers cancer risk. Several studies have demonstrated an inverse relationship between sunlight exposure and the incidence of colon and prostate cancers. Studies correlating the measured plasma levels of vitamin D metabolites with cancer incidence have shown an inverse relationship between plasma 25(OH)D levels and colorectal cancer whereas in the case of prostate cancer, the results have been variable. Several studies have also examined the association between polymorphisms in the VDR gene and the risk for colon and prostate cancers and the results have also been variable but suggestive that some forms of VDR alter the risk of developing cancer.

Characteristics Vitamin D exists in two forms, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). When written without a subscript the designation vitamin D denotes either D2 or D3. Sunlight, in the form of UV-B rays, cleaves the B ring between carbon-9 and 10 to open the ring and create a secosteroid structure. By this process the precursor (provitamin) molecules, 7-dehydrocholesterol in animals and ergosterol in plants, are converted to the secosteroids, vitamin D3 and vitamin D2, respectively. The two secosteroids differ only in the presence of a methyl group at carbon 28 and a double bond between carbon 22 and 23 on the side chain of vitamin D2. Vitamin D2 and vitamin D3 are handled identically in the body and converted, via two hydroxylation steps, first in the liver and then in the kidney to the active hormones, 1,25(OH)2D2 or 1,25 (OH)2D3 (calcitriol). Calcitriol then acts in multiple target tissues throughout the body by binding to its nuclear receptor, the vitamin D receptor (VDR) to regulate gene expression. Since few dietary sources contain high levels of vitamin D, sunlight exposure or ingestion of supplements or vitamin D-supplemented food is essential to maintain adequate vitamin D levels. In recent years it is being increasingly recognized that vitamin D deficiency in many people is related to inadequate sunlight exposure, dark skin, living in

Mechanisms VDR, the receptor through which calcitriol exerts its actions, is expressed in many normal and malignant cell types indicating a wide array of previously unrecognized potential targets for calcitriol action. In many of these normal and malignant cells, calcitriol and its analogs exert pleiotropic actions to inhibit cell proliferation and promote differentiation. A number of important mechanisms have been implicated in calcitriol-mediated growth inhibition. A primary mechanism appears to be the induction of cell cycle arrest in the G1/G0 phase, due to an increase in the expression of cyclin-dependent kinase inhibitors such as p21Waf/Cip1 and p27Kip, inhibition of cyclin-dependent kinase activity and regulation of the phosphorylation status of the retinoblastoma protein (pRb). As the loss of the expression of cell cycle regulators has been associated with a more aggressive cancer phenotype and decreased prognosis and poorer survival, these observations suggest that calcitriol may be a suitable therapy to inhibit cancer progression. In addition, calcitriol induces apoptosis in some cancer cells and down-regulates anti-apoptotic genes like bcl-2. Other mechanisms include the stimulation of differentiation, modulation of growth factor actions and regulation of the expression and function of oncogenes and tumor

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suppressor genes. The inhibition of invasion and metastasis of tumor cells as well as the suppression of angiogenesis have also been shown to contribute to the anti-tumor effects of calcitriol. Recent studies in prostate cancer have revealed anti-inflammatory effects of calcitriol through the inhibition of prostaglandin synthesis and actions as well as the inactivation of stressinduced kinase signaling and down-stream production of inflammatory cytokines. Since inflammation and prostaglandins are associated with carcinogenesis and cancer progression, these anti-inflammatory actions suggest yet another role for vitamin D in cancer chemoprevention and treatment. Role of Vitamin D or Calcitriol in Cancer Prevention or Therapy Because of its actions to inhibit cell proliferation and promote differentiation, calcitriol has been considered a good candidate for possible “chemoprevention” or “differentiation” therapy in a number of malignant cell types that possess VDR. Colon Cancer VDR are present in the colon, in colon cancer cell lines as well as in surgically removed colon cancers. The possibility that calcium and/or vitamin D may be active in decreasing colon cancer has been examined by several groups and an adequate intake of calcium (in the range of 1,800 mg/day) and vitamin D (800–1,000 IU/ day) has been found in some studies to have a protective effect against the development of colon cancer. Studies in a number of colon cancer models have demonstrated the tumor inhibitory and pro-differentiation effects of calcitriol or its analogs both in vitro and in vivo. A recent study in the APC(min) mouse model has demonstrated that both vitamin D and calcium individually exert inhibitory effects on the development of precancerous polyps and exhibit a synergistic effect when used together. VDR expression correlates with colon cancer prognosis: high VDR levels are associated with favorable prognosis and VDR expression is down-regulated in high grade tumors. Breast Cancer VDRs are present in normal breast and breast cancer cell lines and in many human cancer specimens. Adequate calcium and vitamin D intake has been shown to enhance survival rates among breast cancer patients in some studies. Calcitriol suppresses the growth of human breast cancer cell lines in culture and also in vivo in xenografts of human breast cancer cells in nude mice and carcinogen-induced breast cancer in rats. A number of investigators have shown that calcitriol or its analogs exhibit antiproliferative effects in cultured breast cancer cells through a number of different mechanisms. Calcitriol has also been

shown to decrease estrogen receptor-alpha levels in breast cancer cells and inhibit estrogen stimulation of breast cancer cell growth. In addition to its antiproliferative effects, calcitriol stimulates apoptosis in some breast cancer cells and may enhance the responsiveness of breast cancer cells to conventional cytotoxic agents. Studies in VDR null mice (mice in which the VDR was genetically deleted) reveal that calcitriol participates in the negative growth control of normal mammary gland. Disruption of VDR signaling results in abnormal morphology of the mammary ducts, an increase in preneoplastic lesions and accelerated mammary tumor development suggesting that vitamin D compounds may play a beneficial role in the chemoprevention of breast cancer. Prostate Cancer In a prediagnostic study with stored sera, calcitriol blood levels were found to be an important predictor for palpable and anaplastic tumors in men over 57 years of age but not for incidentally discovered or well differentiated tumors. VDR are present in prostate cancer cell lines and in normal prostate. Calcitriol inhibits the growth of all these cell types in culture. Calcitriol and vitamin D analogs exert antiproliferative effects in multiple prostate cancer models and several mechanisms mediate these effects. The induction of apoptosis may also play some role in the growth inhibitory activity of calcitriol in some prostate cancer cells. One of the recently discovered molecular mechanisms mediating calcitriol effects in prostate cells is the inhibition of the synthesis and actions of growth-stimulatory prostaglandins, through multiple calcitriol actions including a decrease in the expression of the pro-inflammatory molecule, cyclooxygenase2 (COX-2). Moreover calcitriol has been shown to cause synergistic inhibition of prostate cell growth when combined with non-steroidal anti-inflammatory drugs (NSAIDs), suggesting that a combination of vitamin D or its analogs with NSAIDs may be useful in prostate cancer therapy. Calcitriol also induces the expression of MAP kinase phosphatase-5 in primary prostate cells leading to the inactivation of the stress kinase p38 and inhibition of interleukin-6 production. These new mechanisms of action support an antiinflammatory role for calcitriol in prostate cancer and suggest that it may have beneficial prostate cancer chemopreventive effects. The efficacy of calcitriol as a chemopreventive agent has recently been evaluated using mutant mice, that recapitulate stages of prostate carcinogenesis from the pre-cancerous lesion known as prostate intraepithelial neoplasia (PIN), to high-grade PIN, to adenocarcinoma. The findings reveal that calcitriol is beneficial at the early-stage preventing the development of high-grade PIN, providing support for its use in the chemoprevention of prostate cancer.

Calcium-Binding Proteins

Several vitamin D analogs exhibit greater antiproliferative potency than calcitriol, raising the possibility of the therapeutic potential of these drugs in the treatment of prostate cancer. Clinical trials have begun to address the utility of calcitriol or its analogs in treating prostate cancer patients. Recent studies have demonstrated that intermittent administration of very high doses of calcitriol are well tolerated by prostate cancer patients without significant toxicity or renal stones. In combination with the chemotherapy drug docetaxel, calcitriol given at extremely high doses once weekly, produced favorable effects on the time to disease progression and survival. An unanticipated benefit of the combination therapy was decreased side-effects of docetaxel. A phase III placebo-controlled randomized trial is currently under way testing the safety and efficacy of this combination in prostate cancer patients.

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4. Krishnan AV, Peehl DM, Feldman D (2005) Vitamin D and prostate cancer. In: Feldman D, Pike JW, Glorieux FH (eds) Vitamin D, 2nd ed., Vol. 2. Elsevier Academic Press, San Diego, pp 1679–1707 5. Feldman D, Pike JW, Glorieux FH (eds.) (2005) Vitamin D, 2nd ed. Elsevier Academic Press, San Diego, pp 3–1843

Calcium-Binding Proteins M EENAKSHI DWIVEDI , J OOHONG A HNN Department of Life Science, Hanyang University, Seoul, Korea

Definition Other Malignancies Calcitriol or other related vitamin D compounds have been shown to exhibit anti-cancer effects in multiple other malignancies as well. The growth inhibitory action of calcitriol on tumor cells was first demonstrated in human melanoma cells. Since then a large body of evidence has accumulated indicating the antiproliferative and pro-differentiation effects of calcitriol in melanocytes as well as malignant melanoma cells and melanoma xenografts. Evidence for a potential beneficial role of vitamin D compounds in hematologic, ovarian, pancreatic, and lung cancers has also been developed. Clinical trials employing calcitriol or vitamin D analogs are currently under way to evaluate the benefits of vitamin D therapy in chemoprevention or therapy of a number of cancer types. Note Added in Proof After this article was written the phase III trial in advanced prostate cancer patients to compare docetaxel plus calcitriol with docetaxel plus placebo was halted because of safety concerns in the clacitriol arm of the study. The details are not yet available to explain the nature of the problem.

References 1. Haussler MR, Whitfield GK, Haussler CA et al. (1998) The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13:325–349 2. Malloy PJ, Pike JW, Feldman D (1999) The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocr Rev 20(2):156–188 3. Nagpal S, Na S, Rathnachalam R (2005) Noncalcemic actions of vitamin D receptor ligands. Endocr Rev 26 (5):662–687

Calcium-binding proteins are ▶proteins that participate in calcium signaling pathways by binding to Ca2+. The most ubiquitous Ca2+-binding protein, found in all eukaryotic organisms including yeasts, is calmodulin. With their role in signal transduction, Ca2+-binding proteins contribute to all aspects of the cell’s functioning, from homeostasis to ▶cancer.

Characteristics Normal cell cycle division is a highly coordinated progression of molecular events that is subject to control mechanisms from both outside and inside the cell. Commitment to cell cycle initiation is made from outside and occurs as a response to extracellular signals such as growth factors. Inside the cell, control mechanisms exist to determine the timing of intracellular events such as nuclear and cytoplasmic cleavage. Under normal conditions, growth-regulating mechanisms endeavor to maintain homeostasis. Homeostasis within a cell is regulated by the balance between proliferation, growth arrest, and ▶apoptosis. Intracellular Ca2+ is an important modulator of a variety of biochemical processes associated with cell cycle progression. With few exceptions, the controls exerted by intracellular Ca2+ are transduced through sitespecific interactions with specialized Ca2+-binding proteins. There exist at least three main families of Ca2+-binding proteins. The first of these is represented by proteins that possess one or more EF-hand helix– loop–helix structural motifs predicting Ca2+-binding domains as typically found within calmodulin. The second class of Ca2+-binding proteins is known generically as annexins. A possible third family of Ca2+-binding proteins is the “calreticulin-like” group of proteins that include ▶calreticulin, Grp78, endoplasmin, and protein disulfide isomerase. Ca2+ has also been implicated in cell growth under pathological states. An

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altered cellular response to extracellular calcium ion concentration is one of the earliest changes induced in mouse epidermal cells by chemical carcinogens. However, whereas some human breast cancer cell lines and leukemia cell lines exhibit Ca2+-induced cell proliferation, other carcinoma cell lines exhibit retarded growth in the presence of Ca2+ or no sensitivity to Ca2+ at all. In a human breast cancer cell line, which is sensitive to Ca 2+, the administration of calcium channel antagonists lowered intracellular (Ca2+) and inhibited cell proliferation. A sustained physiological elevation of intracellular calcium ion concentration (Ca2+i) may be responsible for a loss of proliferative potential in neoplasmic keratinocytes. It appears from preliminary evidence that Ca2+ is not only important to cell cycling and growth in normal cells, but the abnormal regulation of Ca2+ may also contribute to changes in these processes in disease conditions like cancer.

EF-Hand Motif Calcium-Binding Proteins The ▶EF-hand protein structural motif was first discovered in the crystal structure of parvalbumin. It consists of two alpha helices positioned roughly perpendicular to one another and linked by a short loop region (usually about 12 amino acids) that often binds calcium ions. A consensus amino acid sequence for this motif has aided the identification of new members of this family that now has over 200 members. A few of these proteins are present in all cells, whereas the vast majorities are expressed in a tissue-specific fashion. Some members, like S100 family, calcineurin, calmodulin, etc. have proved to be useful therapeutic markers for a variety of cancers. Calcium-Binding Proteins. Table 1 S100 protein S100A2

S100A3

Previous name CAN19, S100L

S100E

S100 family: The S100 (“Soluble in 100% saturated solution with ammonium sulfate”) family, the largest family within the EF-hand protein, comprises at least 26 members, 19 of which (S100A1–16, profillagrin, trychohyalin, and repetin) are located in the epidermal differentiation complex situated at 1q21, while S100B, S100G, S100P, S100Z, and S100A7L2–S100A7L4 are present at other genomic locations (21q22, xp22, 4p16, 5q14, and 1q22, respectively). Their gene structure is highly conserved, in general comprising three exons and two introns, of which the first exon is noncoding. The S100 family is a remarkable group of proteins that acquired highly specialized functions during their evolution, even though they are small proteins (9–13 kDa acidic proteins) with a single functional domain. S100 proteins, which exhibit dramatic changes in the expression, are involved in tumor progression and include S100A1, S100A4, S100A6, S100A7, and S100B (11–14), whereas S100A2 has been postulated to be a tumor suppressor (Table 1). Such changes might be caused by rearrangements and deletions in chromosomal region, which are frequently observed in tumor cells. Although in most cases, the function of S100 proteins in cancer cells is still unknown, the specific expression patterns of these proteins can be used as a valuable prognostic tool. The other members of EF-hand motif Ca2+-binding family involved in cancer are calcineurin, recoverin, calretinin, oncomodulin, etc. (Table 2). Annexins and Other non-EF-Hand Motif Proteins Annexin (AnxA1) is a Ca2+-binding and acidic phospholipid binding protein with antiinflammatory properties. AnxA1 has been found in leukocytes, tissue

S100 proteins involved in cancer with characteristic features Cancer type Esophageal SCC Thyroid Oral SCC Laryngeal SCC Melanoma Skin tumors (other) NSCLC Lung SCC Gastric Lymphoma Prostate Ovarian Breast Astrocytomas

Characteristic features Function in carcinogenesis is dependent upon the context of tissue and associated tumor type as well as stage of malignancy

Marker of patient prognosis

Level of S100A3 protein expression identified pilocytic astrocytomas

Calcium-Binding Proteins Calcium-Binding Proteins. Table 1 S100 protein S100A4

Previous name CAPL, Calvasculin, MTS1, Metastasin, p9Ka, FSP1

S100 proteins involved in cancer with characteristic features (Continued) Cancer type Thyroid carcinoma NSCLC Colorectal Gastric Prostate Breast Gallbladder Bladder Pancreas Esophageal SCC Melanoma Oral SCC Meningioma

S100A5

S100D

S100A6

CABP, CACY, Calcyclin, MLN 4, Thyroid PRA, Prolactin receptorPancreas associated protein Breast Lung Melanoma Colorectal PSOR1, Psoriasin Breast Esophageal SCC Bladder SCC Melanoma Skin SCC Gastric Calgranulin A, MRP-8, MIF, NIF, Prostate P8, CFAG, CGLA, CP-10, Breast Calprotectin Esophageal SCC HNSCC Gastric Pancreas Bladder TCC Endometrial Ovarian Colorectal Calgranulin B, MRP-14, P14, Prostate Calprotectin Breast HNSCC Esophageal SCC Liver (hepatocellular) Gastric Lung Ovarian

S100A7

S100A8

S100A9

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Characteristic features Stimulate angiogenesis Overexpression is associated with many different cancer types Poor patient prognosis in breast, colorectal, NSCLC and bladder cancers

Expression can be associated with prognostic value in recurrence of meningiomas Upregulation of S100A6 appears to be an early event in progression towards pancreatic cancer

Potentially act as a predictor of clinical outcome Expression is restricted to keratinocytes and breast epithelial cells Overexpression has a role in early breast tumor progression Upregulated in PIN and in prostatic adenocarcinomas

Early involvement of the proteins in prostate cancer

Upregulated in PIN and in prostatic adenocarcinomas

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Calcium-Binding Proteins. Table 1 S100 protein

S100 proteins involved in cancer with characteristic features (Continued)

Previous name

Cancer type

S100A10 CAL1L, CLP11, GP11, p10, ANX2LG

S100A11 Calgizzarin, MLN70, S100C

S100A12 Calgranulin C, MRP6, p6, CAAF1, ENRAGE S100A14 BCMP84, S100A15, S114

NSCLC Gastric Renal cell carcinoma Lymphoma Breast Bladder Prostate Thyroid Lymphoma Gastric Colon

Characteristic features Annexin 2 protein ligand Overexpressed in human renal cell carcinoma

Downregulated at transcriptional level in malignant bladder cells Expression may be involved in tumor suppression and better prognosis

Esophageal SCC

Esophageal SCC Circulating tumor cells S100A16 S100F, DT1P1A7 Circulating tumor cells S100B S100, S100 protein (beta chain), Melanoma NEF S100P S100E, MIG9 NSCLC Pancreas Prostate Breast Colon

Cytoplasmic staining pattern in Papillary carcinomas Downregulated Used for CTC monitoring in peripheral blood

Used for CTC monitoring in peripheral blood Putative cancer biomarker Isolated from placenta Upregulation is early event in pancreatic cancer valuable marker for the prediction of clinically relevant early pancreatic lesions

SCC, squamous cell carcinoma; HNSCC, head and neck SCC; NSCLC, nonsmall cell lung carcinoma; TCC, transitional cell carcinoma; CTCC, circulating tumor cells.

Calcium-Binding Proteins. Table 2 EF-hand motif protein Recoverin Calretinin Sorcin Calcineurin B Oncomodulin/ Parvalbumin Calmodulin

Other EF-hand motif family members

Cancer type

Characteristic features

Cancer-associated retinopathy Colon adenocarcinoma and mesothelioma of epithelial type Ovarian carcinoma Squamous cell carcinoma of cervix, pancreatic cancer Carcinoma cell lines characterized by translocative activity Osteoclast apoptosis

Autoimmune response Belongs to the calbindin subgroup, autoantigen in a paraneoplastic disease Overexpression leads to paclitaxel resistance Calcineurin B subunit appears to be a significant biological response modifier due to its anticancer effects Related to motile behavior of carcinoma cells, can be a possible candidate for tumor marker Calcium ion receptor in neoplastic cells

macrophages, T-lymphocytes, and epithelial cells of the respiratory and urinary systems. Cellular functions of AnxA1 include regulation of membrane trafficking, cellular adhesion, cell signaling, and membrane fusion

in exocytosis and endocytosis. The AnxA1 protein is involved in maintaining normal breast biology. The AnxA1 gene expression may provide data about the future therapeutic plan of breast carcinomas. The

Calnexin

decreased expression of ANXA1 gene in normal histological sections of breast may warn the clinician that a malignant version of the cancer is about to form from the benign gland. This observation carries an important prognostic clinical value on microscopic reading of the surgical specimen, especially if these normal glands are adjacent to surgical margins. Similar result was reported as a prognostic factor with downregulation of AnxA1 and other Anxs in the development of the lethal prostatic carcinoma phenotype. The other major protein that belongs to this category is clusterin (CLU). CLU is a disulfide-linked heterodimeric protein associated with the clearance of cellular debris and apoptosis. In prostate, breast, and colorectal cancers, the CLU was found to have antior proapoptotic activity regulated by calcium homeostasis. Reports so far suggest “two faces” of CLU activity: the calcium-dependent cytoplasmic localization of CLU positively correlates with cell survival, whereas nuclear translocation of this protein promotes cell death in calcium-deprived cells. The cytoplasmic retention and high level of the 50-kDa CLU protect tumor cells from apoptotic stimuli induced by chemotherapeutic drugs or natural ligands, such as FasL, whereas its nuclear localization (nCLU) enhances cell apoptosis. The 50-kDa CLU isoform is mainly overexpressed in cancer cells and retained in the cytoplasm, promoting cancer progression and aggressiveness. Cytoplasmic CLU could easily translocate into the nucleus in the presence of various inducers, such as IR, chemotherapy, hormones, or cytokines, or depletion of cellular calcium. These findings support CLU as a valid therapeutic target in strategies employing novel multimodality therapy for advanced prostate cancer.

Calreticulin-like Proteins Calreticulin is a 46-kDa Ca2+-binding chaperone protein found across a diverse range of species. The human gene for calreticulin is located on chromosome 19 at locus p13.3–p13.2 and the homologous gene in the mouse maps to chromosome 8. Calreticulin at the cell surface may play a role in cell adhesion, cell–cell communication, and apoptosis. Calreticulin has also been implicated in the pathology of some cancers. The protein also plays an important role in autoimmunity and cancer. For example, it appears that calreticulin might be an excellent molecular marker for prostate cancer. The expression of calreticulin is downregulated in metastatic melanoma and ▶squamous cell carcinoma, whereas significantly upregulated in colon cancer. Further, the N-domain of the protein has been reported to have inhibitory effects on tumors and to inhibit ▶angiogenesis on endothelial cells. This observation is of great interest because the development of angiogenesis inhibitors is

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currently a highly promising approach in anticancer therapy.

References 1. Donato R (2003) Intracellular and extracellular roles of S100 proteins. Microsc Res Tech 60:540–551 2. Kretsinger RH, Tolbert D, Nakayama S et al. (1991) The EF-hand, homologs and analogs. In: Heizmann CW (ed) Novel calcium-binding proteins: fundamentals and clinical implications. Springer-Verlag, New York, pp 17–37 3. Pfyffer GE, Haemmerlit G, Heizmann CW (1984) Calcium-binding proteins in human carcinoma cell lines. Proc Natl Acad Sci USA 81:6632–6636 4. Pfyffer GE, Humbel B, Strauli P et al. (1987) Calciumbinding proteins in carcinoma, neuroblastoma and glioma cell lines. Virchows Archiv 412:135–144

Calcium-binding Reticuloplasmin of Molecular Weight 55 kDa ▶Calreticulin

CALI ▶Chromophore-Assisted Laser Inactivation

Calnexin Definition CNX is an 88-kDa type I membrane protein in the Endoplasmic Reticulum. CNX and ▶calreticulin (CRT) are ▶paralogs and both function as molecular chaperones for glycoproteins by binding through monoglucosylated N-linked oligosaccharides (Glc1Man5– 9GlcNAc2). ▶Calreticulin ▶Endoplasmic Reticulum Stress

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Calpain

Calpain N EIL O. C ARRAGHER Advanced Science and Technology Laboratory, AstraZeneca R&D Charnwood, Loughborough, England, UK

Definition The calpains represent a unique class of intracellular protein degrading enzymes. This class of ▶proteases was named “calpains” to reflect their dependency upon calcium ions for proteolytic activity, and homology to the ▶papain family of cysteine proteases. In mammalian species, the calpain protein family is comprised of 15 members, of which nine are ubiquitously expressed in all tissues and the remainder are expressed in a tissuespecific manner. The ubiquitously expressed calpain 1 and calpain 2 are the most well characterized isoforms. Calpain 1 and calpain 2 function as heterodimeric enzymes composed of a large catalytic subunit (calpain 1 and calpain 2) bound to a small regulatory subunit (calpain 4). Calpain activity in vivo is tightly regulated by the ubiquitously expressed endogenous inhibitor ▶calpastatin. Calpains result in the proteolysis of a broad spectrum of cellular proteins. No unique consensus amino-acid sequence has been identified as a calpain-binding or -cleavage site, rather, it appears that calpains target substrates for cleavage by recognition of unidentified tertiary structure motifs. Another distinguishing feature of calpain proteases is their ability to confer limited cleavage of protein substrates into stable fragments, rather than complete proteolytic digestion. Thus, the calpain-calpastatin proteolytic system represents a major pathway of post-translational modification of proteins, that influences various aspects of cellular physiology. The recent application of pharmacological and molecular intervention strategies against calpain activity demonstrates a broad role for this class of proteases in the control of proliferation, ▶migration and ▶apoptosis in most cell types.

Characteristics Cell proliferation, migration and apoptosis are key processes that have to be tightly regulated in order to maintain optimal tissue homeostasis, required for development and viability of multicellular organisms. Deregulation of any of these cellular processes will ultimately result in pathological outcomes, such as cancer. A number of studies have identified a correlative link between modulation of calpain gene expression and/or activity with cancer development and progression in vivo. For example, in human renal cell carcinomas, significantly higher levels of calpain 1

expression are found in tumors that metastasized to peripheral lymph nodes relative to tumors that had not metastasized. In addition, elevated calpain activity was detected in breast cancer tissues relative to normal breast tissues and was determined to be greater in estrogen receptor (▶ER)-positive tumors than ER negative tumors. Calpain-mediated proteolysis of the tumor suppressor protein neurofibromatosis type 2 (NF2 or ▶Merlin) is associated with the development of schwannomas and menigiomas. Experimental studies performed in vitro demonstrate that total cellular calpain activity is elevated upon transformation induced by the v-src, v-jun, v-myc, k-ras and v-fos oncogenes. Furthermore, calpain activity is necessary for full cellular transformation induced by such oncogenes. A number of intervention studies utilizing small molecule inhibitors or oligonucleotides that impair calpain activity have demonstrated a role for calpain during tumor cell progression in vitro and in vivo. Cell proliferation, migration and apoptosis are controlled by a plethora of regulatory proteins that participate in complex biochemical signaling cascades, of which calpain is a pivotal regulator. Thus, targeting calpain activity may represent an effective strategy for cancer prevention and/or treatment. Calpain and Cell Proliferation Studies using pharmacological inhibitors of calpain activity, overexpression of calpastatin and cells expressing depleted levels of calpain activity have all implicated calpain in the promotion of cell proliferation. Sequential progression through G1, S, G2 and M phases of the cell-cycle is required for mitosis and cell proliferation. Several studies indicate that calpain can cleave a number of cell-cycle control proteins such as ▶cyclin D, cyclin E and p27kip1 all of which regulate progression through G1 and S phase. Calpain has also been demonstrated to cleave upstream regulators of cell-cycle control proteins such as p53 and p107. Consequently, elevated calpain levels and activity in tumors may contribute to cancer cell proliferation through cleavage of cell-cycle control proteins and deregulation of normal cell cycle control. More detailed mechanistic studies also demonstrate that calpain 2 is an important downstream component of many growth factor receptor and non-receptor tyrosine kinase signaling pathways that include signaling kinases such as, epidermal growth factor receptor (▶EGFr), platelet derived growth factor receptor (▶PDGFr), Src and ▶focal adhesion kinase (▶FAK). Such signaling molecules play an important role in transmitting extracellular signals to intracellular mediators that control cell proliferation, and are often constitutively activated in cancer cells. Activation of receptor and non-receptor kinases subsequently leads to activation of the Ras/▶MAPK pathway, which results in

Calpain

▶ERK-mediated phosphorylation of calpain 2 on a serine residue (Ser 50). Phosphorylation of calpain 2 on Ser 50 initiates a conformational switch culminating in enhanced proteolytic activity. This evidence, together with pharmacological and molecular intervention studies targeting calpain activity, suggests that activation of calpain, in part, mediates growth factor receptor and non-receptor induced cell proliferation and migration of cancer cells. Calpain and Cell Migration The interaction between cell surface adhesion receptors known as ▶integrins and their ▶extracellular matrix substrates controls the migration of all cells. Integrinlinked ▶focal adhesions are large complexes of structural and signaling proteins that provide both a structural and biochemical link between the extracellular environment and intracellular proteins. Dynamic spatial and temporal regulation of focal adhesion assembly and disassembly is required for optimal cell motility. Several studies indicate that calpains localize to integrinassociated adhesions. Furthermore, many of the protein components of focal adhesions are known substrates of calpain. Calpain-mediated cleavage of the focal adhesion components, FAK, paxillin, talin and possibly others promotes the disassembly of these complexes, contributing to reduced cell adhesion and increased migration. In fact calpain-mediated cleavage of talin has been reported to represent the rate-limiting step in adhesion turnover. In addition to mediating focal adhesion turnover, emerging evidence suggests a role for calpain in regulating components of the actin cytoskeleton involved in cell spreading and membrane protrusion, mechanisms that are also essential for persistent and directed cell migration. It is likely that calpain cleavage of actin-binding and -regulatory proteins such as, ezrin, rhoA and cortactin influences the dynamic formation and retraction of membrane structures known as ▶filipodia and ▶lamellipodia thereby influencing cancer cell migration and ▶invasion. Pharmacological and molecular inhibition of calpain activity has been shown to impair cancer cell migration across experimental two-dimensional substrates and invasion into threedimensional extracellular matrix substrates in vitro. Furthermore, a recent intervention study demonstrates that antisense-mediated suppression of calpain 2 gene expression reduced the invasion of prostate cancer cells both in vitro and in a mouse model in vivo. Thus, evidence strongly indicates that calpain activity contributes to the invasion and ▶metastasis of cancer cells. Calpain and Apoptosis Apoptosis is defined as the process of programmed cell death. Apoptosis often follows activation of the caspase family of cysteine proteases, which degrade numerous proteins that are essential for cell viability. Regulated

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apoptosis is critical for the development of multicellular organisms and also restricts the growth and spread of malignant cancer cells. Conflicting roles for calpain activity in the promotion and suppression of cell apoptosis have been proposed. Calpain activity has previously been shown to play a pro-apoptotic role through the activation of caspase 3 and caspase 12 and cleavage of Bax and ▶Bid proteins to their pro-apoptotic forms. Enhanced calpain activity has also been implicated as the major proteolytic pathway resulting in breakdown of essential proteins during caspase-independent mechanisms of apoptosis. Conversely, calpain-mediated cleavage of caspase 7 and caspase 9 has been found to suppress their activity and subsequent apoptosis. In addition, calpain-mediated cleavage of IκBα can lead to activation of the NFκB transcription factor resulting in subsequent expression of anti-apoptotic survival proteins. Many chemotherapeutic agents such as cisplatin induce their tumoricidal effect via inducing apoptosis of cancer cells. Tumor cell resistance to cisplatin-induced apoptosis is a common feature frequently encountered during chemotherapy of cancer patients. Inhibition of calpain activity has been shown to sensitize resistant tumor cells to cisplatin-induced death, whereas other studies suggest that calpain potentiates cisplatin induced cell death. Thus, the role of calpain during cell apoptosis is context dependent and determined by cell type, the apoptotic stimuli and status of intrinsic regulators of cell apoptosis. In contrast to the aforementioned studies suggesting a pro-tumorigenic role for calpain activity, an antitumorigenic role is also supported by studies indicating that calpain degrades a number of oncogene-generated protein products such as PDGFr, EGFr, c-Jun, c-Fos, c-Src, and c-Mos. Also, calpain-mediated cleavage of protein kinase C (▶PKC), a downstream effecter for tumor promoting phorbal esters, inhibits malignant transformation. Furthermore, specifically calpain 9 (nCL-4) activity contributes to the suppression of cell transformation in vitro and gastric tumors in vivo. Although the calpain 9 substrates that mediate this antitumor effect remain to be determined. A substantial body of evidence has accumulated demonstrating that activity of the calpain family of proteases plays a broad and important role in the physiology of both normal and cancer cells. Further investigation into the complex and multifaceted role of calpain in cancer may lead to the discovery of novel therapeutic approaches targeting calpain activity that may impact on the development, progression and prevention of cancer.

References 1. Goll DE, Thompson VF, Li H et al. (2003) The calpain system. Physiol Rev 83:731–801

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Calpastatin

2. Carragher NO, Frame MC (2002) Calpain: a role in cell transformation and migration. Int J Biochem Cell Biol 34:1539–1543 3. Franco SJ, Huttenlocher A (2005) Regulating cell migration: calpains make the cut. J Cell Sci 118(17):3829–3838

Calpastatin Definition Is an endogenous protease inhibitor that acts specifically on calpain. It consists of four repetitive sequences of 120–140 amino acid residues (domains I, II, III and IV), and an N-terminal non-homologous sequence (L). ▶Calpains

Calreticulin Definition Is the molecular chaperone that binds initially to ▶MHC class I, MHC class II, and other proteins that contain immunoglobulin-like domains, such as the T-cell and B-cell antigen receptors. ▶Sjögren Syndrome ▶Molecular Chaperones

Calreticulin YOSHITO I HARA Department of Biochemistry, School of Medicine, Wakayama Medical University, Wakayama, Japan

Synonyms CRP55; calcium-binding reticuloplasmin of molecular weight 55 kDa; Calsequestrin-like protein; CaBP3; ERp60; HACBP; high affinity Ca2+ -binding protein; Reticulin; CRT

Definition

Calreticulin (CRT) is a Ca2+ -binding multifunctional ▶molecular chaperone in the endoplasmic reticulum

(ER). CRT is a 46-kDa soluble protein with a cleavable N-terminal amino acid signal sequence and the C-terminal sequence Lys-Asp-Glu-Leu (KDEL), a retrieval signal in the ER. ▶Calnexin (CNX), a membrane-binding paralog of CRT, shares the ▶chaperone function in the ER. CRT is expressed in a variety of tissues and organs, but its levels are particularly high in the pancreas, liver, and testis. It is also a highly conserved protein with over 90% amino acid identity in mammals including humans, rabbits, rats and mice. The CRT gene has been mapped to human chromosome 19 at p13.2, and its expression is upregulated by ER stress such as unfolded protein responses and deprivation of Ca2+ in the ER.

Characteristics Structure of CRT Based on structural and functional studies, CRT can be divided into three distinct domains; N-terminal [N], proline-rich [P], and C-terminal [C]. The prolinerich P-domain shows a characteristic structure with an extended and curved arm connected to a globular N-domain. The N-terminal region encompassing the N and P-domains of CRT interacts with misfolded proteins and glycoproteins, binds ATP, Zn2+, and Ca2+ with high affinity and low capacity, and is likely to be involved in the chaperone function of the protein. The C-domain binds Ca2+ with high capacity and plays a role in the storage of Ca2+ in the ER in vivo, though no structural information is available at present (Fig. 1). Functions of CRT in the Cell CRT is involved in a number of biological processes including the regulation of glycoprotein folding, Ca2+ homeostasis and intracellular signaling, cell adhesion, gene expression, and nuclear transport (Fig. 2). CRT, a Lectin-like Molecular Chaperone in the ER A molecular chaperone function of CRT has been reported for several protein substrates. In the biosynthesis of glycoproteins bearing ▶N-linked glycans in the ER, the oligosaccharide Glc3Man9GlcNAc2 (Glc, glucose; Man, mannose; GlcNAc, N-acetylglucosamine) is attached to the Asn residue contained in the consensus sequence Asn-X-Ser/Thr, of newly synthesized polypeptides. CRT or CNX binds the Glc1Man5– 9GlcNAc in glycoproteins after the processing of sugar chains. The N-domain of CRT and CNX is speculated to be the oligosaccharide-binding site (▶lectin site). If the glycoprotein is completely folded in the ER, the terminal glucose is removed by glucosidase-II and the glycoprotein is released from the CNX/CRT chaperone cycle. However, if the glycoprotein is not properly folded, the terminal glucose is once again attached by

Calreticulin

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Calreticulin. Figure 1 Schematic structure of calreticulin.

Calreticulin. Figure 2 Functions of calreticulin in the cell. CRT is involved in a variety of cellular processes including the quality control of glycoprotein synthesis in the ER, Ca2+ homeostasis, intracellular signaling, gene expression, and nuclear transport. In cancer cells, the altered expression of CRT may lead to alterations in cellular characteristics, such as growth, adhesion, motility, immune responses, and susceptibility to apoptosis. Furthermore, extracellular CRT fragments (i.e., vasostatin) elicit antiangiogenic or tumor-suppressing activities.

the action of UDP-Glc: glycoprotein glucosyltransferase, which discriminates between folded and unfolded substrates. Together, CRT and CNX form a specific chaperone cycle for the biosynthesis of glycoproteins in the ER. Because of the preference of CNX/CRT for oligosaccharides as substrates, CNX and CRT are called “lectin-like chaperones.” CRT and CNX function with the help of other chaperones such as ▶ERp57 and ▶BiP/GRp78. The binding site for ERp57 has been identified in the P-domain of CRT or CNX. As a chaperone, CRT plays an important role in the formation of major histo-compatibility complex (▶MHC) class I to aid in antigen presentation.

CRT, a Regulator of Ca2+ Homeostasis in the ER The ER is the main reservoir of intracellular Ca2+ and plays an important role in Ca2+ homeostasis. CRT has two Ca2+ -binding sites and this characteristic contributes to the function of the ER as a Ca2+ reservoir. Ca2+ is released from the ER by receptors for inositol-1,4,5-trisphosphate (IP3) and ryanodine, and taken up into the ER by sarcoplasmic and endoplasmic reticulum Ca2+ -ATPase (SERCA). With respect to the regulation of the Ca2+ level, the involvement of CRT and SERCA2b or the IP3receptor has been reported. Furthermore, the storeoperated release of Ca2+ from the ER was shown to be suppressed by overexpression of CRT protein. These

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Calreticulin

findings indicate that CRT is not only a reservoir of Ca2+ but also a regulator of Ca2+ -homeostasis in the ER.

Other Miscellaneous Functions of CRT In and Out of the ER CRT is involved in cell ▶adhesion by affecting integrinrelated cell signaling. In CRT-deficient embryonic stem cells, integrin-mediated Ca2+ influx was impaired leading to a decrease in cell adhesion to fibronectin and laminin. It is still not clear whether CRT affects integrin directly or indirectly to regulate cell adhesion signaling. Cell surface expression of CRT has also been reported in various cell types, and may be related with cell adhesion and ▶migration. The cell surface CRT may modulate cell adhesion by binding with extracellular matrix proteins, such as ▶fibrinogen, ▶laminin, and ▶thrombospondin. Furthermore, extracellular CRT is implicated in the pathological processes of autoimmune diseases. ▶Autoantibodies against CRT were found in 40% of patients with systemic lupus erythematosus, patients with secondary Sjogrens syndrome, rheumatoid arthritis, celiac disease, complete congenital heart block, and halothane hepatitis. CRT is known to bind to complement, C1q, and compete with antibodies for binding to C1q and inhibition of C1q-dependent hemolysis. In autoimmune diseases, impairment of the classical pathway of compliment causes a failure to clear immune complex, resulting in progression of the disease. Therefore, extracellular CRT may contribute to the progression of autoimmune diseases by preventing the clearance of immune complex. Furthermore, it has been reported that cellsurface CRT is involved in the mechanism for clearance of viable or apoptotic cells through the trans-activation of LDL-receptor-related protein (LRP) on phagocytes. However, it is still controversial whether CRT is exported from necrotic cells or apoptotic cells under pathologic conditions. Cytosolic CRT functions as an export factor for multiple nuclear hormone receptors, such as steroid hormone, non-steroid hormone, and orphan receptors. This function is consistent with previous findings that CRT suppresses the transactivation of nuclear hormone receptors including ▶androgen receptor and vitamin D. However, the mechanisms by which CRT molecules are transported into, and retained in, the cytosol/nucleus are not fully defined.

CRT and Development CRT is essential for cardiac and neural development in mice. CRT-deficient embryonic cells showed an impaired nuclear import of nuclear factor of activated T cell (NF-AT3), a transcription factor, indicating that CRT functions in cardiac development as a component

of the Ca2+/▶calcineurin/NF-AT/GATA-4 transcription pathway. Actually, cardiac-specific expression of calcineurin reversed the embryonic lethality of CRTdeficient mouse. CRT transgenic mice suffer a complete heart block and sudden death, and CRT-dependent cardiac block involves an impairment of both the L-type Ca2+ channel and gap junction ▶connexins (Cx40 and Cx43). Phosphorylated Cx43 was also decreased in CRT transgenic heart, suggesting that the functions of protein kinases are altered via the regulation of Ca2+ homeostasis. Collectively, CRT plays a vital role in cardiac differentiation and function, though how has not been fully clarified. CRT and Cancer Expression of CRT in Cancer In terms of the relationship between CRT and cancer, proteomic analysis has revealed a new functional role of CRT in the early diagnosis of cancers. CRT is proposed to be a new tumor marker of bladder cancer. In addition, it was reported that the expression of CRT is up-regulated in a variety of malignant cells or tissues including progressive fibrosarcoma cells, colorectal cancer cells, and pituitary adenomas. Furthermore, autoantibodies to CRT isoforms have utility for the early diagnosis of pancreatic cancer. These reactions are not indicative of malignant properties of CRT, but rather are markers of immunogenicity and anticancer responses. On the other hand, another report demonstrated that CRT is over-expressed in the nuclear matrix in ▶hepatocellular carcinoma, compared with normal liver tissue, suggesting a relationship between overexpressed CRT and malignant transformation. In contrast, it was also reported that CRT expression correlates with the differentiation of ▶neuroblastomas to predict favorable patient survival. Pathophysiological Relevance of CRT in Malignant Disease Susceptibility to ▶apoptosis is important in terms of cancer treatments including the use of antibiotics and irradiation. In embryonic fibroblasts from CRT knockout mice, susceptibility to apoptosis was significantly suppressed, indicating that CRT functions in the regulation of apoptosis. Furthermore, it was found that overexpression of CRT modulates the ▶radiation sensitivity of human glioma U251MG cells by suppressing ▶Akt/protein kinase B signaling for cell survival via alterations of cellular Ca2+ homeostasis. These findings suggest that the expression level of CRT is well correlated with the susceptibility to apoptosis. In contrast, overexpression of CRT provides resistance to oxidant-induced cells death in renal epithelial LLC-PK1 cells. The function of CRT in the regulation of apoptosis may differ in specific cell types, and is still controversial.

cAMP

As for cell adhesion, it was reported that CRT expression modulates cell adhesion by coordinating upregulation of N-cadherin and vinculin. Recently, it has been reported that overexpression of CRT induces ▶epithelial-mesenchymal transition (EMT)-like morphological changes and enhances cellular invasiveness in renal epithelial MDCK cells. The enhanced invasiveness mediated through ▶E-cadherin gene repression was regulated by the gene repressor, ▶Slug, via altered Ca2+ homeostasis caused by overexpression of CRT in MDCK cells. This study suggests that expression of CRT may play some causative role in the gain of invasiveness during the process of malignant transformation. In addition, it has been reported that cellular migration and binding to collagen type V are apparently suppressed in embryonic fibroblasts from CRT knock-out mice, indicating that the cellular level of CRT is important for the regulation of cell motility. Furthermore, CRT protein binds to GCN repeats in mRNA of the myeloid transcription factor ▶CCAAT/ enhancer-binding protein α (CEBPA), and thereby impedes translation of the CEBPA mRNA, suggesting that CRT plays a functional role in the differentiation block in ▶acute myeloid leukemia through suppression of CEBPA by the leukemic ▶fusion gene AML1-MDS1-EVl1. Together, these findings suggest that CRT is involved in the regulation of cancer characteristics, although the overall mechanisms are still not clear.

CRT as a Tool for Cancer Therapy CRT can form complexes with peptides in vitro to elicit peptide-specific CD8+ ▶T cell responses. In addition, peptide-bound CRT purified from tumor extracts elicits an antitumor effect specific to the source tumor. Antigen-specific cancer immunotherapy is an attractive approach to the eradication of systemic tumors at multiple sites in the body. It has been reported that vaccination with DNA encoding chimera for CRT and a tumor antigen, ▶human papilloma virus type-16 (HPV16) E7 [CRT/E7], resulted in a significant reduction in the number of lung tumor nodules in immunocompromised mice. All together, the use of CRT represents a feasible approach for enhancing tumor-specific T cellmediated immune responses. Therapeutic agents that target the tumor vasculature may prevent or delay tumor growth and even promote tumor regression or dormancy. As another approach to cancer therapy, CRT or a fragment thereof (amino acids 1–180) (i.e., ▶vasostatin) inhibits ▶angiogenesis and suppresses tumor growth. The combination of vasostatin and IL-12 as well as vasostatin and interferoninducible protein-10 had a suppressing effect on the cell growth of Burkitt lymphoma and colon carcinoma in

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mouse metastasis models. Although this suggests some potential for use in cancer therapy, the molecular mechanism of CRT actions at the cell surface is not fully understood.

C References 1. Eggleton P, Michalak M (2003) Introduction to calreticulin. In: Eggleton P, Michalak M, (eds) Calreticulin, 2nd edn. Landes Biosciences/Eurekah.com, Georgetown, TX, or Kluwer Academic/Plenum Publishers, New York, NY, pp 1–8 2. Gelebart P, Opas M, Michalak M (2005) Calreticulin, a Ca2+ -binding chaperone of the endoplasmic reticulum. Int J Biochem Cell Biol 37:260–266 3. Johnson S, Michalak M, Opas M et al. (2001) The ins and outs of calreticulin: from the ER lumen to the extracellular space. Trends Cell Biol 11:122–129 4. Michalak M, Corbett EF, Mesaeli N et al. (1999) Calreticulin: one protein, one gene, many functions. Biochem J 344:281–292 5. Williams DB (2006) Beyond lectins: the calnexin/ calreticulin chaperone system of the endoplasmic reticulum. J Cell Sci 119:615–623

Calsequestrin-like Protein ▶Calreticulin

CAM Definition Complementary alternative medicines are popular all over the world. The general concept that natural products are harmless by definition should be changed into a more realistic and responsible attitude.

cAMP Definition Cyclic adenosine monophosphate, second messenger induced in cells treated with various peptide hormones. ▶Suppressors of Cytokine Signaling

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cAMP Response Element Binding Protein (CREB)

cAMP Response Element Binding Protein (CREB)

CAMTA1 K AI -O LIVER H ENRICH , F RANK W ESTERMANN

Definition A transcription factor that is activated by serine phosphorylation triggered by increased intracellular levels of cAMP or calcium. ▶Signal Transduction

Campath-1H Definition A chimeric anti-CD52 monoclonal antibody regimen that has been successfully used for the treatment of refractory ▶Chronic Lymphocytic Leukemia. ▶Mcl Family

Camptothecin Definition Is a plant alkaloid isolated from Camptotheca acuminata (family Nyssaceae) with cytotoxic potential. Various (semi-)synthetic and water-soluble anticancer drugs, including 9-aminocamptothecin and 9-nitrocamptothecin, diflomotecan, topotecan, lurtotecan, and the prodrug ▶irinotecan have been derived from camptothecin. ▶Irinotecan ▶Membrane Transporters ▶Topoisomerases

CAMs ▶Cell Adhesion Molecules

DKFZ, German Cancer Research Center, Heidelberg, Germany

Definition

CAMTA1 is a candidate ▶tumor suppressor gene encoding a member of a protein family designated as calmodulin-binding transcription activators (CAMTAs). It resides within a distal portion of chromosomal arm 1p that is frequently deleted in a wide range of human malignancies.

Characteristics CAMTA1 maps to 1p36.31-p36.23 and its 23 exons are spread over 982.5 kb. The 6,582 bp cDNA encodes a protein of 1,673 amino acids. The protein’s primary structure contains a nuclear localization signal, two DNA-binding domains (CG-1 and TIG), a transcription activation domain, calmodulin binding motifs (IQ motifs), and ankyrin domains. Although the expression of CAMTA1 is seen in various organs, highest levels are found in neuronal tissues. Information on the physiologic roles of CAMTAs is scarce and most data derive from plant and drosophila studies. CAMTAs are transcription factors that typically bind to CGCG boxes via their CG-1 domain. An alternative mechanism of transcriptional activation has been described for CAMTA2, the second human CAMTA homolog. It acts as a coactivator of another transcription factor, Nkx2-5, to stimulate gene expression. This function is inhibited by binding of class II ▶histone deacetylases to the ankyrin-repeat region of CAMTA2. Upstream signaling components can activate CAMTA2 by promoting the export of class II histone deacetylases to the cytoplasm, relieving their repressive influence on CAMTA2. The sole fly homolog of CAMTA1 induces the expression of an ▶F-box gene, the product of which inhibits a Ca2+-stimulating ▶G-protein-coupled receptor (GPCR). The controlled deactivation of Ca2+-stimulating GPCRs is needed to tune Ca2+-mediated signaling and prevent abnormal cell proliferation. As CAMTA activity is increased by the Ca2+-sensor calmodulin, the Ca2+/ calmodulin/CAMTA/F-box protein pathway may mediate a negative feedback loop controlling the activity of Ca2+-stimulating GPCRs. This regulatory loop is of special interest taking into account the fundamental links between GPCR-mediated pathways and cancer biology. Clinical Relevance Deletions within 1p occur in various types of human malignancies, ranging from virtually all types of

Cancer

solid cancers to leukemias and myeloproliferative disorders. Functional evidence for a role of 1p in tumor suppression derives from experiments in which the introduction of 1p chromosomal material into ▶neuroblastoma cells resulted in reduced tumorigenicity. In neuroblastoma and other cancers, deletion of 1p36 is a predictor of poor patient outcome. Therefore, it is widely assumed that distal 1p harbors a gene (or genes) with tumor suppressive properties. To define the DNA, deleted from 1p, more precisely in pursuit of identifying the gene(s) of interest, substantial mapping efforts have been undertaken with the most detailed picture being worked out for neuroblastoma. In this tumor entity, the combination of ▶loss of heterozygosity (LOH) fine mapping studies allowed to considerably narrow down a smallest region of consistent deletion spanning only 261 kb at 1p36.3 and pinpointing the CAMTA1 locus. Sequence analysis revealed no evidence for somatic mutations in the remaining CAMTA1 copy of neuroblastomas with 1p deletion. However, a rare sequence variant leading to amino acid substitution within the ankyrin domain was seen in a subgroup of neuroblastomas. More importantly, low CAMTA1 expression is significantly associated with markers of unfavorable tumor biology and is itself a marker of poor neuroblastoma patient outcome. Moreover, CAMTA1 expression is a neuroblastoma predictor variable that is independent of the established molecular markers including 1p deletion. Thus, the measurement of this variable should allow an additional biological stratification of neuroblastomas and help to assign patients to the appropriate therapy. Additional evidence for a role of CAMTA1 in tumor development comes from ▶glioma and ▶colon cancer in which 1p is frequently deleted. In glioma, a 1p minimal deleted region spans 150 kb and resides entirely within CAMTA1. In colorectal cancer, a genome-wide analysis of genomic alterations revealed that loss of a 2 Mb recurrently deleted genomic region encompassing CAMTA1 has the strongest impact on survival when compared with other genomic changes. Furthermore, as in neuroblastoma, low expression of CAMTA1 is an independent marker of poor patient outcome. The high prevalence of CAMTA1 deletion in neuroblastoma, glioma, and colorectal cancer together with the independent predictive power of low CAMTA1 expression for neuroblastoma and colorectal cancer outcome are consistent with the idea that low CAMTA1 levels mediate a selective advantage for developing tumor cells.

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as a candidate tumor suppressor gene. Clin Cancer Res 11(3):1119–1128 Bouche N, Scharlat A, Snedden W et al. (2002) A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 277 (24):21851–21861 Henrich KO, Claas A, Praml C et al. (2007) Allelic variants of CAMTA1 and FLJ10737 within a commonly deleted region at 1p36 in neuroblastoma. Eur J Cancer 43 (3):607–616 Henrich KO, Fischer M, Mertens D et al. (2006) Reduced expression of CAMTA1 correlates with adverse outcome in neuroblastoma patients. Clin Cancer Res 12(1):131–138 Kim MY, Yim SH, Kwon MS et al. (2006) Recurrent genomic alterations with impact on survival in colorectal cancer identified by genome-wide array comparative genomic hybridization. Gastroenterology 131(6): 1913–1924

Canale-Smith Syndrome Definition

▶Autoimmune Lymphoproliferative Syndrome

Canals of Hering Definition Hepatocytes secrete bile into bile canaliculi which in turn drain into the canals of Hering – small ductules lined in part by cholangiocytes and in part by hepatocytes. ▶Cholangiocarcinoma

Cancer M ANFRED S CHWAB DKFZ, Tumour Genetics, Heidelberg, Germany

References 1. Barbashina V, Salazar P, Holland EC et al. (2005) Allelic losses at 1p36 and 19q13 in gliomas: correlation with histologic classification, definition of a 150-kb minimal deleted region on 1p36, and evaluation of CAMTA1

Definition Cancer is a deregulated multiplication of cells with the consequence of an abnormal increase of the cell number in particular organs. Initial stages of the developing

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Cancer

cancer are usually confined to the organ of origin whereas advanced cancers grow beyond the tissue of origin. Advanced cancers invade the surrounding tissues that are initially connected to the primary cancer. At a later stage, they are distributed via the hematopoetic and lymphatic systems throughout the body where they can colonize in distant tissues and form ▶metastasis. The development of cancers is thought to result from the damage of the cellular genome, either due to random endogenous mechanisms or caused by environmental influences. The origin of cancers can be traced back to alterations of cellular genes. Genetic damage can be of different sorts: . Recessive mutations in ▶tumor suppressor genes . Dominant mutations of ▶oncogenes . Loss-of-function mutations in genes, involved in maintaining genomic stability and ▶repair of DNA (resulting in ▶genomic instability) History Human cancer is probably as old as the human race. It is obvious that cancer did not suddenly start appearing after modernization or industrial revolution. The world’s oldest documented case of cancer comes from ancient Egypt, in 1500 BC. The details were recorded on a papyrus, documenting eight cases of tumors occurring on the breast. It was treated by cauterization, a method to destroy tissue with a hot instrument called “the fire drill.” It was also recorded that there was no treatment for the disease, only palliative treatment. The word cancer came from the father of medicine, Hippocrates, a Greek physician (460–370 BC). Hippocrates used the Greek words, carcinos and carcinoma to describe tumors, thus calling cancer “karkinos.” The Greek terms actually were words to describe a crab, which Hippocrates thought a tumor resembled. Hippocrates believed that the body was composed of four fluids: blood, phlegm, yellow bile and black bile. He believed that an excess of black bile in any given site in the body caused cancer. This was the general thought of the cause of cancer for the next 1,400 years. Autopsies done by Harvey in 1628 paved the way to learning more about human anatomy and physiology. By about the same time period, Gaspare Aselli discovered the lymphatic system, and this led to the end of the old theory of black bile as the cause of cancer. The new theory suggested that abnormalities in the lymph and lymphatic system as the primary cause of cancer. The lymph theory replaced Hippocrates’ black bile theory on the cause of cancer. The discovery of the lymph system gave new insight to what may cause cancer, it was believed that abnormalities in the lymphatic system was the cause. Other theories surfaced, such as cancer being caused by trauma, or by parasites, and it was thought that cancer

may spread “like a liquid” (Bentekoe, 1687; Heinrich Vierling, personal communication). The belief that cancer was composed of fermenting and degenerating lymph fluid was predominant. The discovery of the microscope by Leeuwenhoek in the late seventeenth century added momentum to the quest for the cause of cancer. By late nineteenth century, with the development of better microscopes to study cancer tissues, scientists gained more knowledge about the cancer process. It wasn’t until the late nineteenth century that Rudolph Virchow, the founder of cellular pathology, recognized that cells, even cancerous cells, derived from other cells. The early twentieth century saw great progress in our understanding of microscopic structure and functioning of the living cells. Researchers pursued different theories to the origin of cancer, subjecting their hypotheses to systematic research and experimentation. John Hill first recognized an environmental cause from the dangers of tobacco use in 1761 and published a book “Cautions Against the Immoderate Use of Snuff.” Percivall Pott of London in 1775 described an occupational cancer of the scrotum in chimney sweeps caused by soot collecting under their scrotum. This led to identification of a number of occupational carcinogenic exposures and public health measures to reduce cancer risk. This was the beginning of understanding that there may be an environmental cause to certain cancers. A virus causing cancer in chickens was identified in 1911 (Rous sarcoma virus). Existence of many chemical and physical carcinogens were conclusively identified during later part of the twentieth century. The later part of the twentieth century showed tremendous improvement in our understanding of the cellular mechanisms related to cell growth and division. The identification of ▶transduction of oncogenes with the discovery of the ▶SRC gene, the transforming gene of Rous sarcoma virus, led to formulating the oncogene concept of tumorigenesis and can be viewed as the birth of modern molecular understanding of cancer development. Subsequently, tumor suppressor genes were identified. Many genes that suppress or activate the cell growth and division are known to date, their number is ever growing. It is conceivable that in the end the confusing situation may arise to recognize that all genes of the human genome, in one way or another, take part in signaling normal or cancerous cellular growth.

Characteristics A large proportion of genetic changes appears to arise by mechanisms endogenous to the cell, such as by errors occurring during the replication of the ~3 × 109 base pairs present in the human genome. Environmental factors have a major role as well, predominantly as:

Cancer

. Chemical carcinogens (e.g. aflatoxin B1 in liver cancer (▶liver cancer, molecular biology), tobacco smoke in lung cancer; ▶tobacco carcinogenesis) . Radiation . Viruses (such as ▶hepatitis B virus (▶Hepatitis viruses) in liver cancer, or ▶human papillomavirus in cervical cancer) Types of Genetic Damage Damage to oncogenes and tumor suppressor genes can be of different sorts: . Point mutations resulting in the activation of a latent oncogenic potential of a cellular gene (e.g. ▶RAS) or in the functional inactivation of a tumor suppressor gene by generating an intragenic stop codon that leads to premature translation termination with the consequence of an incomplete truncated protein (e.g. ▶p53) or the failure for maintaining genomic stability (▶mismatch repair genes in ▶HNPCC) . ▶Amplification leading to an increase of the gene copy number beyond the two alleles normally present in the cell (copy number can reach 500 and more; example: ▶MYCN in human ▶neuroblastoma) . Translocation, which is defined as an illegitimate recombination between non-homologous chromosomes, the result being either a fusion protein (where recombination occurs between two different genes such as BCR-ABL in Chronic Myclogenous Leukemia) or in the disruption of normal gene regulation (where the regulatory region of a cellular gene is perturbed by the introduction of the distant genetic material such as ▶MYC in Burkitt lymphoma (▶Epstein-Barr virus)) . Viral insertion by the integration of viral DNA into the regulatory region of a cellular gene. This integration can occur after a virus has infected a cell. Viral insertion is well documented in animal tumors (HBV integration in the vicinity of ▶MYCN in liver cancer in experimental animals; liver cancer, molecular biology) Cellular Aspects Cancer in solid tissues (solid cancer) usually develops over long periods (often 20–30 years latency period) of time. An exception are solid cancers (such as neuroblastoma) in children, which often are diagnosed shortly after birth. Malignant cancers are characterized by their ability to develop metastasis (i.e. secondary cancers at distance from the primary tumor), often they also show multidrug resistance, which means that they hardly react to conventional chemotherapy. It is thought that the development of a normal cell to a metastatic cell is a continuous process driven by genetic damage and genomic instability, with the progressive selection of cells that have acquired a selective advantage within the particular tissue environment

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(▶multistep development). Studies of colorectal cancers have identified 6–7 genetic events required for the conversion of a normal cell to a cell with metastatic ability. This is in contrast to leukemias, which usually require one genetic event, most often a translocation, for disease development. Sporadic Versus Familial Cancer The vast majority of cancers are “sporadic,” which simply means that they develop in an individual. Descendants of this individual do not have an increased risk because the cellular changes that have resulted in cancer development are confined to this individual. In contrast, ~10% of cancer cases have a hereditary background, they show familial clustering (prominent examples include retinoblastoma (▶Retinoblastoma, ▶cancer genetics), ▶breast cancer, FAP (▶APC gene in Familial Adenomatous Polyposis) and HNPCC as familial forms of colorectal cancer (▶colon cancer), ▶melanoma). Familial cancers have been identified to result from germline mutation of genes. These germ line mutations do not always directly dictate cancer development, although they are considered “strong” hereditary determinants. They represent susceptibility genes that confer a high risk for cancer development to the gene carrier. The relative risk of the individual carrying the mutant gene can vary considerably. For instance, the risk of carriers of one of the breast cancer susceptibility genes ▶BRCA1 or ▶BRCA2 for breast cancer development can vary between approximately 60 and 90%. In reality this means that the risk for cancer development is difficult to predict, and individuals may not develop cancer at all in spite of the presence of a mutated gene in their germ line. The molecular basis for the differences in risk are unknown. Formally the activity of modifying factors, either environmental or genetic, has been suggested. Such modifying factors appear to be less important for some other familial cancers, such as retinoblastoma, where the risk is constant between 90 and 95% for gene carriers. Polygenic Determinants of Risk The relative risk of the individual for cancer development can also be determined by so called “weak” genetic factors. Normal cells contain a number of genes involved in ▶detoxification reactions. Different allelic variants of these genes exist in the human population that encode proteins with slightly different enzymatic activities. Although the exact contribution of individual allelic variants to cancer development is difficult to assess, it is reasonable to assume that individuals that have inherited “weak” enzymatic activities in different detoxification systems are likely to have a higher risk. It is likely, therefore, that the risk for such cancers is “polygenic.” ▶Toxicological Carcinogenesis

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Cancer and Cadmium

Cancer and Cadmium T IM S. N AWROT, J AN A. S TAESSEN Division of Lung Toxicology, Department of Occupational and Environmental Medicine (T.S.N.) and the Studies Coordinating Centre (J.A.S.), Division of Hypertension and Cardiovascular Rehabilitation, Department of Cardiovascular Diseases, University of Leuven, Leuven, Belgium

Definition Cadmium is a metal that has the symbol Cd and atomic number 48 in the periodic table. Cadmium has high toxic effects, an elimination half-life of 10–30 years, and accumulates in the human body, particularly the kidney. Roughly 15,000 tons of cadmium is produced worldwide each year for nickel-cadmium batteries, pigments, chemical stabilizers, metal coatings, and alloys.

Characteristics Urinary excretion of cadmium over 24 h is a biomarker of lifetime exposure. Exposure to cadmium occurs through intake of contaminated food or water, or by inhalation of tobacco smoke or polluted air. Occupational exposures can be found in industries such as electroplating, welding, smelting, pigment production, and battery manufacturing. Other exposures to cadmium can occur through inhalation of cigarette smoker. Gastrointestinal absorption of cadmium is estimated to be around 5–8%. Inhalation absorption is generally higher, ranging from 15 to 30%. Absorption after inhalation of cadmium fume, such as cigarette smoke, can be as high as 50%. Once absorbed, cadmium is highly bound to the metal-binding protein, metallothionein. Cadmium is stored mainly in the kidneys and also the liver and testes, with a half-life in the body of 10–30 years. In general, nonsmokers have urinary cadmium concentrations of 0.02–0.7 μg/g creatinine, which increase with age in parallel with the accumulation of cadmium in the kidney. Cadmium is a global environmental contaminant. Populations worldwide have a low-level intake through their food, causing an age-related cumulative increase in the body burden of this toxic metal. Environmental exposure levels to cadmium, that are substantially above the background, occur in areas with current or historical industrial contamination for instance in regions of Belgium, Sweden, UK, Japan, and China. As an environmental carcinogen, cadmium could have substantial health implications. Three lines of evidence explain why the International Agency for the Research on Cancer classified cadmium as a human carcinogen. First, as reviewed by Verougstaete and colleagues, several, albeit not all studies in workers showed a positive

association between the risk of lung cancer and occupational exposure to cadmium; discrepancies between these studies should not be ascribed to the better design of the more recent studies. Verougstraete and colleagues suggested that such inconsistencies might be attributed to the high relative risk of cancer in the presence of coexposure to ▶arsenic, nickel, or toxic fumes, and that the increasingly stringent regulations with regard to levels of exposure permissible at work might be a confounding factor (▶Lead exposure, nickel carcinogenesis). Second, data from rats showed that the pulmonary system is a target site for carcinogenesis after cadmium inhalation. However, exposure to toxic metals in animal studies have usually been much higher than those reported in environmentally exposed humans to toxic metals. Third, several studies done in vitro have shown plausible pathways, such as increased oxidative stress, modified activity of transcription factors, and inhibition of DNA repair. Most errors that arise during DNA replication can be corrected by DNA polymerase proof reading or by postreplication mismatch repair. In fact, inactivation of the DNA repair machinery is an important primary effect, because repair systems are required to deal with the constant DNA damage associated with normal cell functions. The latter mechanism might indeed be relevant for environmental exposure because Jin et al. found that chronic exposure of yeast to environmentally relevant concentrations of cadmium can result in extreme hypermutability. In this study the DNA-mismatch repair system is already inhibited by 28% at cadmium concentrations as low as 5 μM. For example, the prostate of healthy unexposed humans accumulates cadmium to concentrations of 12−28 μM and human lungs of nonsmokers accumulate cadmium to concentrations of 0.9−6 μM. Further, in vitro studies provide evidence that cadmium may act like an estrogen, forming high-affinity complexes with estrogen receptors, suggesting a positive role in breast cancer carcinogenesis. Along with this experimental evidence, two epidemiological studies in 2006 gave important positive input into the discussion on the role of exposure to environmental cadmium in the development of cancer in human beings. First, the results of a population-based case-control study noticed a significant twofold increased risk of breast cancer in women in the highest quartile of cadmium exposure compared with those in the lowest quartile. Second, we conducted a populationbased prospective cohort study with a median follow-up of 17.2 years in an area close to three zinc smelters. Cadmium concentration in soil ranged from 0.8 to 17.0 mg/kg. At baseline, geometric mean urinary cadmium excretion was 12.3 nmol/day for people in the high-exposure area, compared with 7.7 nmol/day for those in the reference (i.e, low exposure) area. The risk of lung cancer was 3.58 higher than in a reference population from an area with low exposure. 24-h urinary

Cancer of B-lymphocytes

excretion is a biomarker of lifetime exposure to cadmium. The risk for lung cancer was increased by 70% for a doubling of 24-h urinary cadmium excretion. Confounding by coexposure by arsenic could not explain the observed association. Epidemiological studies did not convincingly imply cadmium as a cause of prostate cancer. Of 11 cohort studies, only 3 (33%) found a positive association. In conclusion, recent experimental and epidemiological studies strongly suggest environmental exposure to cadmium as a causal factor in the development of cancer of the lung and breast.

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cancers. Increased levels may be associated with pregnancy and lactation, benign breast or ovarian disease, endometriosis, pelvic inflammatory disease, and hepatitis. ▶Serum Biomarkers

Cancer Associated Antigen 19-9 (CA 19-9)

References 1. Verougstraete V, Lison D, Hotz P (2003) Cadmium, lung and prostate cancer: a systematic review of recent epidemiological data. J Toxicol Environ Health B Crit Rev 6(3):227–255 2. Jin YH, Clark AB, Slebos RJ et al. (2003) Cadmium is a mutagen that acts by inhibiting mismatch repair. Nat Genet 34(3):326–329 3. Jarup L, Berglund M, Elinder CG et al. (1998) Health effects of cadmium exposure – a review of the literature and a risk estimate. Scand J Work Environ Health 24 (Suppl 1):1–51 4. McElroy JA, Shafer MM, Trentham-Dietz A et al. (2006) Cadmium exposure and breast cancer risk. J Natl Cancer Inst 98(12):869–873 5. Nawrot T, Plusquin M, Hogervorst J et al. (2006) Environmental exposure to cadmium and risk of cancer: a prospective population-based study. Lancet Oncol7(2):119–126

Cancer Antigen Definition Cell surface proteins specific for cancer cells. ▶Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy

Definition Serum levels of CA 19-9, an intercellular adhesion molecule, was initially found in patients with colorectal cancer, and subsequently also identified in patients with pancreatic and biliary tract cancers, and less often in gastric, ovarian, lung, breast and uterine cancer. Noncancerous conditions that may elevate CA 19-9 include gallstones, cholecystitis, pancreatitis and cirrhosis of the liver. ▶Serum Biomarkers

Cancer Associated Antigen 27-29 (CA 27-29) Definition Cancer antigen 27-29 (synonym: BR 27-29) is a normal epithelial cell mucin-1 (MUC1) apical surface glycoprotein. Elevated serum levels are highly associated with breast cancer. However they can also be found in cancers of colon, stomach, kidney, lung, ovary, pancreas, uterus and liver and in a number of noncancerous conditions, including first trimester pregnancy, ▶endometriosis, ovarian cyst, benign kidney, liver and breast disease. ▶Serum Biomarkers

Cancer Antigen 15-3 (CA 15-3) Definition Carbohydrate antigen 15-3 is a tumor marker associated with breast cancer, and has much less specificity and sensitivity in patients with ovarian, lung, or prostate

Cancer of B-lymphocytes ▶B-cell Tumors

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Cancer Cachexia Syndrome (CCS)

Cancer Cachexia Syndrome (CCS) Definition Loss of weight in the form of lean body mass and fat which results from a complex interaction between cytokines and tumor factors. ▶Nutrition Status

Cancer Causes and Control G RAHAM A. C OLDITZ Washington University in St. Louis, St. Louis, MO, USA

Synonyms Etiology; Prevention

Definition The process of identifying causes of cancer and developing strategies to change cancer risk through health care providers, regulations that reduce risk, or individual and community level changes.

Characteristics Over 6 million people around the world die from cancer each year. There is overwhelming evidence that lifestyle factors impact cancer risk and that positive, populationwide changes can significantly reduce the cancer burden. Current epidemiologic evidence links behavioral factors to a variety of diseases, including the most common cancers diagnosed in the developed world – ▶lung cancer, ▶colorectal cancer, ▶prostate cancer and ▶breast cancer. These four cancers account for over 50% of all cancers diagnosed on western countries. As summarized in Fig. 1, ▶tobacco causes some 30% of cancer, lack of physical activity 5%, obesity 15%, diet 10%, ▶alcohol 5%, ▶viral infections 5%, and ▶UV light by excess sun exposure 3%. Because of the tremendous impact of modifiable factors on cancer risk, especially for the most common cancers, it has been estimated that at least 50% of cancer is preventable. Currently in the US not all risk factors are equally distributed across race and social class. Trends in risk factors should also be considered when assessing potential for prevention. To bring about dramatic reductions in cancer incidence, widespread lifestyle changes will be necessary.

Cancer Causes and Control. Figure 1 Causes of cancer.

Rose advocates the need for population approaches for prevention of chronic disease. He emphasizes that when the relation between a lifestyle factor or biological predictor of risk is continuous, the majority of cases attributable to the exposure will likely arise in those who are not classified as being at high risk. He illustrates this with examples of blood pressure and rates of coronary heart disease. Specifically, even small changes in blood pressure at the population level can translate into large reduction in the rates of coronary disease and stroke. To reduce the risk of disease in the populationsubstantial benefits can be achieved by a small reduction for all members of the society rather than just focusing on the high-risk groups. Because population wide trends in cardiovascular risk factors show continuing improvement, the rate of coronary heart disease incidence and mortality continues to decrease. When we consider population approaches to cancer prevention, we must address the etiologic process, which covers a different time course and sequence from coronary heart disease. Although cardiovascular disease is the end point of the chronic process of atherosclerosis, treatment focuses on the reversal and subsequent prevention of the acute thrombotic process of myocardial infarction. Cancer, on the other hand, is the result of a long process of accumulating DNA damage (▶multistep development), leading ultimately to clinically detectable lesions such as in situ and invasive cancer. For example, studies of the progression in ▶colon cancer from first mutation to invading malignancy suggest that DNA changes accumulate over a period of as long as 40 years. The goal of cancer prevention is to arrest this progression; different interventions interrupt carcinogenesis at different points in the process. Further, most cancers do not have a late “acute” event, analogous to thrombosis, which can be prevented with medical interventions. The benefits of cancer prevention and control programs take time to be observed. The fact that different

Cancer Causes and Control

interventions will impact at different points along the pathway to cancer, that can stretch over nearly half a century, has implications for when we can expect to see pay-off in terms of lower cancer rates. Research has demonstrated that those who initiate smoking during early adolescence greatly increase risk of lung cancer even when one takes into account both the dose and duration of smoking. If we could delay the age at which most adolescents first start to smoke, we would probably substantially reduce lung cancer rates, but this benefit will not be observable for 20–40 years after the intervention. Adult cessation, on the other hand, reduces risk more rapidly, but fails to address the continuing recruitment of the next generation of smokers. Recent declines in the incidence of lung cancer among younger men and women in the United States reflect reductions in the rate of smoking among younger adults. Other lifestyle interventions may act as preventive early in the DNA pathway to cancer. For example, ▶aspirin and ▶folate appear to act early in the pathway inhibiting colon cancer. Population-wide prevention strategies for cancer do work. For example, reductions in lung cancer rates in the United States mirror changes in cigarette smoking patters, with marked decreases seen first in young men, then older men, and finally in women. Introduction of the Papaniculou test for cervical cancer in the 1950s was followed by a dramatic decline in cervical cancer in those countries that made wide-spread ▶screening available. The decline in Australian ▶melanoma mortality for those born after 1950 is an additional example of effective intervention at the population level. Behavior change is possible and offers great potential for cancer prevention. The recommendations for cancer risk reduction include reducing tobacco use, increasing physical activity, maintaining a healthy weight, improving diet, limiting alcohol, avoiding excess sun exposure, utilizing safer sex practices, and obtaining routine cancer screening tests. Age is the dominant factor that drives cancer risk; for all major malignancies, risk rises markedly with age. The importance of age is exemplified by the fact that the aging U.S. population together with projected population growth will result in a doubling of the total number of cancer cases diagnosed each year by the year 2050, assuming that incidence rates remain constant. With this estimated growth in cancer from 1.3 million to 2.6 million cases per year, it is expected that that both the number and proportion of older persons with cancer will also rise dramatically. Tobacco Tobacco is the major cause of premature death around the world accounting for some 5M deaths each year. In the United States, adult smokers lose an average of 13 years of life because of smoking, and approximately

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half of all smokers die of tobacco-related disease. Smoking is well known to cause over 90% of lung cancers in addition to a range of other malignancies (▶tobacco carcinogenesis). It causes about 30% of all the cancer in the developed world, including lung cancer, ▶mouth cancer, larynx cancer, ▶esophagus cancer, ▶pancreas cancer, ▶cervix cancer, ▶kidney cancer, and ▶bladder cancer. Smoking also increased risk of cancers of the colon, stomach, cervix, liver, and prostate, as well as to leukemia. In addition, smoking leads to many other health problems, including heart disease, stroke, lung infections, emphysema, and pregnancy complications. Tobacco may act on multiple stages of carcinogenesis; it delivers a variety of carcinogens, causes irritation and inflammation, and interferes with the body’s natural protective barriers. The health risks of tobacco use are not limited to cigarette smoking. Cigar and pipe use increase the risk of disease, as does exposure to second-hand smoke and smokeless tobacco use. Avoiding initiation of tobacco use clearly offers the greatest potential for disease prevention. However, for those who use tobacco products, there are substantial health benefits that come with quitting. There are numerous effective cessation methods, and in the past 25 years, 50% of all living Americans who have ever smoked, have successfully quit. Quitting smoking has immediate and significant health benefits for men and women of all ages. For example, former smokers live longer than individuals who continue smoking. Those who quit before age 50 have approximately half the risk of dying in the next 15 years. This decline in mortality risk is measurable shortly after cessation and continues for at least 10–15 years. Strategies to assist smoking cessation and decreasing youth initiation from both a population and clinical perspective are essential steps to reducing the burden of cancer. Trends Current smoking among US adults has remained steady over the past decade. Once quite pronounced, gender disparity in smoking rates is now relatively small and has been stable since 1990. In 2002, 25.7% of men were current smokers compared to 20.8% of women. Given the profound impact of smoking on cancer, disparities in smoking rates and in access to effective cessation methods will continue to translate directly into differences in the burden of smoking-related cancers. Physical Activity Lack of physical activity causes over 2M deaths each year around the world. People in the US and in other developed nations are extremely inactive – over 60% of

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the US adult population does not participate in regular physical activity, which includes 25% of adults who are almost entirely sedentary. Fortunately, the negative effects of a sedentary lifestyle are reversible: increasing one’s level of physical activity, even after years of inactivity, can reduce mortality risk. Lack of physical activity increases the risk of colon and breast cancer and likely endometrial cancer, as well as diabetes, osteoporosis, stroke, and coronary heart disease. Overall, sedentary lifestyles have been linked to 5% of deaths from cancer. Among both men and women, high levels of physical activity may decrease the risk of colon cancer by as much as half. Using a variety of measures of activity, studies have consistently shown higher physical activity lowers risk of colon cancer. Physical activity also appears to lower the risk of large adenomatous polyps, precursor lesions for colon cancer, suggesting that it may influence the early stages of the adenoma-carcinoma sequence. In addition, the relationship between physical activity and breast and colon cancer are seen across levels of obesity, indicating that physical activity and obesity have separate or independent effects on cancer incidence. Growing evidence suggests that physical activity may also be protective against lung and prostate cancer. Several mechanisms have been proposed to explain these associations. Physical activity reduces circulating levels of insulin, a growth factor for colonic epithelial cells. Additionally, it is postulated that cancer risk is reduced through alterations in prostaglandin levels, improvement in immune function, and modification of bile acid metabolism. Potential mechanisms for the reduction of breast cancer risk include physical activity’s lowering of the cumulative lifetime exposure to circulating estrogens and improving immune pathways. The benefits of physical activity include the prevention of cancer and a large number of other chronic diseases. Increasing levels of physical activity, even after years of inactivity, reduces mortality risk. As little as 30 min of moderate physical activity (such as brisk walking) per day significantly reduces disease risk.

Trends One major determinant of activity level that has changed over time is the amount of activity required for work and daily living. With advances in technology and the development of labor-saving devices, there is now a greatly reduced need for physical activity for transportation, household tasks, and occupational requirements. Overall, the prevalence of physical inactivity in the United States is remarkably high; in 1996, about 28% of Americans reported absolutely no participation in leisure-time physical activity. In addition, physical activity in schools has declined, and almost half of

young Americans between the ages of 12–21 are not vigorously active on a routine basis. Given the trends in our society, it is unlikely that this decreasing energy expenditure will reverse rapidly. Accordingly, the burden of cancer due to lack of physical activity will increase in the years ahead unless new strategies to promote activity are rapidly implemented.

Weight Control and Obesity Prevention Overweight and obesity is increasing at epidemic rates in the United States, around the world, and is estimated to account for 2.6M deaths each year. Currently almost 65% of American adults are overweight (body mass index (BMI) ≥25 kg/m2), and over 30% are considered obese (BMI ≥ 25 kg/m2). Overweight and obesity cause a variety of cancers; colon, postmenopausal breast, endometrial, renal, and esophageal. The proportion of cancer caused by obesity ranges from 9% for postmenopausal breast cancer to 39% for endometrial cancer. One large US study suggested that obesity influences an even broader range of cancers, increasing the risk of death from cancers of the colon and rectum, prostate, breast, esophagus, liver, gallbladder, pancreas, kidney, stomach, uterus, and cervix in addition to non-Hodgkin’s lymphoma and multiple myeloma. Overall, obesity causes 14% of cancer deaths among men and 20% of cancer deaths among women. Excess body fat may act by altering levels of hormones and tumor growth factors. It is clear that excess weight has severe health consequences. In addition to raising the risk of cancer, overweight and obesity also increase the risk of a multitude of other diseases and chronic conditions, such as stroke, cardiovascular disease, type 2 diabetes, osteoarthritis, and pregnancy complications. The International Agency for Research on Cancer has proposed a comprehensive set of recommendations to address the issue of weight control at multiple levels, including steps by health care providers, regulatory approaches to create adequate access to safe places for exercise (including school, worksite, and community), and family and community level actions.

Trends In the U.S., the prevalence of overweight and obesity has increased so dramatically and so rapidly, it is frequently referred to as an obesity epidemic. The trend is also being seen among children and adolescents. This epidemic has affected people of all ages, races, ethnicities, socioeconomic levels, and geographic regions. Given limited long-term success in weight reduction programs, the cancer burden due to obesity will likely continue to follow the rising prevalence of this risk factor in the coming years.


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Dietary Improvements Fruit and vegetable intake has been most consistently evaluated as a cancer prevention strategy. The global burden of inadequate intake is estimated to account for over 2M deaths each year. While evidence for cardiovascular benefits and reduced risk of diabetes are clear, evidence for cancer risk reduction has become less convincing with the results of numerous prospective cohort studies showing weaker associations with cancer risk. Low intake of fruits and vegetables are probably related to increased risk of pancreas, bladder, lung, colon, mouth, pharynx, larynx, esophagus and stomach cancer. Although the effect of fruit and vegetable consumption on the risk of prostate cancer has been examined in nearly twenty studies, data remain inconsistent. The majority of studies suggest that overall fruit and vegetable intake has little effect if any on the risk of prostate cancer. However, individual fruits and vegetables may offer the potential for greater risk reduction, with tomatoes being the most promising, with a 40–50% reduction in risk among men who consumed large amounts of tomatoes and tomato products. The carotenoid ▶lycopene is hypothesized to be responsible for the protective effect. A number of mechanisms have been suggested to explain the protective effect of fruits and vegetables, but it is not known if specific agents, such as ▶carotenoids, folic acid, and vitamin C, or a special combination of factors create anticarcinogenic effects. It is also possible that diet in childhood and adolescence is more important than later in life in driving risk of cancer. A number of studies have found that as folate intake increases, the risk of colorectal cancer (as well as polyps) decreases. The Nurses’ Health Study found that a high intake of folate from fruits and vegetables was sufficient to lower risk but that supplementation with a multivitamin that contained folate offered even greater reductions. The underlying biologic role of folate and its interaction with the MTHFR gene add support to the causal relation between low folate and colon cancer. In addition to the reduction in risk of colon cancer, growing evidence points to folate also reducing the adverse effect of alcohol on breast cancer. Based on this evidence and the benefits for prevention of neural tube defect and cardiovascular disease, use of a daily vitamin supplement containing folate is recommended.

Fiber has been shown to reduce the risk of heart disease and diabetes, but it does not appear to offer protection against cancer. Long believed to help prevent colon cancer, the data do not support this hypothesis.

Dietary Fat Variations in international cancer rates have often been attributed to differences in total fat intake, yet evaluation has shown no clear link between dietary fat and breast, colon or prostate cancer. Although dietary fat overall does not appear to impact cancer risk, there is some evidence to suggest that certain types of fat, such as animal fat, may increase risk.

Vitamin A and Carotenoids Isolated ▶vitamin A and carotenoids are not likely to play a large role in cancer prevention. Some observational data support a probable inverse relation with lung cancer risk, but randomized trials of beta-carotene intake found either no effect or an increased risk of lung cancer. It has also been suggested that beta-carotene impacts breast cancer risk, however, it seems that at

Red Meat High intake of red meat, including beef, pork, veal and lamb is associated with an elevated risk of colorectal cancer. The mechanism of this increased risk is not well understood, but it may be related to the high concentrations of animal fat or to carcinogens such as heterocyclic amines produced when the meat is cooked at high temperatures. Calcium Higher calcium intake has been linked to a reduced risk of colorectal adenomas and colorectal cancer. However, increased dietary calcium is also associated with an increased risk of prostate cancer. Research indicates that there may be a moderate intake of calcium that provides protection against colorectal cancer risk without causing a large increase in prostate cancer risk. Excess Caloric Intake One consistent dietary finding is that excess calories from any source result in weight gain and increased cancer risk. As the obesity epidemic continues to spread, the importance of balancing caloric intake with caloric expenditure becomes even more evident for the prevention of cancer and other chronic diseases. Whole Grains Although grain products in general have not been shown to affect cancer risk, whole-grain foods may provide some protection against stomach cancer. Grains such as wheat, rice, and corn form the basis for most diets worldwide. Some grain products, such as wholewheat bread and brown rice, are consumed in the “whole-grain” form, while others, like white bread and white rice, are more refined. During the process of refining grain, most of the fiber, vitamins, and minerals are removed, thus whole-grain foods tend to be more nutrient-rich than refined foods and may offer more in terms of disease prevention. The benefits of wholegrain foods in reducing cardiovascular disease and ischemic stroke are well established.

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best, there is only a small decrease in breast cancer risk associated with a high intake of carotenoids.

drink per day for women and less than two drinks per day for men.

Selenium Ecological studies have suggested that increased ▶selenium intake is associated with decrease risk of colon and breast cancer. A randomized control trial of selenium for skin cancer prevention showed no effect of selenium on skin cancer incidence, however it did show a reduction in incidence of lung, colon and prostate cancer. Despite these promising results, the impact of selenium remains unclear. Fortification of the soil in Finland in the mid-1980s led to higher blood selenium levels, but no decline in incidence or mortality has been noted for prostate or colon cancer.

Safer Sex and Decreased Viral Transmission ▶Unsafe sex is responsible for 2.9M deaths each year, primarily due to the transmission of ▶HIV. However, unprotected sexual contact also results in the spread of multiple other sexually transmitted infections including oncogenic viruses. Some of these viruses may also be spread through exposure to blood and blood products. ▶Human papillomavirus causes cervical cancer, vulvar, penile and anal cancer; ▶hepatitis B virus and ▶hepatitis C virus cause ▶hepatocellular cancer; human lymphotropic virus-type 1 is associated with adult T cell leukemia (▶human T-cell leukemia virus); human immunodeficiency virus-type 1 causes ▶Kaposi sarcoma and non-Hodgkin lymphoma; and human herpes virus causes Kaposi sarcoma and body cavity lymphoma. Prevention strategies to contain the spread of these viruses should include behavioral and educational interventions to modify sexual behavior, and structural and regulatory changes to promote safer sex and make condoms readily available. Biomedical interventions to administer vaccines are also needed. For example, it is estimated that vaccination programs could reduce the global burden of liver cancer by 60%. Additional strategies to prevent viral spread include needle exchange programs for intravenous drug users; regulation of tattooing and acupuncture; screening of blood donors, and the development of artificial blood products.

Vitamin D Growing evidence relates lower levels of ▶vitamin D to increased risk of cancer and to poor survival after diagnosis. Trends The proportion of adults consuming the recommended five servings of fruits and vegetables a day varies between 8 and 32%. While these estimates are clearly low for the entire population, certain groups, as defined by gender, race/ethnicity, education, and income, are of particular concern. Given constraints due to both financial resources and physical access to markets that provide fresh fruit and vegetables, it remains likely that SES gradients in diet will continue. Interventions are needed to overcome these existing barriers and make healthy foods readily available to all. Limitation of Alcohol Use Globally, alcohol intake in excess is responsible for 1.8M deaths each year. Clear benefits of moderate alcohol intake have been shown in terms of reducing cardiac and diabetes risk, but alcohol remains a risk factor for cancer mortality. Alcohol is a known carcinogen that may raise cancer risk in several ways. For example, it may act as an irritant, directly causing increased cell turnover, or it may allow for improved transport and penetration of other carcinogens into cells. Alcohol use is a primary cause of esophageal and oral cancer, and it is associated with an increased risk of breast, liver, and colorectal cancer. Multiple other risks are also associated with alcohol use, including the risk of hypertension, addiction, suicide, accident, and pregnancy complication. To balance the cardiovascular benefits with the risks of cancer and other negative consequences, it is recommended that those who drink alcohol should do so only in moderation. Intake should be limited to less than one

Trends and Disparities Current U.S. data on the prevalence of these different viruses is not adequate to predict trends in cancer incidence. In addition, the recent development of new technologies such as vaccines against HPV, suggest a new era in prevention of cervical cancer. However, for success, such vaccines must be available and accessible to the entire population. Assuring access remains a policy priority to maximize the potential benefit of this cancer prevention strategy. Sun Protection The American Cancer Society estimates over 50,000 melanoma diagnoses each year in the U.S. The incidence of melanoma is rising more rapidly than that of any cancer in this country. Exposure to the sun (▶UV radiation) is the major modifiable cause of melanoma and other skin cancers. For most people, the majority of lifetime sun exposure occurs during childhood and adolescence, and migrant studies clearly show that age at migration to high-risk countries has a strong impact on risk of this malignancy. For this reason, early intervention has the greatest potential for prevention.


The risk of melanoma and other less aggressive forms of skin cancer exists for all racial and ethnic groups, but skin cancers occur predominantly in the non-Hispanic white population. Constitutional characteristics including hair color, mole count, and family history contribute to risk of melanoma. However, studies show that established risk factors alone do not identify a sufficient proportion of cases to focus prevention efforts on only a subset of the population. Because identifying high-risk individuals will miss the majority of cases, population-based efforts provide greater protection. There is tremendous potential to substantially reduce the burden of this common malignancy through effective prevention efforts.

Screening Screening for cancer can provide protection in several ways. In the case of colorectal and cervical cancers, screening can detect premalignant changes that can be treated to prevent cancer from developing. This primary prevention has the potential to substantially reduce the burden of cancer. With colorectal screening the mortality from colon cancer is reduced by a half or more. If cancer is already present, screening can act as a secondary prevention (▶early detection), such as mammography for breast cancer, facilitating early diagnosis and treatment, thereby decreasing morbidity and mortality. This type of prevention is an added benefit of colorectal and cervical screening, and is the main goal of breast cancer and prostate cancer screening.

Trends and Disparities Trends in cervical cancer screening have been impacted by the breast cancer cervical cancer screening act which provided resources to states, via the Centers for Disease Control and Prevention, to bring screening services to low income women. Despite these efforts, national data suggest that low income and Hispanic women are less likely to be current with screening recommendations. Lack of access to care, defined as not having a usual source of health care, was associated with significantly lower compliance with cervical screening. Evidence from the 1998 Health Interview Survey indicates that all US born women have comparable and high compliance with screening for cervical cancer. Foreign born women, however, appear to be underscreened, accounting for the disparity among Hispanic women and suggesting a priority area for prevention as the U.S. continues to have a large immigrant population at risk of cancer. Surveys of colorectal screening suggest that the rates of screening have been rising and that Caucasians are more likely to be up to date with screening than other racial or ethnic groups.

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Conclusion Lifestyle changes offer tremendous potential for prevention of cancer and multiple other chronic conditions. This potential is often underestimated. To achieve the maximal benefit through behavioral change, interventions are necessary at multiple levels. Societal changes are needed to support and encourage the behavior modification of individuals. Approaches are needed to target individuals, communities, and systems, and create an environment less inductive to high-risk lifestyles. Social systems and regulatory efforts must complement individual behavior changes if these changes are to be sustained and the benefits of reduced disease burden realized. Overall, the major lifestyle factors considered here account for the majority of cancer and could be modified to prevent at least half of all cancers. However, the burden of cancer is not limited to just the major lifestyle factors considered here. For example, occupational and environmental exposures also account for a relatively small number of cancer cases compared to the lifestyle factors considered above. Yet the burden of exposure to these harmful agents may be disproportionately high among low-income populations, accentuating their cancer risk. In large part, these exposures can be prevented through adequate enforcement of regulatory changes, and this should remain a high priority. Small individual changes can result in large population benefits, but efforts to create prevention programs for only certain members of our society limits the potential for prevention. We must largely reframe our approach to the issue. Identifying risk factors and setting goals for reduction is only the beginning. Research and policy must now focus on bringing about population-wide lifestyle change, addressing the issues of disparities, and leaving no group or community behind.

References 1. Colditz GA, DeJong D, Emmons K et al. (1997) Harvard Report on Cancer Prevention. Volume 2. Prevention of Human Cancer. Cancer Causes Control 8:1–50 2. Curry S, Byers T, Hewitt M (2003) Fulfilling the potential of cancer prevention and early detection. National Academy Press, Washington, DC 3. International Agency for Research on Cancer (2002) Weight control and physical activity (Vol. 6). International Agency for Research on Cancer, Lyon 4. Rose G (1981) Strategy of prevention: lessons from cardiovascular disease. Br Med J (Clin Res) 282:1847–1851 5. U.S. Department of Health and Human Services (1996) Physical activity and health: A Report of the Surgeon General. US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Atlanta, GA

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Cancer Cell-platelet Microemboli

Cancer Cell-platelet Microemboli ▶Tumor Cell-Induced Platelet Aggregation

Cancer Epidemiology

useful distinction among etiological studies concerns the nature of the information on exposure: while some studies use information routinely collected for other purposes, such as censuses and medical records, in other circumstances exposure data are collected ad-hoc following a variety of approaches including questionnaires, pedigrees, environmental measurements, measurement of biological markers. A method-oriented (rather than subject- or design-oriented) approach has lead to the identification of specific sub-disciplines such as ▶molecular epidemiology.

PAOLO B OFFETTA International Agency for Research on Cancer, Lyon, France

Synonyms Population-based cancer research

Definition Knowledge about causes and preventive strategies for malignant neoplasms has greatly advanced during the last decades. This is largely attributable to the development of cancer epidemiology. In parallel to the identification of the causes of cancer, primary and secondary preventive strategies have been developed. A careful consideration of the achievements of cancer research, however, suggests that the advancements in knowledge about causes and mechanisms have not been followed by an equally important reduction in the burden of cancer. Part of this paradox is explained by the long latency occurring between exposure to carcinogens and development of the clinical disease. In addition, the most important risk factors of cancer are linked to lifestyle, and their modification entails cultural, societal and economic consequences. The failure to identify valid biomarkers of cancer risk is another reason of the limited success in cancer control. Cancer epidemiology investigates the distribution and determinants of cancer in human populations. Although the main tool in cancer epidemiology is the ▶observational study, ▶the intervention study, of experimental nature, is conducted to evaluate the efficacy of prevention strategies, such as screening programs and chemoprevention trials (clinical trials are usually considered outside the scope of epidemiology). Intervention studies follow the randomized trial design. Observational epidemiology can be broadly divided in ▶descriptive epidemiology and analytical studies. Analytical studies can be based on data collected at the individual or population level. The former consist of ▶cohort study, ▶case-control study and ▶cross-sectional study (and a few variations on these themes), the latter of so-called ▶ecological study. Family-based studies are used in ▶genetic epidemiology to identify hereditary factors. An additional

Characteristics A distinctive feature of cancer epidemiology is the availability in many countries of a population-based ▶cancer registry, which allow the calculation of valid and reliable estimate of the occurrence of cancer (incidence, mortality, prevalence, survival). Typically, registries collect routinely demographic data of patients, which are used to generate statistics according to period of diagnosis, age, sex, and other characteristics. These studies of descriptive epidemiology have been critical in developing etiological hypotheses. One particular type of descriptive studies concerns migrants from low- to high-risk areas, or vice-versa: the repeated demonstration of rapid changes in the risk of many cancers among migrants (from that prevalent in the area of origin towards that of the host area) provided very strong evidence of a predominant role of modifiable factors in the etiology of human cancer. While cancer registries were initially established in high-resource countries, a growing number of population s in middle- and low-resource countries are now covered by good-quality registries, thus providing a solid infrastructure for most ambitious research projects. Other routinely collected data are used in epidemiology. Mortality statistics are available in many countries of the world, and provide a good approximation of the incidence of the most fatal cancers. In a growing number of populations, automatic linkage is possible between incidence or mortality data and other population-based registries (e.g., hospital discharges, use of medications). ▶Record-linkage study may represent an efficient alternative to investigations based on ad-hoc collection of data. The number of new cases of cancer which occurred worldwide in 2002 has been estimated at about 10,800,000. Of them, 5,800,000 occurred in men and 5,000,000 in women. About 5,000,000 cases occurred in developed countries (North America, Japan, Europe including Russia, Australia and New Zealand) and 5,800,000 in developing countries. Among men, lung, prostate, stomach, colorectal, and liver cancers are the most common malignant neoplasms, while breast, cervical, colorectal, lung and stomach cancers are the

Cancer Epidemiology

most common neoplasms among women. The number of deaths from cancer in 2002 was estimated at about 6,700,000 and that of 5-year prevalent cases at about 24,600,000. Epidemiology has been instrumental to identify the causes of human cancer. In several cases, the epidemiological results preceded the elucidation of the underlying mechanisms. In other areas, however, epidemiological techniques are not sufficiently sensitive and specific to lead to conclusive evidence on the presence or absence of an increased risk. As for other branches of the discipline, the observational nature of epidemiology represents an opportunity for bias, including that generated by confounding, to generate spurious results. Techniques have been developed to prevent, control and assessment the presence and extent of bias in epidemiological studies. Cancer epidemiology has lead to the identification of tobacco smoking and use of smokeless tobacco products, chronic infections, overweight, alcohol drinking and reproductive factors as major causes of human cancer. Other important causes include medical conditions, some drugs, perinatal factors, physical activity, occupational exposures and ultraviolet and ionizing radiation. A role of diet in cancer risk has been suggested, but for very few dietary factors there is conclusive evidence of an effect on cancer risk. With a few exception of little relevance in most populations, the role of pollutants on cancer is not established. Tobacco smoking is the main single cause of human cancer worldwide. It is a cause of cancers of the oral cavity, pharynx, esophagus, stomach, liver, pancreas, nasal cavity, larynx, lung, cervix, kidney and bladder, and of myeloid leukemia. The proportion of cancers in a population attributable to tobacco smoking depends on the distribution of the habit a few decades earlier. Therefore, in populations in which the tobacco epidemic has not fully matured (e.g., men in many low-income countries and women in most European countries), the full effect of tobacco smoking on cancer burden is not yet observed. The notion that genetic susceptibility plays an important role in human cancer is old, and genetic epidemiology studies have characterized familial conditions entailing a very high risk of cancer have been identified, such as the Li-Fraumeni syndrome and the familial polyposis of the colon, and have identified high-risk cancer genes responsible for these syndromes. However, such high-risk conditions explain only a small fraction of the role of inherited susceptibility to cancer. The remaining fraction of genetic predisposition is likely explained by the combination of common variants in genes involved in one or more steps in the carcinogenic process, such as preservation of genomic integrity, repair of DNA damage. The identification of such low-penetrance susceptibility genes and of their

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interactions with exogenous factors (so-called ▶geneenvironment interactions) represents a challenge to genetic epidemiology. Advances in molecular biology and genetics offer new tools for epidemiological investigations, and have lead to the development of new methodological approaches, broadly defined as molecular epidemiology. The application of biomarkers to epidemiology has lead to advances in the identification of human carcinogens (e.g., the role of aflatoxin in liver cancer) and in the elucidation of mechanisms of carcinogenesis (e.g., TP53 mutations in tobacco-related carcinogenesis). Exposure to most known carcinogens – at least in theory – be avoided or reduced. This is true in particular for tobacco smoking and chronic infections, the two major known causes of cancer. Tobacco control measures have been implemented in most countries, and effective vaccination is today available against two of the main carcinogenic viruses, Hepatits B and Human Papilloma. Control of workplace exposure to known and suspected carcinogens in high-resource countries is another example of successful primary prevention of cancer. In many instances, however, primary prevention of cancer would require major changes in lifestyle, which are difficult to achieve. Detection of preclinical neoplastic lesions before they have developed the full malignant phenotype, and notably the ability to metastasize is a highly appealing approach to control cancer. The effectiveness of screening has been demonstrated via epidemiological studies for cervical cancer (cytological smear), breast cancer (mammography) and colorectal cancer (colonoscopy). The development of effective strategies for the early detection of other neoplasms is an active area of research. Cancer epidemiology exemplifies the strengths and the weaknesses of the discipline at large. Cancer epidemiology has the privilege of using complete and good quality disease registries available in many populations and covering a broad spectrum of rates and exposures. In many occasions, cancer epidemiology has been the key tool to demonstrate the causal role of important cancer risk factors. The best example is the association between tobacco smoking and lung cancer, which lead in the early 1960s to the establishment of criteria for causality in observational research. These findings have brought important regulatory and public health initiatives as well as lifestyle changes in many countries of the world. These epidemiological “discoveries” share two important characteristics: they involve potent carcinogens and methods are available to reduce misclassification of exposure to the risk factor of interest and to major possible confounders. It has been therefore possible to demonstrate consistently an association in different human populations. Note that it is not necessary for the prevalence of exposure to

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be high (although this obviously has an impact on the population ▶attributable risk): examples are the many occupational exposures and medical treatments for which conclusive evidence of carcinogenicity has been established on the basis of epidemiological studies conducted in small populations of individuals with well characterized exposure. When these conditions are not met, however, the evidence accumulated from epidemiological studies is typically inconsistent and difficult to interpret. The history of cancer epidemiology presents many examples of premature conclusions, which have not been confirmed by subsequent investigations and have damaged the reputation of the discipline. Exposure misclassification, uncontrolled confounding, emphasis of positive findings generated by chance, and inadequate statistical power are the most common limitations encountered in epidemiological studies. Several solutions have been proposed to overcome these problems. First, epidemiological studies should be very large in size. This is achieved either by conducting multicentric studies including thousands of cases of cancer, or by performing pooled and meta-analyses of independently investigations. Second, as mentioned above, the use of biological markers of exposure and early effect might contribute to reduce exposure misclassification, increase the prevalence of the relevant outcomes and shed light on the underlying mechanisms. Finally, guidelines have been developed to improve and standardize the conduct are report of observational epidemiological studies. Although relatively young, epidemiology has become a key component in cancer research. Most cancer centers have an epidemiological research group, and cancer is a major subject of research in most academic departments of epidemiology. Epidemiologists are more and more often invited to meetings of clinicians and basic researchers not only to provide an introduction to the distribution and the risk factors of a given cancer, but to participate in interdisciplinary discussions on clinical, preventive or mechanistic aspects of the disease. The strongest cancer epidemiology groups in the world are those combining different lines of expertise, from biostatistics to molecular biology and genetics to medical oncology. Despite its limitations, cancer epidemiology remains one of the most powerful tools at the disposal of the research community to combat cancer at all levels.

3. Doll R, Peto R (2003) Epidemiology of cancer. In: Warrell DA, Cox TM, Firth TD, (eds) Oxford Textbook of Medicine. Fourth Edition. Oxford University Press, London: pp 193–218 4. STROBE Statement. STrengthening the Reporting of OBservational studies in Epidemiology (http://www. strobe-statement.org/)

Cancer Epigenetics B ERNA D EMIRCAN , K EVIN B ROWN University of Florida College of Medicine, Gainesville, FL, USA

Definition

▶Epigenetics is defined as chromatin modifications that can alter gene expression, are heritable during cell division, but do not involve a change in DNA coding sequence.

Characteristics

References

In the context of normal biological processes, epigenetic mechanisms establish regions within the genome containing transcriptionally active (termed euchromatin) and silent (termed heterochromatin) DNA. Further, epigenetic mechanisms are responsible for stably inherited patterns of gene expression such as X chromosome inactivation and genomic imprinting (i.e., selective expression of maternal or paternal alleles). Chromatin modifications that alter gene expression are both changes to the methylation state of DNA and post-translational modifications to histone complexes. It is well recognized that genetic mutations occur in cancer cells and that these events can exert profound and disease-associated changes in gene expression and/ or function. However, it is becoming widely accepted that cancer cells also exhibit aberrant epigenetic alterations and that these changes can play a prominent role in disease initiation and progression. Epigenetic changes are potentially as important as genetic mutations in causing cancer since chromatin alterations can exert an influence regional gene expression, thereby changing the transcriptional profile of multiple genes. In this chapter we summarize the principal epigenetic alterations that occur in cancer cells: regional DNA hypermethylation and ▶histone modifications, and global DNA hypomethylation.

1. Last JM (1983) A dictionary of epidemiology. Oxford University Press, New York 2. Ferlay J, Bray F, Pisani P et al. (2004) Globocan 2002 – cancer incidence, mortality and prevalence worldwide. IARC CancerBase No. 5, version 2.0. Lyon, IARC Press

DNA Hypermethylation Chromatin structure is influenced by cytosine ▶methylation, the only known naturally occurring base

Cancer Epigenetics

modification in DNA. Cytosine methylation occurs at 5′-CG-3′ dinucleotides (referred to as CpGs) and is catalyzed by a class of enzymes termed ▶DNA methyltransferases (DNMTs). Several DNMTs have been characterized in mammalian cells including DNMT1, DNMT3a and DNMT3b. These enzymes catalyze the transfer of a methyl group from Sadenosylmethionine (SAM) to the 5-carbon position of cytosine, forming 5-methycytosine. DNMT3a and DNMT3b appear to be principally involved in methylating previously unmodified cytosines (termed de novo methylation). In contrast, DNMT1 preferentially methylates hemimethylated DNA and is thus viewed as the DNA methyltransferase principally responsible for continuation of DNA methylation patterns in daughter cells (termed maintenance methylation). From a statistical standpoint, the human genome is depleted in CpG dinucleotides; however, 60% of genes in our genome are associated with regions ranging from 200 to 4,000 bases in length containing high density of CpG dinucleotides relative to the bulk genome. These regions are referred to as ▶CpG islands and are usually located within upstream promoter regions or gene transcriptional start sites. In normal somatic cells, gene-associated CpG islands are usually unmethylated and associated with genes in a transcriptionally active euchromatic state. In cancer cells, hypermethylation of such CpG islands is strongly correlated with the transcriptional silencing of genes. Thus, through this epigenetic mechanism, tumor cells can dramatically down regulate expression of numerous genes, including ▶tumor suppressor genes (TSGs). At present, numerous TSGs have been characterized as targets for epigenetic silencing through hypermethylation of associated CpG islands. Table 1 is a partial listing of characterized TSG whose promoter regions have been shown to be hypermethylated in various tumor types. Given the wide spectrum of tumor types that display

Cancer Epigenetics. Table 1 Gene

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epigenetic silencing of TSGs, mounting evidence clearly supports the assertion that epigenetic silencing is a prominent mechanism driving the process of tumorigenesis. Cancer cells often over express DNMTs. Compared to normal tissues, the expression of DNMT1 is almost always increased in tumors. However, since DNMT1 expression is normally regulated during the cell cycle with increased abundance paralleling entry into S-phase, much of this increased expression may simply reflect increased cell proliferation within the tumor. Although demonstrable in model experiments, it remains unresolved if increased expression of DNMT1 is responsible for aberrant methylation in cancer cells. In contrast, increased expression of DNMT3a and DNMT3b observed in some tumors is likely significant, since these enzymes are normally expressed at low levels in somatic cells. However, it is still unclear to what extent over expression of these enzymes is responsible for cancer-associated DNA hypermethylation especially when one considers that cancer cells exhibit overall genome hypomethylation (discussed below). Thus, it remains unclear how CpG islands associated with specific TSG are targeted for hypermethylation during the process of tumorigenesis. One mechanism by which DNA methylation can negatively impact gene expression is by simply blocking the binding of essential transcription factors to gene promoter sequences. While several examples of this are documented, it is also apparent that CpG methylation is also capable of directing transcriptional repression through promoting additional layers of chromatin alteration. Specifically, several proteins have been characterized that bind to methylated CpG dinucleotides and are capable of promoting further chromatin condensation and consequential transcriptional repression through recruitment of chromatin-modifying activities.

Genes subject to epigenetic silencing in cancer Function

Tumor types

APC BRCA1 CDH1 (E-cadherin) MGMT MLH1 CDKN2A (p16)

Regulation of β-catenin, cell adhesion DNA repair Homotypic epithelial cell–cell adhesion

Colorectal, gastrointestinal Breast, ovarian Bladder, breast, colon, liver

DNA repair DNA repair Cell cycle control

PTEN VHL

Regulation of cell growth and apoptosis Inhibits angiogenesis, regulates transcription DNA damage response

Brain, colorectal, lung, head and neck Colorectal, endometrial, ovarian Lung, brain, breast, colon, bladder, melanoma prostate Prostate, brain, endometrial, melanoma Renal cell carcinoma

ATM

Breast, colorectal, head and neck

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Histone Modification Owing to technical considerations, DNA methylation is the most widely analyzed type of epigenetic alteration in human tumors. However, another extremely important epigenetic modification capable of altering gene expression during carcinogenesis involves various types of histone modifications. The fundamental packing unit of chromatin within the nucleus is termed the ▶nucleosome. A single nucleosome unit contains 146 base pairs of DNA wrapped around eight histone subunits (histone octamer). The histone octamer contains two copies each of histones H2A, H2B, H3 and H4. Structural studies have determined that each histone possesses an aminoterminal tail rich in the amino acid lysine. These lysine residues can undergo a variety of post-translational modifications including acetylation, methylation, phosphorylation, and ubiquitination. Such modifications are recognized by various proteins and protein complexes and combinations of histone modifications constitute a proposed histone code important in establishing a given gene’s transcriptional profile. Perhaps the best-studied histone modifications is acetylation of the ε-amino group of lysine residues within the amino-terminal tail of H3 and H4 although acetylation of both H2A and H2B occurs as well. This is a reversible modification that is carefully controlled by two large enzyme families: histone ▶acetyltransferases (HATs) and ▶histone deacetylases (HDACs). The net positive charges carried by these lysine residues are proposed to contribute to the high affinity of histones for negatively charged DNA. Acetylation of lysine residues by HATs neutralizes this positive charge thus decreasing histone/DNA interaction. This raises molecular access to DNA and promotes gene transcription. Conversely, HDACs promote transcriptional repression by supporting chromatin condensation into a heterochromatic conformation. Several proteins that bind specifically to methylated CpG through a conserved methyl-binding domain (MBD) motifs have been discovered. The first such protein to be characterized, termed MeCP2, is capable of recruiting the co-repressor molecule mSin3 to the sites of methylated DNA. In turn, mSin3 binds to HDAC1 and HDAC2, thus promoting localized histone deacetylation. The importance of HDAC activity in transcriptional repression is underscored by the observation that repression of several gene promoters that can be partially relieved by HDAC inhibitors. A functionally similar complex termed MeCP1 binds to methylated DNA via an associated protein termed MBD2. The MeCP1 complex contains multiple subunits besides MBD2, including components of the NuRD complex, a characterized repressor complex containing both chromatin remodeling and HDAC activities.

In addition to acetylation, lysine residues within histone tails can be methylated and exist in either mono-, di-, or trimethylated states. Similar to the effects of histone acetylation/deacetylation, methylated lysine 4 of H3 (H3K4) is associated with transcriptionally active chromatin, while transcriptionally silent chromatin generally contains methylated lysine 9 of H3 (H3K9). H3K9 is methylated by a number of histone methyltransferases including ESET, Eu-HMTase, G9a, and the closely related methyltransferases SUV39-H1 and SUV39-H2. Histone methylation is likely a dynamic process since a histone demethylase, termed LSD1, was recently characterized. Methylated H3K9 binds to the chromodomain protein heterochromatin protein 1 (HP1) which promotes heterochromatin formation and gene silencing. Moreover, since H3K9 methylation cannot occur when this position is acetylated, it is clear that H3K9 acetylation and methylation represent opposing forces in determining chromatin conformation. In a broad view, it is reasonable to propose that CpG methylation and histone acetylation/deacetylation act synergistically in the progressive silencing of genes. One model that accounts for tumor-suppressor gene silencing by epigenetic mechanisms invoke abnormal hypermethylation of the promoter CpG island followed by recruitment of MBD proteins, including complexes such as MeCP2 and MeCP1 that recruit HDACs to the area of hypermethylation and promote further transcriptional repression through histone modification. An alternate model proposes that transcriptionally repressive histone modifications are the first event in gene silencing and subsequently promote CpG methylation resulting in further transcriptional repression. Experimental evidence supports both of these models and may be reflective of the variety of gene promoters and model systems used for study. Less equivocal is the fact that DNA methylation appears the dominant silencing mechanism since inhibition of DNA methylation generally restores gene expression while HDAC inhibitors generally exert more modest effects on gene silencing. DNA Hypomethylation While CpG islands associated with gene promoters are generally unmethylated in normal adult somatic cells, the majority of CpG dinucleotides elsewhere in the genome are generally methylated. Moreover, despite the fact that many CpG islands are subject to hypermethylation in cancer cells, it is equally well documented that tumor cells display an overall loss of methylated cytosines compared to normal tissue. This tumor-associated global DNA hypomethylation predominantly occurs in repetitive DNA sequences within the human genome although the molecular mechanisms responsible for this loss of DNA methylation are poorly understood.

Cancer Genome Project

Recent work on a human genetic disorder has underscored an important role for DNA methylation in maintenance of genome stability. The immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disease characterized by germline mutation of the DNMT3B gene. Loss of DNMT3B activity in ICF leads to hypomethylation of repetitive satellite DNA sequences within heterochromatin adjacent to the centromeric region of chromosomes. The loss of methylation is most prominent within the pericentric regions of human chromosomes 1 and 16 and leads to multiple chromosomal abnormalities including chromatin decondensation, chromosomal translocations and deletion, and multiradial chromosomal structures. These observations, as well as those made on cells with engineered disruption of DNMTs, clearly support the view that DNA methylation is critical in maintaining normal chromosome structure. Since cancer cells often show chromosomal rearrangements, it is likely that cancer-associated DNA hypomethylation allows for heightened rates of chromosomal instability. Retrotransposon sequences of the LINE (long interspersed nuclear element) and SINE (short interspersed nuclear element) classes as well as human endogenous retroviruses (HERVs) are major targets of tumorassociated DNA hypomethylation. Mobility of these DNA elements is kept in check in normal tissues owing, in part, to dense methylation of CpG dinucleotides within their genomic structure. It follows that increased mobility of these dormant mobile elements occurs as a result of cancer-associated DNA hypomethylation and there have been reports of retrotranspositionlike insertions involving LINE-1 sequences in tumors although retrotransposition of endogenous elements seemingly occurs more often in rodents than humans. In addition to the hypomethylation of CpG dinucleotides present within repetitive DNA elements, cancer-associated hypomethylation also occurs in regions of the genome encoding single-copy genes. Dysregulation of allele-specific methylation will result in the loss of imprinting (LOI) and allow for both maternal and paternal gene expression. Perhaps the best-studied example of this is the insulin-like growth factor 2 (IGF2) gene where LOI occurs in primary tumors and in patients with the inherited, cancer-prone ▶Beck-with-Wiedemann. While not as well-studied as gene silencing due to DNA hypermethylation, it is likely that additional examples of cancer-promoting increased gene expression stemming from DNA hypomethylation will be uncovered in the future. Epigenetic Alterations as Targets for Diagnosis and Therapeutic Intervention Sequencing data obtained from the human genome project are currently undergoing analysis to construct a

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human epigenetic map based on CpG content. This knowledge coupled with cross-species comparisons of the epigenome will be invaluable in deciphering the epigenetic elements involved in gene regulation. Epigenetic alterations typically occur early during the oncogenic process, and detection of such early abnormalities may aid in early diagnosis and/or preventing cancer progression through dietary alterations or pharmacological intervention. With increasing awareness of the importance of epigenetics in tumorigenesis, and the advent of sensitive laboratory approaches to analyze epigenetic alterations, it is likely that epigenetic profiles will ultimately be used in the clinical setting to provide information useful in predicting an individual’s predisposition to cancer, assisting in tumor staging, and guiding optimal therapeutic approaches. A promising feature of alterations in DNA methylation patterns and chromatin structure in cancer cells is their potential for reversibility, because these modifications occur without changing the primary nucleotide sequence. At present, two major pharmacological targets associated with these epigenetic changes are DNMTs and HDACs. The DNMT inhibitor 5-azadeoxycytidine (5-azadC) and related compounds cause transcriptional reactivation of endogenous genes with hypermethylated promoters. This drug, also termed Decitabine, is currently used to treat certain types of hematological malignancies, especially advanced ▶myelodysplastic syndromes (MDS). HDAC inhibitors, such as trichostatin A and sodium butyrate, have been shown to increase the level of histone acetylation in cultured cells, and to cause growth arrest, differentiation and apoptosis. Based on these observations, the potent HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) is in clinical trials.

Cancer Genome Project Definition The Cancer Genome Project (CGP) was founded at the Wellcome Trust Sanger Institute in 2000 and aims to establish an unbiased catalogue of mutations involved in human tumorigenesis by complete sequencing of candidate genes. By sequencing the human BRAF gene, the CGP discovered a hitherto unknown mechanism of oncogenic activation of B-Raf that is not only found in 7% of human cancers, but also provided more insight into the complex process of B-Raf activation. ▶B-Raf Somatic Alterations

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Cancer Germline Antigens

Cancer Germline Antigens A DAM R. K ARPF Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA

Synonyms Cancer-testis (CT) antigens; CG antigens

Definition

CG ▶antigens (Cancer-testis (CT) antigens) are a class of immunogenic tumor antigens encoded by genes expressed in ▶gametogenic cells of the testis and/or ovary and in human cancer.

Characteristics Identification The main criterion for classification of a gene as a CG antigen pertains to its expression pattern in gametogenic, somatic, and tumor tissues. A gene is generally considered to be a CG antigen if it is expressed in the gametogenic cells of the testis or ovary (including fetal ovary) and in some proportion of human cancers, but is expressed in two or fewer normal somatic tissues. CG antigen genes are also commonly expressed in ▶trophoblast tissue. CG antigens were originally identified from searches for auto-antigens expressed in human cancer. The original method for antigen screening used ▶autologous typing, in which ▶T-cells (T-lymphocyte) from a ▶melanoma patient were screened for reactivity with tumor cells from the same patient; this method led to the identification of MAGE-A (named for ▶melanoma antigen)/CT1 genes. In later studies, another immunological assay was developed to identify tumor antigens and this method, ▶SEREX (Serological analysis of recombinant cDNA expression libraries), was used to successfully identify a variety of important CG antigen genes, including NY-ESO-1/CT6 and SSX/CT5. Recognition of the unique expression pattern of CG antigen genes has led to the use of gene expression analyses (including EST or SAGE database searching) to identify CG antigen genes. This method has led to the identification of additional CG genes, including XAGE-1/CT12 and SCP-1/CT8. Although formally classified as CG antigen genes, genes identified by the latter non-immunological method may not be antigenic in cancer patients. Nomenclature A nomenclature system for CG antigens has been devised, which is based on their chronology of discovery, and also accounts for the numerous family

members that exist for certain CG antigens. In this system, CG antigen genes are referred to by their original given names and also are assigned a separate CT identifier or CT#. Currently, over 40 CG antigen gene families are recognized, comprising more than 89 distinct mRNA transcripts. CG antigen genes have been assigned into two groups on the basis of chromosomal localization. ▶CG-X antigens: These genes reside on the X-chromosome where, interestingly, close to 10% of the total number of genes encode CG antigens. CG-X genes are typically members of large multigene families, e.g. MAGE-A/CT1, MAGE-B/CT3, and MAGE-C/CT7. In normal tissues, CG-X genes are often expressed in pre-meiotic spermatocytes in the testis. All of the current important targets of CG antigen ▶cancer vaccines are members of this group, including MAGE-A1/CT1.1, MAGE-A3/CT1.3, and NY-ESO-1/CT6.1. ▶Non-X CG antigens: A number of CG antigen genes are located on autosomal chromosomes. Unlike CG-X genes, these genes are highly dispersed in the genome and do not exist in multigene families. In normal tissues, non-X CG genes are often expressed during meiosis, where some members play roles in DNA recombination, including SCP-1/CT8 and SPO11/CT35. The members of this gene group do not include any currently validated cancer antigens, although certain members are expressed at high levels in cancer.

Regulation of Expression Certain cancer types appear to expresses CG antigen genes frequently, while others rarely express them. Tumor types that frequently express CG antigens include melanoma, lung, ovary, and ▶bladder cancer; tumors that rarely express CG antigens include ▶colon cancer, renal cancer, and leukemia/lymphoma. CG antigen genes show coordinate expression in human cancer. That is, the great majority of tumors either do not express CG antigen genes or express two or more CG antigen genes simultaneously, while relatively few tumors express only one CG antigen gene. Another characteristic of CG antigen gene expression in cancer (revealed by immunohistochemical staining) is that tumors that express CG antigens show heterogeneous expression within the tumor: often only focal staining is observed. The coordinate but heterogeneous expression of CG antigens in cancer has led to the intriguing hypothesis that CG antigen expression is indicative of the activation of a normally dormant gametogenic program in tumor cells (possibly corresponding to tumor stem cells). The observation of coordinate expression of CG antigen genes suggests that CG antigen gene activation may be controlled by a common molecular mechanism. Supporting this idea, a number of studies

Cancer Germline Antigens

have suggested a key role for ▶DNA methylation in regulating CG antigen gene expression. Promoter DNA hypermethylation has been observed to correlate with CG antigen gene repression in normal tissues and nonexpressing tumors, while treatment of tumor cell lines in vitro with DNA methyltransferase inhibitors such as ▶5-aza-2′-deoxycytidine (DAC) leads to CG antigen gene activation, coincident with promoter DNA ▶hypomethylation. Conversely, tumor cell lines and tissues that endogenously express CG antigen genes often display promoter DNA hypomethylation. Many CG antigen genes have CpG-rich promoter regions that serve as targets for regulation by DNA methylation. Other studies have shown that ▶histone deacetylase (HDAC) inhibitors can either augment DAC-mediated CG antigen gene activation or can activate CG antigen genes on their own. As DNA ▶methylation and ▶chromatin structure (in the form of histone modification status) are intimately linked, it is not surprising that both of these ▶epigenetic mechanisms serve as important regulators of CG antigen gene expression. Consistent with the model that epigenetic mechanisms regulate CG antigen gene expression is the observation that DNA hypomethylation occurs during gametogenesis, which is the normal setting for CG antigen gene expression.

Function CG antigens are a rare group of genes in that clinical studies designed to target these antigens for ▶immunotherapy of cancer are more advanced than is our basic knowledge of the function of the gene products. However, some information about CG antigen gene function has recently come to light. As mentioned earlier, many non-X CG antigens have roles in germ cell maturation, including mediating the structure of synaptonemal complexes (SCP1/CT8), facilitating DNA recombination during meiosis (SPO11/CT35), and contributing to spermatid function (ADAM2/CT15, OY-TES-1/CT23). In tumors, the function of CG antigen genes is less clear, but recent studies of the MAGE-type antigens, which share a region referred to as the MAGE homology domain (MHD), indicate that these proteins might serve as transcriptional repressors via interactions with other transcriptional regulatory proteins that themselves recruit co-repressors such as HDACs. CG antigen genes have also been reported to play a role in the evolution of ▶chemotherapy resistance in cancer cell lines, suggesting that the CG antigen gene products could serve as viable targets for anticancer therapy. An recent report appears to link these two observations by showing that MAGEA2/CT1.2 disrupts ▶p53 function by recruiting HDAC3 to p53, leading to chemotherapy resistance in cancer cells.

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Clinical Studies The identification of CG antigens as tumor-specific antigens has led to a great deal of interest in treating cancer by targeting CG antigens via vaccine-based immunotherapy. In particular, MAGE-A1/CT1.1, MAGE-A3/CT1.3, and NY-ESO-1/CT6.1 have been developed as targets for this approach. In early studies, the antigenic peptides from CG antigens that elicited ▶T-cell dependent responses were mapped, and these peptides were utilized for vaccination. Because responses to peptide-based vaccine formulations are limited by patient ▶HLA type, more recent vaccination approaches targeting CG antigens have utilized full-length ▶recombinant proteins. These recombinant proteins can be introduced using viral vectors, including vaccinia and fowlpox viruses. Alternatively, recombinant CG antigen proteins can be assembled with ▶adjuvants such as ISOMATRIX, which further enhances immune responses. A common finding in CG antigen vaccine clinical studies is that the treatment is safe and elicits both ▶antibody and T-cell mediated immune responses in vivo. In particular, NY-ESO-1/CT6.1 vaccine trials have shown encouraging results, with durable and multifaceted immune responses, as well as suggestive data indicating clinical benefit, in terms of disease stabilization and prolonged time to recurrence. Many of the patients targeted in these clinical trials have had malignant melanoma, and a proportion of these patients displayed evidence of immune recognition to the target antigen prior to vaccine therapy. In virtually all cases, patients have been selected for inclusion in CG antigen vaccine trials based on the expression of the antigenic target in tumor biopsies. To expand the patient population that would benefit from this immunotherapy approach, a number of investigators have proposed using DNA methyltransferase and/or HDAC inhibitors (which are FDA approved and known to augment CG antigen gene expression) in combination with CG antigen directed vaccines. The potential benefit of this multi-modality approach awaits clinical testing.

References 1. Simpson AJG, Caballero OL, Jungbluth A et al. (2005) Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 5:615–625 2. Scanlan MJ, Simpson AJG, Old LJ (2004) The cancer/ testis genes: review, standardization, and commentary. Cancer Immun 4:1–15 3. Scanlan MJ, Gure AO, Jungbluth AA et al. (2002) Cancer/ testis antigens: an expanding family of targets for cancer immunotherapy. Immunol Rev 188:22–32 4. Davis ID, Chen W, Jackson H et al. (2004) Recombinant NY-ESO-1 protein with ISOMATRIX adjuvant induces broad integrated antibody and CD4+ and CD8+ T cell responses in humans. Proc Natl Acad Sci USA 101:10697–10702

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Cancer of the Large Intestine ▶Colon Cancer

Cancer-mediated Bone Loss ▶Bone Loss, Cancer Mediated

Cancer Networks

cancer registries typically contain demographic and clinical characteristics. ▶Cancer Epidemiology

Cancer Stem Cells Definition A minor population of cells within a tumor that is thought to be necessary for tumor growth and propagation. Like all stem cells, cancer stem cells are characterized by their capacity for self renewal and unlimited replication. ▶Stem Cells and Cancer

Definition Local management teams, responsible for population areas of about 500,000, whose role is to co-ordinate cancer service within the National Health Service in England and Wales. ▶National Institute for Health and Clinical Excellence (NICE)

Cancer Stem-Like Cells G AETANO F INOCCHIARO Istituto Nazionale Neurologico Besta, Unit of Experimental Neuro-Oncology, Milano, Italy

Synonyms

Cancer-Prone Genetically Modified Mouse Models Definition Transgenic or knockout mice prone to cancer development as a consequence of ▶oncogene activation or ▶tumor suppressor gene inactivation. ▶Oncomouse ▶Immunoprevention of Cancer

Cancer Registry Definition System of on-going registration of all newly diagnosed cases of cancer in a given population. The files of

Tumor initiating cells

Definition Cancer stem(-like) cells are those cells that possess the capacity for self-renewal and for causing the heterogeneous lineages of cancer cells that comprise the tumor.

Characteristics The definition follows a consensus at a workshop on cancer stem(-like) cells (CSC) organized by the American Association for Cancer Research (AACR). There is considerable debate and some controversy on the CSC concept, so that a consensus definition is required. The importance of the debate is proportional to its relevance to the change in our perception of cancer, intirinsic to the CSC paradigm, implying that not all cancer cells are equal but that only a small fraction of them is endowed with the properties of perpetuating the disease. This hierarchical model has not only important biological consequences but also relevant therapeutic implications, as we discuss in this essay.

Cancer Stem-Like Cells

The CSC paradigm fits in a model of cancer as a caricature of an organ that is already present in the literature as suggested by data published 30 to 40 years ago. In particular, Hamburger and Salmon established growth conditions for cancer cells in soft agar medium and found that tumor stem cell colonies, arising from different types of cancer with 0.001–0.1% efficiency, had differing growth characteristics and colony morphology. Studies by Dick and co-workers in the 1990s showed that in several forms of acute myeloid leukemia (AML) cells that could engraft in immunodeficient mice are restricted to a minority subpopulation defined as [CD34+/CD38neg]: these cells, therefore, shared a cell surface phenotype with normal human primitive hematopoietic progenitors, suggesting that they may have originated from normal stem cells rather than from committed progenitors. Also of interest was the observation that leukemic cells engrafted in NODSCID mice (non obese diabetic-severe combined immunodeficiency: an immunodeficient mouse strain characterized by lack of B, T and NK lymphocytes) showed similar phenotypic heterogeneity to the original donor: thus, [CD34+, CD38neg] retain the differentiating capacity necessary to give rise to CD38+ and Lin+ cells (lineage positive). The presence of CSC has also been demonstrated in chronic myeloid leukemia (CML). This disease has a chronic phase and a terminal stage; the blast crisis and molecular events underlying this evolution are not completely understood. In the chronic phase, the chromosomal translocation t(9:22)BCR-ABL, a diagnostic marker of CML, can be detected in most circulating mature lineages. In the blast crisis, however, highly undifferentiated BCR-ABL+ cells accumulate in the blood. In particular, an expansion of granulocytemacrophage progenitors (GMP) is present in blast cells, showing aberrant acquisition of self-renewal properties and nuclear expression (i.e. activation) of beta-catenin, a key, positive regulator of stem cell self-renewal. These observations imply that during progression of CML the GMP subfraction of leukemic progenitors acquire stem

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cell characteristics. Thus, the functional hierarchy of CSC can be modified during the natural history of this tumor as a result of its progression. The requirement for a periodical renovation is not only present in blood but also in the skin and epithelia of the respiratory, gastrointestinal, reproductive and genito-urinary systems. Other tissues like brain, previously considered as exclusively post-mitotic, contain stem cells that can be mobilized and activated under conditions of stress, such as hypoxia. Thus the CSC model could also be applied to solid tumors, and a series of recent papers report data supporting the identification of a stem cell population in different cancers (see Table 1). Initial data were gained in breast cancer where a small population of cells with a CD44+ /CD24 neg-low phenotype appears exclusively capable of tumor initiation. The most malignant of brain tumors, glioblastoma multiforme (GBM), was also found to contain a fraction of neoplastic cells identified and selected on the basis of CD133 expression. Not only could CD133+ cells self-renew and differentiate into different neural lineages but also, in vivo, only the CD133+ cells were able to reinitiate malignant gliomas with phenotype similar to the original tumor. The CSC paradigm may also help to explain intratumor heterogeneity, a frequent finding in most cancers: heterogeneity could be consequent to functional diversity of cells at different states of differentiation. On the other hand, the patterns of tumor heterogeneity and gene expression profiles can be highly similar in the original tumor and in distant metastasis. It is rather obvious that the existence of cancer stem cells may provide novel therapeutic targets of increased effectiveness in contrasting or even eliminating cancer. Brain tumors have provided a highly fertile ground to start verifying this hypothesis, as outlined in Table 2. Data are piling up indicating that CD133+ GBM CSC are highly pro-angiogenic, because of the high levels of VEGF expression, and have greater resistance to chemotherapy and radiotherapy. As a consequence,

Cancer Stem-Like Cells. Table 1 Tumor Acute Myeloid Leukemia Breast cancer Glioblastoma Myeloma Prostate cancer Melanoma Lung cancer Colon cancer Pancreatic cancer

Markers

Reference

CD34+/CD38neg CD44+/CD24-/neg CD133+ CD138neg CD44+/alpha2beta1 integrin high/ CD133+ CD20+ Sca-1+/ CD45neg/ Pecam neg/CD34pos CD133+ CD44+/CD24+/ESA (epithelial specific antigen)+

Bonnet and Dick 1997 Al-Hajj et al 2003 Singh et al 2003 Matsui et al 2004 Collins et al 2005 Fang et al 2005 Kim et al 2005 O’Brien et al 2007, Ricci-Vitiani et al 2007 Li et al 2007

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Cancer Stem-Like Cells. Table 2 Pathway/mechanism in CSC Angiogenesis-Increased production of VEGF Increased resistance to radiation Specific patterns of expression Cell cycle deregulation Resistance to chemotherapy

Potential treatment

Reference

Bevacizumab Chk1 and Chk2 inhibitor Dendritic cell targeting Bone Morphogenetic Protein 4 BCRP1/MGMT inhibition (?)

Bao et al Cancer Res 2006 Bao et al Nature 2006 Pellegatta et al 2006 Piccirillo et al 2006 Liu et al 2006

specific therapeutic strategies can be attempted and combined to overcome CSC. Upon radiotherapy CD133+ GBM CSC activate checkpoint kinases 1 and 2 and repair mechanisms more effectively than CD133neg cells. Resistance to chemotherapy can be linked to an intriguing aspect of the CSC phenotype, the side population (SP) phenotype. SP cells have the ability to extrude the DNA binding dye Hoechst 33342 via the drug transporter BCRP1/ABCG2. Interestingly, the BCRP1/ABCG2 pump can also effectively extrude chemotherapeutic drugs such as mitoxantrone. Also related, although of less immediate relevance in the clinical setting, are the observations reported by Pellegatta et al. using glioma neurospheres as a target for dendritic cell (DC, the most potent of antigen presenting cells) immunotherapy. Normal neural stem cells may grow as neurospheres (NS) in the absence of serum and in the presence of two critical growth factors, EGF and bFGF. NS are enriched in neural stem cells but also contain partially committed progenitors as well as a differentiated progeny. Oncospheres with similar characteristics were obtained from GBM but also from other solid tumors like breast or colon carcinomas. Pellegatta et al set up a murine model showing that DC loaded with GBM NS are much more effective in protecting mice against the GBM challenge than DC loaded with GBM cells where CSC are poorly represented. Thus, CSC targeting by immunotherapy is feasible and highly effective, opening new scenarios for clinical immunotherapy and supporting the idea that CSC are at the heart of malignant growth. Also of interest is the observation by Piccirillo et al. that treating GBM CSC with the differentiating factor BMP4 can block growth in vitro and avoid tumor formation in the majority of mice in vivo. Given the increasing number of observations supporting the CSC paradigm in different tumors, it is expected that more therapeutically relevant observations will be proposed in the near future. Together with therapeutic and clinical implications the CSC concept seems to have important consequences for our understanding of tumor biology. Modern genetics and molecular biology have given a definition of cancer as a genetic disease in which a growing

burden of mutations leads to a progressively more aggressive and ultimately lethal phenotype (Fig. 1). A Darwinian selection for these mutations, privileging those that can confer resistance to different challenges, like hypoxic stress or immune attack, appears to be the most plausible rationale for making sense of this evolutionary catastrophe. The hierarchical, CSC model seems to introduce an element of rigidity in this highly flexible scenario, implying that only cells endowed with stem cell properties can afford tumor perpetuation (Fig. 1). Are these two models different or are they compatible? A convincing answer to this tough question will undoubtedly require a lot of robust science in the time to come but comments can be given on the basis of data that are already available. One important issue that the CSC model addresses is that of the cell of origin for cancer(s): stem cells, because they are long-lived and self-renewing, are excellent candidates to play the “cell of origin” role. A stem cell hosting a critical mutation could be quiescent for years and then be engaged in a repair response requiring mobilization and proliferation. For example, hypoxic stress may activate the CXCR4 pathway that not only attracts stem cells but may also favour their proliferation, thus being the spark initiating the cancer fire. However, an initiating mutation could also arise in a more committed progenitor (see the integrated model in Fig. 1): acquisition of a stem-like phenotype could in this context be the consequence of environmental challenges; in vivo, for instance, hypoxia could play an important role in de-differentiation; in vitro, the modification of growth factors could have similar consequences. Epigenetic changes could play important roles in mediating rapid and genome-wide changes that can substitute for genetic mutations and lead to dedifferentiation. In the Darwinian model different mutations (M1 through M4) accumulate during evolution and confer heterogeneity. In the Hierachical model tumor arises in a stem cell, thus becoming a cancer stem cell (CSC): heterogeneity is conferred by asymmetrical divisions creating different types of cancer cells (CC1 through 4). In the Integrated model a first mutation (M1) can arise in a progenitor or even a committed cell. During progression, though, external stimuli may give rise

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Cancer Stem-Like Cells. Figure 1 Biological models for tumor evolution.

to a cancer stem cell that through asymmetric division will create other CSC as well as more differentiated tumor cells. ▶Stem-Like Cancer Cells

undetectable expression in normal tissues except germ cell. ▶BORIS

References 1. Clarke MF, Dick JE, Dirks PB et al. (2006) Cancer stem cells – perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res 66:9339–9344 2. Dalerba P, Cho RW, Clarke MF (2007) Annu Rev Med 58:267–284 3. Jamieson CH, Ailles LE, Dylla SJ et al. (2004) Granulocytemacrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 351:657–667 4. Sanai N, Alvarez-Buylla A, Berger MS (2005) Neural stem cells and the origin of gliomas. N Engl J Med 353:811–822 5. Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33

Cancer (or Tumor) Stroma ▶Tumor Microenvironment

Cancer Vaccines M ALAYA B HATTACHARYA-C HATTERJEE 1 , S UNIL K. C HATTERJEE 1 , A SIM S AHA 1 , K ENNETH A. F OON 2 1

Cancer Testis Antigens

University of Cincinnati and The Barrett Cancer Center, Cincinnati, OH, USA 2 The Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Definition

Definition

Cancer Testis Antigens are genes that can be defined by predominant expression in various types of cancer and

A vaccine should activate a unique lymphocyte (B and/ or T cell) response, which has an immediate anti-tumor

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Cancer Vaccines. Figure 1 T-cell activation. T-cells recognize antigens as fragments of proteins (peptides) presented with major histocompatibility complex (MHC) molecules on the surface of cells. The antigen presenting cell processes exogenous protein from the vaccine or from the lysed tumor cell in to a peptide, and presents the 14/25 mer peptide to CD4 helper-T-cells on a class II molecule. There is also data that suggests that exogenous proteins can be processed into 9/10 mer peptides that may be presented on MHC class I molecules to CD8 cytotoxic T-cells. Activated Th1 CD4 helper T-cells secrete Th1 cytokines such as IL-2 that upregulate CD8 cytotoxic T-cells. Activated Th2 CD4 helper T-cells secrete Th2 cytokines such as IL-4, IL-5 and IL-10 that activate B cells.

effect as well as memory response against future tumor challenge (Fig. 1). The primary role of a cancer vaccine is the treatment of cancer or in prevention of recurrence in a patient with surgically resected cancer, rather than “prevention” of cancer in a person who has never had cancer. Therefore, cancer vaccines are not thought of in the traditional sense of vaccines that are used for infectious diseases. If the current cancer vaccines prove to be useful in the above respects, then they may have a future role in preventing cancer in persons who have never had cancer but are at high-risk for a particular type of cancer.

Characteristics The first and most obvious types of vaccines are prepared from autologous or allogeneic tumor cells. Alternatively, membrane preparations from tumor cells may be used. In some instances, tumor cell vaccines have been combined with cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-2 (IL-2). More recently, with advances in molecular biological approaches, gene modified-tumor cells expressing antigens designed to increase the immune response, or gene modified to secrete cytokines have been an additional tool used in vaccination. In addition, increase in our knowledge of tumor associated antigens (TAA) have led to the use of purified TAAs, DNA-encoding protein antigens, and/or protein derived peptides. All of these approaches are currently being tested in the clinic.

Mechanistically, the ultimate aim of a vaccine is to activate a component of the immune system such as B lymphocytes, which produce antibodies or T lymphocytes, which directly kill tumor cells. Antibodies must recognize antigens in the native protein state on the cell’s surface. Once bound, these molecules can mediate antibody-dependent cellular cytotoxicity or complementmediated cytotoxicity, both mechanisms which are capable of destroying tumor cells. T lymphocytes, on the other hand, recognize proteins as fragments or peptides that vary in size, presented in the context of major histocompatibility (MHC) antigens on the surface of the cells recognized (Fig. 1). The proteins from which the peptides are derived may be cell surface or cytoplasmic proteins. MHC antigens are highly polymorphic, and different alleles have distinct peptide binding capabilities. The sequencing of peptides derived from MHC molecules have led to the discovery of allele-specific motifs that correspond to anchor residues that fit into specific pockets on MHC class I or II molecules. T Lymphocytes There are two types of T lymphocytes, helper T lymphocytes and cytotoxic T lymphocytes (CTLs) that recognize antigens through a specific T cell receptor (TCR) in close conjunction to the CD3 molecules, which is responsible for signaling. CD4 helper T cells recognize antigens in association with class II MHC gene products, and CD8 positive CTLs recognize

Cancer Vaccines

antigens in association with class I MHC gene products. CD4 helper T cells are activated by binding via their TCR to class II molecules that contain 14–25 amino acid peptides in their antigen-binding cleft. Specialized antigen presenting cells (APCs), such as dendritic cells (DCs), macrophages, and B lymphocytes, capture extracellular protein antigens, internalize and process them, and display class II-associated peptides to CD4 helper T cells. The CD8 positive CTLs are activated by binding via their TCR to class I molecules that contain 9–10 amino acid peptides in their antigen-binding cleft. All nucleated cells can present class I-associated peptides, derived from cytosolic proteins such as viral and tumor antigens, to CD8 positive T cells. There are two types of CD4 helper T cells capable of generating either antibody or cell-mediated immune responses, based on the type of signaling they receive. Th1 CD4 helper T cells stimulate cell-mediated immunity by activating CTLs through the release of cytokines such as IL-2. Th2 CD4 helper T cells mediate an antibody response through the release of cytokines such as IL-4 and IL-10. Tumor Cells The most straightforward means of immunization is the use of whole tumor cell preparations (either autologous or allogeneic tumor cells). The advantage of this approach is that the potential TAAs are presented to the immune system for processing and presentation to the appropriate T cell precursors. The difficulty with this approach lies in the availability of fresh autologous tumor material and the sparcity of well-characterized long-term tumor cell lines. Regardless, whole tumor cell vaccines have been an area of intense interest. A variety of trials using autologous tumors for colon cancer and malignant melanoma have been reported. In one trial, freshly thawed autologous colon cancer cells were inactivated with radiation, mixed with ▶BCG (bacille Calmette-Guerin) and injected into patients who had their primary colon cancer resected but were at risk for recurrence. This study did reveal disease-free survival and overall survival trends in favor of the vaccine arm. In a melanoma study, autologous tumor cells were mixed with dinitrophenyl (DNP) and mixed with BCG. Promising results were reported for patients with metastatic disease and for patients with locally resected melanoma. The weakness of autologous cell vaccines can be overcome with the allogeneic approach: First an allogeneic vaccine is generic and developed from cell lines selected to provide multiple TAAs and a broad range of HLA expression. Second, allogeneic cells are more immunogeneic than autologous cells. Third there is no requirement to obtain tumor tissue by surgical resection for a prolonged course of immunotherapy.

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A polyvalent melanoma cell vaccine called CancerVax developed for allogeneic viable melanoma cell lines has demonstrated promising results for patients with resected metastatic disease and for resected local disease. Randomized phase III studies are ongoing in the United States comparing CancerVax plus BCG versus BCG for patients with stage III melanoma. Another variation of cell vaccines is using “shed” antigen vaccines. These are vaccines that are prepared from the material shed by viable tumor cells into culture medium. The potential advantage is that it contains a broad range of antigens expressed on the surface of melanoma cells and the shed antigens are partially purified. Trials of such vaccines in melanoma patients have demonstrated specific humoral and cellular immune responses in patients and promising early clinical results. Another approach to tumor cell vaccines is the introduction of foreign genes encoding cytokines such as IL-2 and GM-CSF into tumor cells. Alternatively, molecules designed to increase the immunogenicity of the tumor cell such as CD80 and CD86. Gene transfer can be accomplished by transfection of plasmid constructs (electroporation) or transduction using a viral vehicle such as a retrovirus or an adenovirus. Another option tested for gene transfer is physical gene delivery in which a plasmid or “naked” DNA is delivered directly into tumor cells. There are a number of mechanisms to carry this out including liposomes as gene carriers, use of a “gene gun,” electroporation and calcium phosphate-mediated gene transfer. In one phase I trial, 21 patients with metastatic melanoma were vaccinated with irradiated autologous melanoma cells engineered to secrete human GM-CSF. Metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction. Peptides and Carbohydrates An advantage to peptide vaccines is that they can be synthetically generated in a reproducible fashion. The major disadvantage is that they are restricted to a single HLA molecule and are not of themselves very immunogenic. To increase their immunogenicity, peptides may be injected with adjuvants, cytokines or liposomes or presented on DCs. Whole proteins have the advantage over peptides in that they can be processed for a wider range of MHC class I and II antigens. Mucins such as MUC I are heavily glycosylated high molecular weight proteins abundantly expressed on human cancers of epithelial origin. The MUC I gene is over-expressed and aberrantly glycosylated in a variety of cancers including colorectal cancer. MUC 1 is being widely used as a focus for vaccine development. Using expression-cloning techniques, several groups have cloned the genes encoding melanoma antigens

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recognized by T cells and have identified the immunogenic epitopes presented on HLA molecules. Ten different melanoma antigens have been identified. Direct immunization using the immunodominant peptides from the tumor antigens or recombinant viruses such as adenovirus, fowlpox and vaccinia virus encoding the relevant genes have been pursued to immunize patients with advanced melanoma. Initial results have demonstrated increased anti-tumor T cell reactivity in patients receiving peptide immunization. Immunization in melanoma patients with melanoma antigens have been reported. One study showed that immunization of melanoma patients with MAGE-1 peptide pulsed on DCs induced melanoma-reactive and peptide specific CTL responses at the vaccination sites and at distant tumor deposits. Administration of the gp-100 molecule in conjunction with high-dose bolus IL-2 to 31 patients with metastatic melanoma revealed an objective response of 42%. This is compared with the typical response of high-dose systemic IL-2 without peptide of only 15%. Based on these data, a randomized trial was initiated to compare the peptide vaccine plus IL-2 versus IL-2 alone in metastatic melanoma patients. Immunization against tumor-associated carbohydrate antigens has also been attempted. Carbohydrate antigens typically bypass T cell help for B cell activation. Investigators demonstrated that some carbohydrates may activate an alternative T cell pathway. Vaccine studies have been reported using the GM-2 ganglioside vaccine. Patients were pretreated with low dose cyclophosphamide. After a minimum follow up of 72 months, there was a 23% increase in disease-free interval and a 17% increase in overall survival in patients who produced antibody against GM-2. This suggested a benefit to the GM-2 ganglioside vaccine which has led to a current phase III trial. Recombinant Vaccines Expressing Tumor Antigens The carcinoembryonic antigen (CEA) is highly expressed on colorectal cancer and on a variety of other epithelial tumors, and is thought to be involved in cell-cell interactions. A recombinant vaccinia virus expressing human CEA (rV-CEA) stimulates specific T cell responses in patients. This was the first vaccine to demonstrate human CTL responses to specific CEA epitopes and class I HLA-2 restricted T cell mediated lysis, and demonstrated the ability of human tumor cells to endogenously process CEA to present a specific CEA peptide in the context of a MHC for T-cell mediated lysis. Anti-idiotype Vaccines The idiotype network offers an elegant approach to transforming epitope structures into idiotypic determinants expressed on the surface of antibodies. According

to the network concept, immunization with a given TAA will generate production of antibodies against these TAA, which are termed Ab1; the Ab1 is then used to generate a series of anti-idiotype antibodies against the Ab1, termed Ab2. Some of these Ab2 molecules can effectively mimic the three-dimensional structure of the TAA identified by the Ab1. These Ab2 can induce specific immune responses similar to those induced by the original TAA and therefore can be used as surrogate TAAs. Immunization with Ab2 can lead to the generation of anti-anti-idiotypic antibodies (Ab3) that recognize the corresponding original tumorassociated antigen identified by Ab1. The anti-idiotype antibody represents an exogenous protein that should be endocytosed by APCs and degraded to 14–25 mer peptides to be presented by class II antigens to activate CD4-helper T cells. Activated Th2 CD4-helper T cells secrete cytokines such as IL-4 that stimulate B cells that have been directly activated by Ab2 to produce antibody that binds to the original antigen identified by Ab1. In addition, activation of Th1 CD4-helper T cells secrete cytokines that activate T cells, macrophages and natural killer cells that directly lyse tumor cells, and in addition, contribute to ADCC. Th1 cytokines such IL-2 also contribute to the activation of a CD8CTL response. This represents a putative pathway of endocytosed anti-idiotype antibody. The anti-idiotype antibody may be degraded to 9/10 mer peptides to present in the context of class I antigens to activate CD8-cytotoxic T cells, which are also stimulated by IL-2 from Th1 CD4-helper T cells. Anti-idiotype antibodies that mimic distinct TAAs expressed by cancer cells of different histology have been used to implement active specific immunotherapy in patients with malignant diseases including colorectal carcinoma, malignant melanoma, breast cancer, B cell lymphoma and leukemia, ovarian cancer, or lung cancer. A murine monoclonal anti-idiotype antibody, 3H1 or CeaVac, which mimics CEA was developed by the authors and was used in a phase I clinical trial. Among 23 patients with advanced colorectal cancer, 17 patients generated anti-anti-idiotypic Ab3 responses, and 13 of these responses were proven to be true anti-CEA responses. The median survival of 23 evaluable patients was 11.3 months, with 44% 1-year survival. Toxicity was limited to local swelling and minimal pain. In another clinical trial, 32 patients with resected colorectal cancer were randomized to treatment with CeaVac. All 32 patients entered into this trial generated high-titer IgG anti-CEA antibodies, and ~75% generated CEA specific T cell responses. These data demonstrated that 5-fluorouracil based chemotherapy regimens did not have any adverse effect on the immune response developed by CeaVac. TriGem, an anti-idiotype monoclonal antibody that mimics the disialoganglioside GD2 was used as a vaccine in

Cancer Vaccines

clinical trial consisting of 47 patients with stage IV melanoma. Forty of 47 patients developed high-titer IgG anti-GD2 antibodies. Seventeen patients were stable on the study from 8 to 34 months. Disease progression occurred in 27 patients on the study from 1 to 9 months. For the 26 patients with soft tissue disease, the median overall survival has not been reached. For 18 patients with visceral metastasis, the median overall survival was 15 months. These results exceed historical controls with stage IV melanoma. Another anti-idiotype monoclonal antibody, TriAb, which mimics the human milk fat globule (HMFG) membrane antigen, is highly overexpressed on breast cancer cells and a variety of other cancer cells, including ovarian cancer, non small-cell lung cancer, and colon cancer. Immunizations with this anti-idiotype antibody elicited both anti-HMFG antibodies and idiotype specific T cell responses in patients with breast cancer in the adjuvant setting as well as in patients with advanced disease following autologous bone marrow transplantation. Although these initial clinical data are promising, active specific immunotherapy with antiidiotype antibodies need to be tested in combination with other conventional and experimental therapies to overcome the multiple mechanisms by which tumor cells escape immune recognition and destruction. The anti-idiotype vaccine therapy for patients with minimal residual disease might be curative in the adjuvant setting and may improve the quality of patients’ life. Dendritic Cell-based Vaccines DCs are the professional APCs of the immune system and are present in peripheral tissues, where they capture antigens. These antigens are subsequently processed into small peptides as the DCs mature and move towards the draining secondary lymphoid organs. There the DCs present the peptides to naïve T cells, thereby inducing a cellular immune response that involves both CD4 T helper 1 (Th1) cells and cytotoxic CD8 T cells. DCs are also important at inducing humoral immune response through their capacity to activate naïve and memory B cells. DCs can also activate natural killer (NK) cells and natural killer T (NKT) cells. Therefore, DCs can conduct all of the elements of the immune orchestra, and they are therefore a fundamental target and tool for vaccination. The development of ex vivo techniques for generating large numbers of DCs in vitro from mouse bone marrow cells supplemented with either GM-CSF alone or GM-CSF plus IL-4 allowed the approach of DC-based tumor vaccination to be fully exploited. Numerous studies in mouse tumor models have shown that DCs pulsed with tumor antigens can induce protective and therapeutic anti-tumor immunity. In 1996, Hsu et al reported the first DC-based clinical trial of follicular B cell lymphoma patients who were treated

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with peripheral blood-derived DCs pulsed with a tumorspecific idiotype (Id) protein. Of these ten patients, eight developed a proliferative cellular response to Id and one patient developed an Id-specific CTL response. However, tumor regression was not reported in these DC-vaccinated patients. In several other trials a correlation between immunological and clinical outcome has been demonstrated. However, the efficacy of therapeutic DC-based vaccination has been modest and these trials have had similar clinical outcome: mainly, immunized patients often demonstrate significant activation of adaptive immunity to the targeted tumor antigen(s) as shown by various methods such as tetramer analysis, IFN-γ ELISPOT, and 51Cr-release assay; but only a limited number of immunized patients demonstrate significant tumor regression. The complexity of the DC system requires rational manipulation of DCs to achieve protective or therapeutic immunity. Further research is needed to analyze the immune responses induced in patients by distinct ex vivo generated DC subsets that are activated through different pathways. These ex vivo strategies should help to identify the parameters for in vivo targeting of DCs. Overall, we remain optimistic that improved cancer vaccines will ultimately yield favorable clinical results, particularly after these approaches have been modified in a manner that integrates recent progress related to the physiology of DCs and our improved understanding of how tumors and the host immune system interact with each other. Conclusion There exist several promising immunologic approaches to vaccine therapy of cancer. The challenge of immunotherapy research is to determine which combination of approaches leads to a favorable clinical response and outcome. Several studies have shown enhanced survival of patients receiving vaccines; however, a randomized phase III clinical trial has yet to show a statistically significant improvement in the survival of such patients. ▶Cancer-Germline (CG) Antigens ▶Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶T-cell Response

References 1. Emens LA (2006) Roadmap to a better therapeutic tumor vaccine. Int Rev Immunol 25:415–443 2. Dalgleish AG, Whelan MA (2006) Cancer vaccines as a therapeutic modality: the long trek. Cancer Immunol Immunother 55:1025–1032 3. Nestle FO, Farkas A, Conrad C (2005) Dendritic-cellbased therapeutic vaccination against cancer. Curr Opin Immunol 17:163–169

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4. Saha A, Chatterjee SK, Mohanty K et al. (2003) Dendritic cell based vaccines for immunotherapy of cancer. Cancer Ther 1:299–314 5. Bhattacharya-Chatterjee M, Chatterjee SK, Foon KA (2002) Anti-idiotype antibody vaccine therapy for cancer. Expert Opin Biol Ther 2:869–881

Cannabinoids G UILLERMO V ELASCO, M ANUEL G UZMA´ N Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain

Synonyms

Cancer Without Disease ▶Dormancy

Cancers of Hormone-responsive Organs or Tissues ▶Endocrine-Related Cancers

Phyto-cannabinoids; Endocannabinoids; cannabinoids; Marijuana

Synthetic

Definition Cannabinoids are a family of lipid molecules that comprises a series of metabolites produced by the hemp plant Cannabis sativa (the phyto-cannabinoids); several fatty-acid derivatives endogenously produced by most animals (the endogenous ligands for cannabinoid receptors), and different synthetic compounds structurally or functionally related with the natural cannabinoids. Activation of cannabinoid receptors by some of these molecules reduce the symptoms associated to cancer chemotherapy and inhibit the growth of tumor cells in culture and in animal models of ▶tumor xenografts.

Characteristics

Cancer-Testis-Antigens Definition Tumor antigens which are only expressed by malignant tumor cells as well as by germ line cells in the testes. The MAGE1 protein was one of the first cancer-testis antigen originally identified in melanoma cells but also expressed by various other tumor types. It belongs to a large family of “MAGE-related” proteins whose biological function is still unclear. It is thought that common epigenetic alterations in tumors lead to re-expression of genes which are normally only expressed in the germ line. ▶Melanoma Vaccines ▶Cancer Germline (GC) Antigens ▶GAGE Proteins

Candidat of Metastasis 1 ▶p8 Protein

The hemp plant Cannabis sativa produces approximately 70 unique compounds known as cannabinoids, of which Δ9-tetrahydrocannabinol (THC) is the most important owing to its high potency and abundance in cannabis. THC exerts a wide variety of biological effects by mimicking endogenous substances – the endocannabinoids anandamide and 2-arachidonoylglycerol – that bind to and activate specific cannabinoid receptors (Fig. 1a and b). So far, two cannabinoid-specific ▶G-protein-coupled receptors have been cloned and characterized from mammalian tissues: The CB1 receptor is particularly abundant in discrete areas of the brain, but is also expressed in peripheral nerve terminals and various extra-neural sites. In contrast, the CB2 receptor was initially described to be present in the immune system, although recently it has been shown that expression of this receptor also occurs in cells from other origins including many types of tumor cells. Signaling Pathways Modulated by Cannabinoid Receptors Most of the physiological, therapeutic and psychotropic actions of cannabinoids rely on the activation of CB1 and CB2 receptors (Fig. 1a and b). Extensive molecular and pharmacological studies have demonstrated that cannabinoids inhibit adenylyl cyclase through CB1 and CB2 receptors. The CB1 receptor also modulates ion channels, inducing, for example, inhibition of N- and

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Cannabinoids. Figure 1 Cannabinoids, cannabinoid receptors and its mechanisms of action. (a) Δ9-tetrahydrocannabinol (THC), the main active component of marijuana, and the endocannabinoids anandamide and 2-Arachidonoylglycerol are ligands of cannabinoid receptors. (b) Both CB1 and CB2 receptors belong to the family of G-protein-coupled receptors. Binding of cannabinoids to cannabinoid receptors leads, among other actions and depending on the cell context, to: inhibition of adenylyl cyclase, modulation of the activity of several ion channels, modulation of phosphatidylionsoitol-3 kinase (PI3K) and of mitogen activated protein kinase cascades, or stimulation of ceramide generation.

P/Q-type voltage-sensitive Ca2+ channels and activation of G protein-activated inwardly rectifying K+ channels. Besides these well-established signaling events that mediate – among others – the neuromodulatory actions of the endocannabinoids, cannabinoid receptors also modulate several pathways that are more directly involved in the control of cell proliferation and survival, including extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, ▶phosphatidylinositol 3-kinase/Akt and focal adhesion kinase. In addition, cannabinoids stimulate the generation of the bioactive lipid second messenger ceramide via two different pathways: sphingomyelin hydrolysis and ▶ceramide synthesis de novo. Palliative Effects of Cannabinoids in Cancer Cannabinoids have been known for several decades to exert palliative effects in cancer patients, and nowadays

capsules of THC (Marinol-TM) and its synthetic analogue nabilone (Cesamet-TM) are approved to treat nausea and emesis associated with cancer chemotherapy. In addition, several clinical trials are testing other potential palliative properties of cannabinoids in oncology such as appetite stimulation and analgesia.

Mechanism Involved in the Antiemetic Effect of Cannabinoids One of the most important physiological functions of the cannabinoid system is to modulate synaptic transmission. Thus, activation of cannabinoid receptors at presynaptic locations leads to reduced neurotransmitter release. As the CB1 receptor is present in cholinergic nerve terminals of the myenteric and submucosal plexus of the stomach, duodenum and colon, it is likely that cannabinoid-induced inhibition of digestive tract motility is due to blockade of acetylcholine release in

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these areas. There is also evidence that cannabinoids act on CB1 receptors localized in the dorsal vagal complex of the brainstem – the region of the brain that controls the vomiting reflex. In addition, endocannabinoids and their inactivating enzymes are present in the gastrointestinal tract and may play a physiological role in the control of emesis.

ganglia, peripheral terminals of primary afferent neurons). Endocannabinoids serve naturally to suppress pain by inhibiting nociceptive neurotransmission. In addition, peripheral CB2 receptors might mediate local analgesia, possibly by inhibiting the release of various mediators of pain and inflammation, which could be important in the management of cancer pain.

Mechanism Involved in Appetite Stimulation by Cannabinoids The endogenous cannabinoid system may serve as a physiological regulator of feeding behavior. For example, endocannabinoids and CB1 receptors are present in the hypothalamus, the area of the brain that controls food intake; hypothalamic endocannabinoid levels are reduced by leptin, one of the most prominent anorexic hormones; and blockade of tonic endocannabinoid signaling with the CB1 antagonist rimonabant – inhibits appetite and induces weight loss. CB1 receptors present in nerve terminals and adipocytes also participate in the regulation of feeding behavior.

Antitumoral Effects of Cannabinoids Cannabinoids have been proposed as potential antitumoral agents on the basis of experiments performed both in cultured cells and in animal models of cancer. A number of plant-derived, synthetic and endogenous cannabinoids are now known to exert antiproliferative actions on a wide spectrum of tumor cells in culture. More importantly, cannabinoid administration to nude mice curbs the growth of various types of tumor xenografts, including lung carcinoma, glioma, thyroid epithelioma, lymphoma, skin carcinoma, pancreatic carcinoma and melanoma. The requirement of cannabinoid receptors for this antitumoral activity has been revealed by various biochemical and pharmacological approaches, in particular by determining cannabinoid receptor expression in the tumors and by using selective cannabinoid receptor agonists and antagonists. Although the downstream events by which cannabinoids exert their antitumoral action have not been completely unraveled, there is substantial evidence for the implication of at least two mechanisms: induction of ▶apoptosis of tumor cells and inhibition of tumor ▶angiogenesis (Fig. 2a).

Mechanism Involved in the Analgesic Effect of Cannabinoids Cannabinoids inhibit pain in animal models of acute and chronic ▶hyperalgesia, ▶allodynia and spontaneous pain. Cannabinoids produce antinociception by activating CB1 receptors in the brain (thalamus, periaqueductal grey matter, rostral ventromedial medulla), the spinal cord (dorsal horn) and nerve terminals (dorsal root

Cannabinoids. Figure 2 Mechanism of cannabinoid antitumoral action. (a) Cannabinoid administration decreases the growth of tumors by several mechanisms, including at least: (i) reduction of tumor angiogenesis, (ii) induction of tumor cell apoptosis, and perhaps (iii) inhibition of tumor cell migration and invasiveness. (b) Cannabinoid treatment induces apoptosis of several types of tumor cells via ceramide accumulation and activation of an ER stress-related pathway. The stress-regulated protein p8 plays a key role in this effect by controlling the expression of ATF-4, CHOP and TRB3. This cascade of events triggers the activation of the mitochondrial intrinsic apoptotic pathway through mechanisms that have not been unraveled as yet. Cannabinoids also decrease the expression of various tumorprogression molecules such as VEGF and MMP2.

Cannabinoids

Induction of Apoptosis Different studies have shown that the pro-apoptotic effect of cannabinoids on tumor cells relies on the stimulation of cannabinoid receptors and a subsequent activation of the proapoptotic ▶mitochondrial intrinsic pathway. In glioma and pancreatic tumor cells, treatment with cannabinoids leads to accumulation of the pro-apoptotic sphingolipid ceramide which in turn leads to up-regulation of the stress-regulated protein ▶p8, which belongs to the family of ▶HMG-I/Y transcription factors. The acute increase of p8 levels after cannabinoid treatment triggers a cascade of events that involves the up-regulation of several genes involved in the ▶endoplasmic reticulum (ER) stress response including the activating transcription factor 4 (ATF-4) and the C/EBP-homologous protein (CHOP). These two transcription factors cooperate in the induction of the ▶tribbles homologue 3 (TRB3), a ▶pseudokinase that is involved in the induction of apoptosis (Fig. 2b). The processes downstream of ER stress activation involved in the execution of cannabinoid-induced apoptosis of tumor cells are not completely understood yet but include inhibition of the anti-apoptotic kinase Akt and activation of the mitochondrial intrinsic pathway. Of interest the pro-apoptotic effect of cannabinoids is selective of tumor cells. For instance, treatment of primary cultured astrocytes with these compounds does not trigger ceramide accumulation, induction of the aforementioned ER stress-related genes or apoptosis. Furthermore, cannabinoids promote the survival of astrocytes, oligodendrocytes and neurons in different models of injury, supporting the notion that cannabinoids activate opposite responses in transformed and non-transformed cells. Inhibition of Tumor Angiogenesis To grow beyond minimal size, tumors must generate a new vascular supply (angiogenesis) for purposes of cell nutrition, gas exchange and waste disposal, and therefore blocking the angiogenic process constitutes one of the most promising antitumoral approaches currently available. Immunohistochemical analyses in mouse models of glioma, skin carcinoma and melanoma have shown that cannabinoid administration turns the vascular hyperplasia characteristic of actively growing tumors to a pattern of blood vessels characterized by small, differentiated and impermeable capillaries. This is associated with a reduced expression of ▶vascular endothelial growth factor (VEGF) and other proangiogenic cytokines such as angiopoietin-2 and placental growth factor, as well as of type 1 and type 2 VEGF receptors, in cannabinoid-treated tumors. Pharmacological inhibition of ceramide synthesis de novo abrogates the antitumoral and antiangiogenic effect of cannabinoids in vivo and decreases VEGF

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production by glioma cells in vitro and by gliomas in vivo, indicating that ceramide plays a general role in cannabinoid antitumoral action. Other reported effects of cannabinoids might be related with the inhibition of tumor angiogenesis and invasiveness by these compounds (Fig. 2a and b). Thus, activation of cannabinoid receptors on vascular endothelial cells in culture inhibits cell migration and survival. In addition, cannabinoid administration to glioma-bearing mice decreases the activity and expression of ▶matrix metalloproteinase-2, a proteolytic enzyme that allows tissue breakdown and remodeling during angiogenesis and metastasis. In line with this notion, cannabinoid intraperitoneal injection reduces the number of metastatic nodes produced from paw injection in lung, breast and melanoma cancer cells in mice. Therapeutic Potential of Cannabinoids as Antitumoral Agents On the basis of these preclinical findings, a pilot clinical study of THC in patients with recurrent ▶glioblastoma multiforme has been recently run. Cannabinoid delivery was safe and could be achieved without significant psychoactive effects. Also, although the limited number of patients involved in the trial did not permit the extraction of statistical conclusions, median survival of the cohort was similar to other studies performed in recurrent glioblastoma multiforme with temozolomide and carmustine, the drugs of reference for the treatment of these tumors. In addition THC administration correlated with decreased tumor cell proliferation and increased tumor cell apoptosis. The significant antiproliferative action of cannabinoids, together with their low toxicity compared with other chemotherapeutic agents and their ability to reduce symptoms associated to standard chemotherapies, might make these compounds promising new antitumoral agents.

References 1. Mackie K (2006) Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol 46:101–122 2. Guzman M (2003) Cannabinoids: potential anticancer agents. Nat Rev Cancer 3:745–755 3. Hall W, Christie M, Currow D (2005) Cannabinoids and cancer: causation, remediation, and palliation. Lancet Oncol 6:35–42 4. Carracedo A, Lorente M, Egia A et al. (2006) The stressregulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells. Cancer Cell 9:301–312 5. Guzman M, Duarte MJ, Blazquez C et al. (2006) A pilot clinical study of Delta9-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme. Br J Cancer 95:197–203

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Cap

Cap Definition Structure added to the 5′-end of nascent eukaryotic mRNA molecules. It aligns eukaryotic mRNAs on the ribosome during translation. ▶Funnel Factors

Cap-Binding Complex Definition A protein complex that recruits messenger RNA to the ribosome. ▶MCT-1 Oncogene

Cap-Dependent Translation Definition In eukaryotes, translation can usually be initiated at the 5′ end “cap” of the mRNA since cap-recognition is required for the assembly of the initiation complex. Most of the transcripts are translated by cap-dependent translation. Alternatively, some transcripts might be translated from an internal ribosome entry site.

family. The subcellular localization of CapG protein is largely nuclear, though other ▶gelsolin family members localize only in cytoplasm, suggesting that CapG may have a function in addition to cytoskeleton modulation. The human CapG gene, mapped at 2p11.2, is ubiquitously expressed in normal tissues, but down-regulation of the expression was observed in roughly one third of melanomas, stomach, and lung cancers. Furthermore, CapG has been shown to possess tumor suppressor activity when transfected into certain cancer cell lines. ▶Gelsolin family

Capillary Hemangioblastoma Definition Low-grade tumor comprised of stromal cells and abundant capillaries, preferentially occurring in the cerebellum. Multiple hemangioblastomas are associated with the Von-Hippel Lindau disease. ▶Uncertain or Unknown Histogenesis Tumors ▶Von Hippel-Lindau Tumor Suppressor Gene

CAR ▶Chimeric Antigen Receptor on T Cells

▶Rapamycin ▶Funnel Factors

Carbonyl Reductases Capecitabine ▶Reductases

Definition

Capecitabine is a 5-fluorouracil (Fu) oral ▶prodrug. ▶Erlotinib (Tarceva®)

CapG Definition Is, a 45-kDa protein composed of 348 amino acids, is a member of the gelsolin/villin actin-modulating protein

Carboplatin Definition Second-generation platinum compound with a broad spectrum of antineoplastic properties. Carboplatin contains a platinum atom complexed with two ammonia groups and a cyclobutane-dicarboxyl residue. This agent is activated intracellularly to form reactive platinum complexes that bind to nucleophilic groups

Carcinoembryonic Antigen

such as GC-rich sites in DNA, thereby inducing intrastrand and interstrand DNA ▶cross-links, as well as DNA-protein cross-links. These carboplatin-induced DNA and protein effects result in ▶apoptosis and cell growth inhibition. This agent possesses tumoricidal activity similar to that of its parent compound, ▶cisplatin, but is more stable and less toxic.

Carboxylesterase Definition Carboxylesterases, present in serum, in the epithelial lining of the intestines, in tumor tissue, and in high content in the liver, are enzymes responsible for metabolizing (hydrolysis) a wide variety of drugs and ▶xenobiotics. The carboxylesterase genes are located on chromosome 16q13-q22 and are supposed to be highly conserved during evolution. ▶Irinotecan

Carboxypeptidase Definition A type of protease or hydrolase that removes the amino acid at the free carboxyl end of a polypeptide chain. ▶Prostate-Specific Membrane Antigen

Carcinoembryonic Antigen P ETER T HOMAS Departments of Surgery and Biomedical Sciences, Creighton University, Omaha, NE, USA

Synonyms CEA; CD66e; CEACAM5

Definition CEA is a glycoprotein of approximately 150–180 kDa. It’s measurement in serum is used clinically as a ▶biomarker for a number of cancers (pancreas, breast, stomach, ovary, lung and medullary carcinoma of the thyroid) but its primary use is in monitoring cancers of the colon and rectum.

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Characteristics Protein Structure CEA was discovered in 1965 in ▶colon cancer and fetal tissue extracts and was described as an ▶oncofetal antigen. Many of the advances in tumor marker research lead directly back to the discovery of CEA The protein component of CEA is 79 kDa in size and the balance of 70–100 kDa is made from up to 28 complex N-linked multi-antennary carbohydrate structures containing N-acetyl-glucosamine, mannose, galactose, fucose and sialic acid. Low resolution X-ray studies have shown an elongated monomeric structure that could be described as a bottle brush. The molecule is composed of a series of six disulphide linked immunoglobulin like domains (IgC2-like) of either 93 (type A) or 85 (type B) amino acids and a seventh Ndomain of 108 amino acids which is an IgV (variable antigen recognition domain) structure without the stabilizing disulphide bridge. CEA can attach to the cell membrane and this is achieved by post translational modification of a small (26 amino acids) hydrophobic C-terminal domain to a ▶glycosyl phosphatidyl inositol linkage (see Fig. 1A). Cleavage of this linkage by ▶phospholipases releases CEA into the lumen of the intestine or other extra-cellular compartments. The CEA Gene Family The complete gene for CEA has been cloned and it includes a promoter region that confers cell type specific expression. The ▶CEA gene family comprises of 29 genes or pseudogenes located between the q13.1 and q13.3 regions of chromosome 19. The family can be divided into three groups: The CEA group of 12 genes, the pregnancy specific glycoprotein group (PSG) of 11 genes and a third group composed of 6 pseudogenes. Only 16 of the 29 genes are expressed. Sequence data has shown that the CEA family is a subset of the Immunoglobulin supergene family. Comparative sequence studies of the CEA gene family from various species suggest that the CEA family have a common ancestry and arose relatively recently in evolution. Function of CEA in Normal and Cancerous Tissue In general members of the CEA family subgroup have a ubiquitous distribution in adult tissues. However CEA itself has a more restricted expression being found only in colon, pyloric mucus cells, epithelial cells of the prostate, in sweat glands and in squamous cells in the tongue, cervix and esophagus. In the colon CEA is located at the apical surface of colonic enterocytes and is associated with the ▶glycocalyx or fuzzy coat. In the normal colon CEA is maximally expressed on columnar cells at the level of the free luminal surface. CEA is also found in goblet cells in association with mucins. The function of CEA in the normal individual is not well understood and has been the subject of much

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Carcinoembryonic Antigen. Figure 1 CEA Structure (a) Showing insertion of CEA into the plasma membrane (down arrow) the Ig domain structure and the position of the N-linked sugar chains. An arrow marks the position of the PELPK receptor recognition sequence. (b). Shows homotypic binding between two CEA molecules with attachment between the N and A3 domains. (Structures are modified from the CEA Homepage http://cea. klinikum.uni-meunchen.de)

speculation. It has been estimated that the normal person can produce 70 mg or more of CEA a day and excrete it in the feces. CEA has been shown to bind to various fimbriated gut pathogens and therefore it has been suggested that it has a function in protecting the gut epithelia. In cancer cells CEA may perform a number of functions. Unlike the normal colonocyte where CEA expression is highly polarized in cancers this polarity is lost and its expression occurs through the whole of the cell surface. It has been shown that CEA

can act as a Ca2+ independent homotypic adhesion molecule binding with itself through an interaction between the N and A3 domains (Fig. 1B) and causing aggregation of tumor cells. This allows the malignant epithelium to adopt a multi-layered structure and may disrupt the normal pattern of differentiation. CEA can also bind heterotypically to other members of the gene family including the ▶nonspecific cross reacting antigen (NCA, CD66c, CEACAM6) and the ▶biliary glycoprotein (BGP, CD66a, ▶CEACAM1) of which seven different forms have been identified. It is unlikely that CEA functions as a ▶cell adhesion molecule in the normal colon because of its apical expression. CEA is cleared from the circulation by the hepatic ▶macrophages (▶Kupffer cells). A cell surface receptor identical to the heterogeneous nuclear RNA binding protein ▶hnRNP M4 recognizes a penta-peptide (Pro– Glu–Leu–Pro–Lys (PELPK)) located at the hinge region between the N and first immunoglobulin loop domain (A1) of CEA. Patients with a mutation in the region coding for this peptide have extremely high circulating CEA levels presumably due to the inability of Kupffer cells to clear the protein from the blood. CEA has also been implicated in the development of hepatic ▶metastasis from colorectal cancers by the induction of a localized inflammatory response that affects retention and implantation in the liver. Cytokines produced also protect the tumor cells against the toxic effects of hypoxia. CEA producing cells therefore have a selective advantage for growth in the liver. Recent studies have also shown that CEA can protect cancer cells from a form of programmed cell death called ▶anoikis and this also seems to involve the PELPK motif and inhibition of Trail-R2 (▶DR5) signaling. CEA is also protective against other forms of ▶apoptosis including drug and UV light induced programmed cell death. The related protein CEACAM-1, however, is a pro-apoptotic protein. Clinical Aspects The main clinical use for CEA is as a tumor marker especially for cancers in the colon and rectum and approximately 90% of these cancers produce CEA. CEA has also been used as a marker for breast and small cell cancer of the lung. Approximately 50% of breast and 70% of small cell cancers express CEA. Accurate immuno-assays are commercially available for its measurement in body fluids. Immunohistochemistry on biopsy or resection specimens is also often carried out, for example the intensity of CEA staining has been associated with a worse prognosis for breast cancer. Normal serum levels are T (promotor) and c.IVS2-105A>G (intron). There are considerable differences in the frequencies and distribution of CDKN2A mutations across the world. Many mutations have been shown to arise from a common founder, and are more frequent in particular geographic locations. For example Sweden and the Netherlands have single predominant founder mutations (p.R112_L113insR, and p16 Leiden respectively) involving over 90% of families tested. The G101W mutation, common in Italy, France and Spain, has been calculated to arise from a single genetic event approximately 93 generations ago. Many additional mutations have been repeatedly reported, and where analysis has been performed these have invariably been shown to be due to common founders. The only exception to this appears to be a 24 bp insertion in exon 1a, that has arisen multiple times, presumably because of DNA slippage over a 24 bp repeat region.

Mutation of ARF Germline mutations affecting ARF but not p16INK4a have been reported in a small number (~3%) of melanoma families. Whereas the distribution of p16 mutation types (approximately 70% missense or nonsense, 23% insertion or deletion, 5% splicing and 2% regulatory) is consistent with that observed in the Human Genome Mutation Database, the reported ARF specific mutations are almost all either splicing mutations (affecting the 3′ splice site of exon 1b) or large deletions.

Penetrance The pattern of susceptibility in melanoma pedigrees is consistent with the inheritance of autosomal dominant genes with incomplete penetrance. The overall penetrance of CDKN2A mutations in melanoma families has been estimated to be 0.30 by age 50 years and 0.67 by age 80 years. There is significant variation in the penetrance of CDKN2A mutations with geographical location. By age 50 years penetrance was estimated to be; 0.13 in Europe, 0.5 in the United States, and 0.32 in Australia, by age 80 years; 0.58 in Europe, 0.76 in the United States, and 0.91 in Australia (Fig. 4). This indicates that the CDKN2A mutation penetrance varies with melanoma population incidence rates, thus the same factors that effect population incidence of melanoma may also mediate CDKN2A penetrance.

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Modifiers of Penetrance of CDKN2A Mutations The MC1R gene (16q24) which encodes for the melanocyte-stimulating hormone has been shown to be a risk factor in families with segregating CDKN2A mutations. MC1R variants have been shown to act as modifier alleles, increasing the penetrance of CDKN2A mutations and reducing the age of onset of melanoma.

CDKN2A. Figure 4 Age specific penetrance estimates for CDKN2A mutations. Penetrance is shown for melanoma pedigrees from Australia, Europe, America (US) and all geographic locations combined.

Multiple Primary Melanoma General characteristics of inherited susceptibility to many types of cancer are early age of onset and the development of multiple primary tumors. Hence the presence of multiple primary melanomas (MPM) in an individual may be a sign of them being a CDKN2A mutation carrier. This is the case for a small proportion (13/133, 10%) of MPM cases without a family history of the disease. In contrast, analysis of MPM cases with a family history of disease yields CDKN2A mutations in 55/139 (40%) of samples tested. The proportion of CDKN2A mutations in sporadic MPM cases increases with increasing number of melanomas (10/119 (8.5%) of cases with two primary melanomas, compared to 11/83 (33%) cases with 3 or more primary tumors). CDKN2A Mutations and Non-Melanoma Cancers Since CDKN2A is a tumor suppressor found to be inactivated in a wide range of different tumors, one might expect individuals carrying germline mutations of CDKN2A to be prone to cancers other than melanoma. ▶Breast, ▶prostate, ▶colon, and ▶lung cancers, have been suggested to be associated with CDKN2A mutations, however, these common cancers may occur in CDKN2A positive pedigrees by chance. Convincing evidence for susceptibility to another tumor type has been shown only for pancreatic cancer, which has been shown to be significantly associated with CDKN2A mutations in all regions except Australia, the reason for this is not yet understood. There appears to be no evidence of an association between neural system tumors (NSTs) and CDKN2A mutations involving p16. However, there is marginal evidence for the association of NSTs with ARF specific mutations.

CDKN2a Polymorphisms as Low Risk Factors The A148T variant, located in exon 2 of the CDKN2A gene has no observed effect on p16 function, and does not segregate with disease in melanoma pedigrees. The contribution of this polymorphism to melanoma risk remains unclear, an association with increase in risk has been seen in some populations, but not in others. The 500 C > G and the 540 C > T polymorphisms in the 3′ untranslated region of the CDKN2A gene have been shown to be associated with melanoma risk. The frequencies of the rare alleles at these loci have been shown to be higher in melanoma cases than in controls. It is possible that these variants might alter the stability of the CDKN2A transcript or the level of transcription, or that they may be in linkage disequilibrium with an unidentified variant which is directly responsible for melanoma predisposition. The contribution of these polymorphisms to melanoma risk is likely to be small in comparison to that of CDKN2A inactivating mutations.

CDKN2A and the Atypical Mole Syndrome Since the description of the “B-K mole syndrome” much debate has ensued regarding the association between melanoma and the ▶atypical mole syndrome (AMS). Several authors have concluded that atypical moles segregate independently of CDKN2A mutations, although individuals with high numbers of naevi in melanoma-prone families are three times more likely to be CDKN2A mutation carriers than those with a low number of naevi. Support for the notion that CDKN2A is naevogenic comes from a study of a large series of 12-year-old twins in which total naevus count was found to be tightly linked to CDKN2A. This finding has recently been corroborated by two independent genome wide association studies that have mapped loci responsible for naevi in twin cohorts. Both studies showed peaks of high linkage scores at 9p21 directly over the CDKN2A gene.

References 1. Bishop JN, Harland M, Randerson-Moor J et al. (2007) Management of familial melanoma. Lancet Oncol 8(1):46–54

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2. Goldstein AM, Chan M, Harland M et al. (2007) Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents. J Med Genet 44(2):99–106 3. Hayward NK (2003) Genetics of melanoma predisposition. Oncogene 22(20):3053–3056 4. Sharpless E, Chin L (2003) The INK4a/ARF locus and melanoma. Oncogene 22(20):3092–3098 5. Sharpless NE (2005) INK4a/ARF: a multifunctional tumor suppressor locus. Mutat Res 576(1–2):22–38

cDNA chips ▶Microarray (cDNA) Technology

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contributes to progression of many ▶epithelial cancers and immune dysfunctions.

Characteristics The CEA gene family encodes a set of 22 genes and 11 pseudogenes clustered in a 1.8 Mb region on human chromosome 19q13.2 between the CY2A and D19S15 marker genes. The CEA genes encompass an N-terminal Ig variable domain followed by one to six Ig constant-like domains. A striking characteristic of these proteins is their extensive ▶glycosylation on asparagine residues with multi-antennary carbohydrate chains. CEA and CEACAM1 are further modified by addition of ▶Lewisx and sialyl-Lewisx high-mannose residues. The proteins differ however, in their C-terminal regions producing either secreted entities such as the pregnancy-specific glycoproteins (▶PSG1–11) or others, tethered to the cell surface by either a glycosyl phosphatidylinositol linkage (CEA, CEACAM6–8) or a bona fide transmembrane domain (CEACAM1, CEACAM3, CEACAM4, CEACAM18–21) (Fig. 1). The CEACAM1 gene is unique in this family in that it produces twelve different splicing variants. More information on the structural features of the CEA gene family members is available at http://cea.klinikum.unimuenchen.de. CEA is a monomeric protein adopting a

CEA Gene Family N ICOLE B EAUCHEMIN McGill Cancer Centre, McGill University, Montreal, Quebec, Canada

Synonyms CEACAM1=BGP, C-CAM, CD66a; CEACAM5=CEA, CD66e; CEACAM6=NCA, CD66c; CEACAM7=CGM2; CEACAM8=CGM6, CD66b

Definition The Carcinoembryonic Antigen (▶CEA) gene family comprises 33 genes, 22 of which are expressed. All family members share similar structural features encompassing immunoglobulin (Ig) variable and/or constant domains and therefore constitute members of the large immunoglobulin superfamily. These proteins are either secreted or membrane-bound. Several CEACAMs function as homophilic or heterophilic intercellular ▶cell adhesion molecules. CEA, ▶CEACAM1, ▶CEACAM6 and ▶CEACAM7 also play a significant role as regulators of tumor cell proliferation and differentiation and their overexpression (CEA and CEACAM6) or their down-regulation (CEACAM1 and CEACAM7)

CEA Gene Family. Figure 1 Schematic representation of some members of the CEA family. Most CEA family members, except the pregnancy-specific glycoproteins (PSG) that are secreted proteins, are associated with the cell membrane (depicted in grey). The immunoglobulin variable-like domains (the N domain) are shown in blue and the immunoglobulin constant-like domains are represented in orange. The N-linked glycosylation sites are indicated by sticks and balls, colored in dark orange. The glycosylphosphatidylinositol membrane anchors are represented by arrows. The CEACAM1 gene expresses many splice variants. However, only the CEACAM-4L isoform containing four Ig domains and the longer cytoplasmic tail is shown here.

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β-barrel cylindrical shape resembling a “bottle brush,” whereas CEACAM1 is present as both a monomeric and dimeric protein. Expression and Functions of CEA Family Members in Normal and Tumor Tissues Although not ubiquitous, CEA family members exhibit a wide tissue distribution. CEA and CEACAM6 are found mainly in columnar epithelial and goblet cells of the colon in the early fetal period and are maintained in adult life. In the colonic brush border, CEA, CEACAM1, 6 and 7 demonstrate maximal expression at the free luminal surface, although CEACAM1 and 7 are also found at the lateral membrane. In addition to its expression in epithelia, CEACAM1 is located on granulocytes, lymphocytes and on endothelial cells, whereas CEACAM6 is also expressed on granulocytes and monocytes. CEACAM3 and 8 are found exclusively on granulocytes. CEA, CEACAM1 and CEACAM6 are recognized as cell adhesion molecules contacting each other by antiparallel self-binding (homophilic). Some associations are exclusive, such as ▶CEACAM8-CEACAM6. The first Ig domain is crucial in these interactions. Various CEA family members also act as heterophilic partners for ▶E-selectin and ▶galectin-3. Another striking feature of CEA family members is their ability to act as pathogen receptors binding to outer membrane proteins of Neisseria gonococci and Haemophilus influenzae as well as fimbriae of Salmonella typhimurium and Escherichia coli. In addition, CEACAM1 is the receptor for the mouse hepatitis viruses. The bacterial and viral adhesin functions of the CEA family members confer strong immunosuppressive activity in T and B lymphocytes, whereas they enhance integrindependent cell adhesion in epithelial cells with concomitant increase of the TGF-β1 receptor CD105. Other functions for CEA and CEACAM6 include inhibition of cellular differentiation as demonstrated in a number of cellular systems and inhibition of the ▶apoptotic process of ▶anoikis by activation of β1 integrins. PSG1–11 are mainly expressed in syncytiotrophoblast during the first trimester of pregnancy where they act as immunomodulators and inhibit cell-matrix interactions. CEA is abundantly expressed in tumors of epithelial origin such as colorectal, lung, mucinous ovarian and ▶endometrial adenocarcinomas. For these reasons, CEA has a long history as a marker of colonic, intestinal, ovarian and breast tumor progression and its high expression is associated with poor prognostic and recurrence of disease post-surgically. High pre-operative CEA levels are indicative of a poor prognosis whereas low levels are associated with increased survival of the patients. The tumorigenic potential of CEA and CEACAM6 was recently clarified by

transgenic overexpression of a bacterial artificial chromosome fragment of 187 kb encoding the full CEA, CEACAM6 and CEACAM7 genes. When the CEABAC transgenic mice were treated with the azoxymethane ▶carcinogen to induce colon cancers, expression of CEA and CEACAM6 was increased by 2–20 fold, a situation reminiscent to that observed in the human cancer. Information on CEACAM7 expression in tumors is more limited. It is down-regulated in colorectal cancers, but increased in gastric tumors. CEACAM6 however, exhibits a broader distribution than in the cancers described above, as it is additionally found in gastric and breast carcinomas, and ▶acute lymphoblastic leukemias. In fact, overexpression of CEACAM6 in ▶pancreatic cancer confers increased resistance to anoikis and increased metastasis. It also modulates chemoresistance to the ▶gemcitabine agent, thereby suggesting that CEACAM6 determines cellular susceptibility to apoptosis. Expression and Functions of CEACAM1 CEACAM1 expression is more complex. It is downregulated in colon, prostate, hepatocellular, bladder, endometrial, renal cell and 30% of breast carcinomas, but overexpressed in gastric and squamous lung cell carcinomas and ▶melanomas. In thyroid carcinomas, CEACAM1 was shown to restrict tumor cell growth. However, it increases the thyroid cancer metastatic potential. Manipulation of CEACAM1 expression levels in colonic, prostatic and bladder tumor cell lines, negative for CEACAM1, has indeed confirmed that expression of the longer variant, CEACAM1-4L, produces reduction of tumorigenic potential in vitro and inhibition of tumor growth in xenograft mouse models. The importance of cell surface CEACAM1 expression for maintenance of normal epithelial cellular behavior has recently been confirmed in vivo; a Ceacam1-null mouse exhibits a significantly increased colon tumor load compared to the wild-type littermates upon carcinogenic induction of colorectal cancer. CEACAM1’s role as a modulator of tumor progression depends on the involvement of its cytoplasmic domain in signaling via its tyrosine and serine phosphorylation. Two Tyr residues are positioned within Immunoreceptor Tyrosine-based Inhibition Motifs (▶ITIM). The membrane-proximal Tyr488 is a phosphorylation substrate of Src-like kinases as well as of the Insulin and Epidermal growth factor receptors. Upon Tyr phosphorylation, CEACAM1-L associates with the tyrosine phosphatases SHP-1 and SHP-2. The SHP-1CEACAM1-L protein complex regulates its function in various tissues such as inhibition of epithelial cell growth, CD4+ T cell activation and insulin clearance from hepatocytes. CEACAM1-L ▶tyrosine phosphorylation also stimulates its association with the cytoskeletal proteins G-actin, tropomyosin and paxillin, thereby

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influencing cell adhesion, and with the ▶β3 integrin, hypothesized to influence cell motility. The CEACAM1L cytoplasmic domain also carries seventeen serine residues most of which lie in consensus sequences recognized by serine kinases. However, little is know about their functional implications apart from the CEACAM1-S Thr/Ser452 and Ser456, shown to modulate direct binding to G- and F-actin, tropomyosin and calmodulin, and CEACAM1-L’s Ser503 whose mutation to an Ala residue enhances colonic or prostatic tumor development in xenograph models. Additionally, Ser503 renders permissive Tyr488 phosphorylation by the insulin receptor. Transgenic mice overexpressing a Ser503Ala CEACAM1-L mutant in the liver developed hyperinsulinemia, secondary insulin resistance and defective insulin clearance. As a consequence of the decreased insulin receptor endocytosis and altered insulin signaling, the transgenic mice became obese demonstrating increased visceral adiposity, elevated serum free fatty acids and plasma and hepatic triglyceride levels. CEACAM1-L also contributes to important functions in the immune system. It functions as an inhibitory coreceptor in T lymphocytes. Its conditional deletion in these cells amplified TCR-CD3 signaling, whereas overexpression in T cells was responsible for decreased proliferation, ▶allogeneic reactivity and cytokine production in vitro, with delayed type hypersensitivity and inflammatory bowel disease in vivo. Regulation of this function involves the ITIM motifs and the SHP-1 tyrosine phosphatase. A similar function and mechanism have been described in B lymphocytes and natural killer cells. Indeed, CEACAM1-mediated intercellular adhesion between melanomas with increased CEACAM1 expression and NK cells allows inhibition of NK-cell-elicited killing, thereby conferring upon CEACAM1 a role in tumor immunosurveillance. Similarly, heterophilic engagement of CEACAM1 with CEA, overexpressed in many tumors, also inhibits lymphocyte-mediated and NK-cell-mediated killing having therefore detrimental effects on immune surveillance. In addition, increased expression of CEACAM1 on endothelial cells present in tumors in response to ▶VEGF activation and/or hypoxia provokes a pro-angiogenic switch with increased endothelial tube formation and invasion. Therefore CEACAM1’s contribution to cancer progression most likely depends on its positive or negative expression and signaling in epithelial tumor cells, on its systemic effects on metabolism and adiposity, on its role in immunosurveillance and most probably on endothelial proliferation and invasion.

and CAAT boxes and are considered members of the housekeeping gene family. Their distal promoter regions (> −500 bp) contain highly repetitive elements, whereas their proximal promoter regions are rich in GC boxes and SP1 binding sites. Five footprinted regions have been identified in the CEA promoter, the first three binding respectively, to the Upstream Stimulatory Factor (USF), and SP1 and SP1-like factors. Similarly, the human CEACAM6 promoter is regulated by the USF1 and USF2 as well as SP1 and SP3 transcription factors. A silencer element has also been located in its first intron. In contrast, the human CEACAM1 promoter does not bind the SP1 factors, but associates with an AP-2-like factor and the USF and HFN-4 transcription factors. The gene is additionally controlled by the hormonal changes (estrogens and androgens) and can be induced by cAMP, retinoids, glucocorticoids and insulin. Moreover, many genes of this large family are triggered by inflammation via interferons, tumor necrosis factors and interleukins. It has been reported more recently that expression of the CEACAM1 gene is influenced by TPA and calcium ionophore in endometrial cancers, the expression of ▶BCR/ABL in leukemias, the expression of the β3 integrin in melanomas and VEGF and hypoxia in angiogenic situations. In prostate cancer, there is an inverse correlation between the down-regulation of CEACAM1 and the increased expression of the transcriptional repressor Sp2 that acts to recruit histone deacetylase to the CEACAM1 promoter.

Transcriptional Regulation The upstream promoters of the CEA and CEACAM1 genes have been dissected to identify important binding sites responsible for their transcriptional regulation. These two genes do not encompass classical TATA

References

The Next Frontier The diversity of functions of the members of the CEA gene family and their dynamic expression patterns in normal and tumor tissues has slowed the development of effective targeted therapies. More recently, effective strategies have been devised using vaccination with CEA peptide-loaded mature dendritic cells that induced potent CEA-specific T cell responses in advanced colorectal cancer patients. Effective protection from tumor development have also been seen with delivery of adenoviral vectors encoding CEA fused to immunoenhancing agents such as tetanus toxin or the Fc portion of IgG1. Likewise, targeting of CEACAM6 in pancreatic cancer may result in decreased tumor load. The therapeutic and selective targeting of CEACAM1 in melanomas, gastric and lung carcinomas as well as its location in tumor endothelia may prove to be a favorable avenue of future interventions.

1. Hammarström S (1999) The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 9:67–81

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2. Horst A, Wagener C (2004) CEA-related CAMs. Handb Exp Pharmacol 165:283–341 3. Gray-Owen SD, Blumberg RS (2006) CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 6:433–446 4. Kuespert K, Pils S, Hauck CR (2006) CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol 18:1–7 5. Leung N, Turbide C, Marcus V et al. (2006) Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) contributes to progression of colon tumors. Oncogene 25:5527–5536

CEACAM5 ▶Carcinoembryonic Antigen

CEACAM5=CEA,CD66e ▶CEA Gene Family

CEA-related Cell Adhesion Molecule 1 ▶CEACAM1 Adhesion Molecule

CEACAM6=NCA, CD66c ▶CEA Gene Family

CEA-Vaccine Virus CEACAM7=CGM2 Definition A vaccine constructed from a recombinant vaccine virus containing the human carcinoembryonic antigen gene.

▶CEA Gene Family

▶Carcinoembryonic Antigen (CEA)

CEACAM8=CGM6, CD66b Ceacam1, Ceacam6, Ceacam7, Ceacam8

▶CEA Gene Family

Definition Carcinoembryonic antigen-related cell adhesion molecules. Members of the CEA family. ▶CEA Gene Family

CEACAM1 Adhesion Molecule A NDREA K RISTINA H ORST, C HRISTOPH WAGENER Institute of Clinical Chemistry, University Medical Center Hamburg Eppendorf, Hamburg, Germany

CEACAM1=BGP, C-CAM, CD66a ▶CEA Gene Family

Synonyms NCA-160, nonspecific cross-reacting antigen with a Mw of 160kD; BGP, biliary glycoprotein; CD66a, cluster of differentiation antigen 66 a; CEACAM1 CEA-related cell adhesion molecule 1

CEACAM1 Adhesion Molecule

Definition CEACAM1 (CEA-related cell adhesion molecule 1) belongs to the CEA (▶Carcinoembryonic antigen, ▶CEA gene family) family of cell surface glycoproteins, a subfamily of the immunoglobulin gene superfamily. The CEA family comprises two major groups, the CEA-related molecules and the PSG (pregnancy-specific glycoprotein)-related molecules. Additionally, a number of pseudogenes have been identified. To date, 29 genes are known, which are clustered on human chromosome 19 (19q13.1-19q13.2). The CEA-related members of the CEA family display a complex expression pattern on human healthy and malignant tissues. They are linked to the cell membrane via GPI anchors, or they are transmembrane proteins with a cytoplasmatic tail. The PSG-related molecules are soluble glycoproteins; their expression is restricted to the placenta, more specifically, to the syncytiotrophoblast, which is the outermost fetal component of the placenta. CEACAM1 has been structurally and functionally conserved in humans and rodents.

Characteristics Properties of CEACAM1 Human CEACAM1 has been originally identified in human bile due to its crossreactivity with CEA-antisera. It was therefore named biliary glycoprotein I or nonspecific cross-reacting antigen at first. Amongst the cluster of differentiation antigens on human leukocytes, CEACAM1 used to be referred as CD66a. However, with the latest revision of the nomenclature for the CEA family, CD66a, BGP, or NCA-160 became CEACAM1. Its structural similarities to CEA and the immunoglobulin superfamily proteins became apparent, once the cDNA sequence for CEACAM1 became available. CEACAM1 displays the broadest expression pattern amongst CEA family members; it has first been described as a cell–cell ▶adhesion molecule on rat hepatocytes. CEACAM1 is expressed on epithelia, endothelia, and on leukocytes. CEACAM1 is a heavily glycosylated molecule that exists in 11 known isoforms emerging from differential splicing and proteolytic processing. The two major isoforms of CEACAM1 consist of four extracellular Ig-like domains, a transmembrane domain, and either a long or a short cytoplasmic tail, referred to as the long (CEACAM1-4L) and the short isoform (CEACAM1-4S), respectively. In addition to these transmembrane isoforms, soluble CEACAM1 isoforms are found in body fluids, for example, in saliva, serum, seminal fluid, and bile. Glycans on the extracellular domains of CEACAM1 are linked to the protein backbone via N-glycosidic linkages. It is presently unknown whether all of the 19 motifs that may render N-linked

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▶glycosylation actually harbor sugar moieties. On human granulocytes, CEACAM1 is a major carrier of Lewisx glycans that are implicated in cellular adhesion to cognate lectins on blood vessels, within the extracellular matrix, or antigen presenting cells. CEACAM1 also elicits cell–cell adhesion via self-association in a homomeric fashion or via formation of heteromers with other CEA-family members and different adhesion molecules that are either located on the same cell or on neighboring cells. The resulting adhesive properties are modulated by differential expression ratios between the long and short CEACAM1 isoform, respectively. Through its long and short cytoplasmic tail, CEACAM1 mediates molecular interactions with cytoskeletal components or adapter proteins, which are integral parts of various key signal transduction pathways (▶Signal transduction, cell biology). These interactions are in part dependent on differential phosphorylation of the CEACAM1-4L cytoplasmic domain on tyrosine and serine residues. The overall phosphorylation status of the CEACAM1-4L cytoplasmic domain relays signals, which contribute to cellular motility and differentiation, and thus determine cell fate by promoting proliferation or cell death. Phosphorylation of CEACAM1-4L cytoplasmic tyrosines that are part of an imperfect ITIM (immune receptor tyrosine-based inhibition motif) and serine residues regulate the interaction with kinases, phosphatases, cellular receptors for insulin (▶Insulin receptor), the epidermal growth factor (▶Epidermal growth factor receptor ligand, epidermal growth factor receptor inhibitor), and other cellular adhesion molecules, for example, integrin αvβ3 (▶Integrin signaling and cancer). These qualities make CEACAM1 an important tool for cellular communication and they illustrate why so many different biological functions have been attributed to CEACAM1 in different biological contexts (Fig. 1).

CEACAM1 in Cancer The first report on CEACAM1, in the context of human pathological conditions, was on elevated serum levels of a biliary glycoprotein in patients with liver or biliary tract disease. Later, aberrant CEACAM1 expression in a broad variety of human malignancies has been reported. In the progression of malignant diseases, two general patterns in the changes of CEACAM1 expression levels have emerged. In the first group of tumors, CEACAM1 expression is downregulated in the course of progressing disease. In the second group of tumors, CEACAM1 expression appears to be upregulated; often, this upregulation of CEACAM1 expression is observed in the context with increased invasiveness (▶Invasion) of the primary tumor or is found on microvessels in progressing (▶Progression) tumor areas (Fig. 2).

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CEACAM1 Adhesion Molecule. Figure 1 Schematic representation of CEACAM1-4L and CEACAM1–4S and their participation in extracellular and intracellular communication. The two major CEACAM1 isoforms consist of four extracellular immunoglobulin-like domains, a transmembrane domain and either a long or a short cytoplasmic tail. The N-terminal domain (N) resembles a variable-like Ig domain but lacks the cystin bond usually found in Ig members. The A1, B1, and A2 domain resemble constant I-type-like Ig domains. Motifs for N-linked glycosylation are represented by lollipops. With its extracellular domains, CEACAM1 mediates recognition of various pathogens, such as Escherichia coli, Salmonella typhimurium, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis. The murine homologue of CEACAM1 is the receptor for the murine hepatitis virus: Additionally, CEACAM1 binds to galectin-3, DC-SIGN (dendritic cell ICAM3-grabbing nonintegrin), and integrin αvβ3. Tyrosine and serine residues involved in relaying CEACAM1-4L-mediated signal transduction are indicated by red and grey circles, respectively. Through its long cytoplasmic tail, CEACAM1-4L interacts with intracellular kinases of the SRC-family ▶(SRC), the tyrosine phosphatases SHP-1 and SHP-2, caspase-3 as well as with paxillin, filamin, and calmodulin. Differential phosphorylation of the CEACAM1-4L cytoplasmic domain is required for its interaction with the insulin receptor, regulating insulin receptor internalization and recycling, and for modulating immune responses elicited by lymphocytes, for example. The short cytoplasmic domain of CEACAM1–4S binds to actin and tropomyosin.

Loss of CEACAM1 Expression in Tumorigenesis and Tumor Progression Human cancers that show downregulation of CEACAM1 expression in the course of tumor progression are carcinomas of the liver (▶Hepatocellular carcinoma), colon (▶Colon cancer, colorectal premalignant lesions), kidney (▶Renal cell carcinoma, renal carcinoma), urinary bladder (▶Bladder cancer, bladder tumors), prostate (▶Prostate cancer, clinical oncology), mammary gland (▶Breast cancer), and the endometrium (▶Endometrial cancer). In general, downregulation and subsequent loss of CEACAM1 expression is more frequent in

high-grade tumors that are poorly differentiated, and often associated with a larger tumor size. On epithelia, especially those that form a lumen, CEACAM1 exhibits a pronounced apical expression, like in the entire gastrointestinal tract, breast, liver, prostate, bladder, and kidney. CEACAM1 expression has been implicated in morphogenesis of lumen formation. In the process of building an asymmetrical epithelium, lateral CEACAM1 expression on neighboring cells is lost and often becomes entirely apical once a lumen or a duct has been formed. The loss of CEACAM1 expression in the context of tumorigenesis


CEACAM1 Adhesion Molecule. Figure 2 Dysregulation of CEACAM1 expression in human cancers. Changes of epithelial CEACAM1 expression in the course tumor progressison: In mammary carcinomas and carcinomas of the liver, colon, endometrium, kidney, bladder, and prostate, CEACAM1 expression is downregulated on tumor epithelium (▶Epithelial cancers). Downregulation of CEACAM1 levels often correlates with dedifferentiation of the tumor and loss of tissue architecture. In carcinomas of the thyroid, ▶non-small cell lung cancer (Lung cancer), pancreatic tumors (▶Pancreas cancer, clinical oncology), and malignant melanomas (▶Melanoma), CEACAM1 is induced or upregulated in the course of tumor growth. Here, CEACAM1 expression is found on the invasive front of the tumors and is related to development of metastatic disease (▶Metastasis) and poor prognosis. In pancreatic cancer, CEACAM1 has been identified as a novel biomarker (▶Biomarker, clinical cancer biomarker) that indicates presence of malignant disease.

has been studied most extensively in the context of breast, colonic, and prostate carcinomas. A hallmark of carcinomatous lesions is the loss of polarity of their epithelial structures. In colonic epithelium, for example, loss of polarity is accompanied by the loss of apical CEACAM1 expression that occurs in early adenomas and carcinomas. In these tumors, presence and absence of CEACAM1 correlate with normal and reduced apoptosis (▶Apoptosis, apoptosis signals), respectively. Furthermore, the naturally occurring process of ▶anoikis, once cells lose contact to their substratum, is compromised. This observation and the fact that the CEACAM1 gene is silenced in the

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course of aberrant cell growth prompted the hypothesis that CEACAM1 acts as a ▶tumor suppressor. In intestinal cells, presence of the long CEACAM1 isoform is required to suppress tumor growth, and lack of CEACAM1-4L expression is accompanied by a decrease in proteins that inhibit cell cycle progression. In human mammary epithelial cells, CEACAM1 expression is causally related to lumen formation and differentiation. In mammary glands, CEACAM1-4S is the predominating isoform, and only the short cytoplasmic tail induces apoptosis of the central cells and subsequently leads to lumen formation in mammary morphogenesis. During tumor progression, CEACAM14S expression is lost and acinar polarity no longer can be observed. However, since particular mutations or allelic loss of the CEACAM1 gene in human cancers has not been described so far, it is likely that dysregulation of CEACAM1 expression rather than irreversible loss of the CEACAM1 gene are linked to tumorigenesis and tumor progression in vivo. Hence, gene silencing may attribute to the loss of the tumor suppressive qualities of CEACAM1. Though there are no changes in promoter ▶methylation of the CEACAM1 gene linked to tumor progression, CEACAM1 promoter activity appears to be regulated by binding of the transcription factor Sp2. In high-grade prostate carcinomas, Sp2 is highly abundant, whereas CEACAM1 expression is lost. Sp2 localizes to the CEACAM1 promoter and imposes repression of gene transcription by recruiting ▶histone deacetylase.

Upregulation of CEACAM1 Expression in Malignant Diseases Opposed to its tumor suppressive functions, certain tumors gain CEACAM1 expression in the course of cancer development. In the case of malignant melanomas and thyroid carcinomas, expression of CEACAM1 correlates with an increase of tumor invasiveness and development of metastatic disease. In primary cutaneous malignant melanomas, for example, CEACAM1 expression is found at the invasive front of the tumors, and its coexpression with integrin αvβ3 indicates that CEACAM1 may directly promote on cellular invasion. In a follow-up study, CEACAM1 was identified as an independent prognostic marker, predicting the development of metastatic disease and poor survival. In this context, it is noteworthy that CEACAM1 on melanoma cells forms homophilic cell–cell contacts with CEACAM1 molecules on tumor-infiltrating lymphocytes, and leads to inhibition of their cytolytic function. Similarly, in human non-small cell lung cancer, CEACAM1 expression correlates with advanced disease, whereas it is not expressed on the normal bronchiolar epithelium; this CEACAM1 neoexpression

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was identified as an independent prognostic marker, indicating lower incidence of relapse-free survival. In pancreatic carcinomas, CEACAM1 has been identified as a novel serum biomarker, with an increased CEACAM1 expression on neoplastic cells of pancreatic adenocarcinomas and elevation of serum levels at the same time. Additionally, significant differences in CEACAM1 serum levels were found in patients with either pancreatic cancer or chronic pancreatitis. Opposed to the classical pancreatic tumor marker CA19-9, CEACAM1 was confirmed as an independent marker to distinguish between the presence of malignant disease and pancreatitis. CEACAM1 and Tumor Angiogenesis CEACAM1 expression on human blood vessels is restricted to newly formed vessels, and usually, no CEACAM1 is found on mature, large vessels. The first indication that CEACAM1 is related to ▶angiogenesis was the description of CEACAM1 neoexpression on newly formed vessels in the human placenta. Furthermore, CEACAM1 is expressed on vessels in wound healing tissues and on tumor vessels of human bladder carcinomas, the prostate, hemangiomas, and ▶neuroblastomas. CEACAM1 expression in endothelia is induced by VEGF (▶Vascular endothelial growth factor)-dependent pathways and appears to favor vessel maturation. In human prostate carcinomas, CEACAM1 shows divergent expression on tumoral blood vessels and the tumor epithelium. The presence of epithelial CEACAM1 is observed in the context of poor tumoral blood vessel growth and loss of epithelial CEACAM1 expression parallels enhanced tumor angiogenesis. Especially in high-grade prostate carcinomas, tumor proximal vessels are expressing CEACAM1. Contrary to prostate carcinomas, microvessels in human neuroblastomas are CEACAM1-positive only during tumor maturation, but absent in undifferentiated, high-grade tumors. In ▶Kaposi sarcomas, CEACAM1 upregulation is observed, indicating that CEACAM1 might be related to lymphatic reprogramming of the vasculature in these tumors. Studying CEACAM1 in Cancer: Animal Models In animal models investigating CEACAM1 function in tumorigenesis in vivo, the observations from human diseases could be confirmed. The focus of the mouse and rat models (▶Mouse model) studied to date was set largely on the tumor-suppressive effects or enhancement of metastatic disease of CEACAM1-4L on the progression of colonic cancer, prostate cancer, hepatocellular carcinomas, and malignant melanomas. In CEACAM1knockout mice, chemically induced colonic tumor growth was significantly increased in terms of tumor numbers and size opposed to CEACAM1-expressing

wild type littermates. In syngeneic and xenotypic transplantation of tumor cells of the colon, prostate, and hepatocellular carcinomas, the tumor-suppressive effects of CEACAM1-4L expression could also be validated. After xenotransplantation of human CEACAM1-expressing melanoma cell lines into immune-deficient mice, enhanced metastasis was observed when compared to transplantation of CEACAM1-negative cell lines.

References 1. Beauchemin N, Draber P, Dveksler G et al. (1999) Redefined nomenclature for members of the carcinoembryonic antigen family. Exp Cell Res 252:243–249 2. Prall F, Nollau P, Neumaier M et al. (1996) CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues. J Histochem Cytochem 44:35–41 3. Gray-Owen SD, Blumberg RS (2006) CEACAM1: contact-dependent control of immunity. Nat Rev Immunol 6:433–446 4. Kuespert K, Pils S, Hauck CR (2006) CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol 18:565–571 5. Singer BB, Lucka LK (2005) CEACAM1. UCSD-nature molecule pages. Nature Publishing Group, doi:10.1038/ mp.a003597.01

C/EBPa Definition The transcription factor CCAAT enhancer binding protein-α is a tumor suppressor gene and a crucial regulator of granulopoiesis through inhibition of cJUN. Disruption of C/EBPα, including dominant negative mutations of C/EBPα, are found in acetyl myeloid leukemia. ▶NUP98-HOXA9 Fusion ▶Tumor Suppressor Gene ▶Chromosomal Translocation t(8;21)

C/EBP Homologous Protein Definition CHOP; synonym growth arrest and DNA damageinducible gene 153; Is a C/EBP family transcription factor which is involved in ▶endoplasmic reticulum stress-mediated ▶apoptosis.

Celastrol

CED ▶Convection Enhanced Delivery

CED-3 Definition One of three genes (Ced-3, -4, and -9) that control the process of programmed cell death in Caenorhabditis elegans. Ced-9 is homologous of mammalian ▶BCL-2 family members and acts upstream of Ced-3 and Ced-4. Ced-4 is homologous of APAF-1 and Ced-3 is homologous of the proapoptotic cysteine proteases known as caspases. ▶APAF-1 Signaling ▶Apoptosis

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9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b, 13,14,14a,14b-tetradecahydropicene-2-carboxylic acid

Definition Celastrol is a natural quione methide friedelane tripterene, widely found in the plant genuses of celastrus, maytenus and tripterygium, all of which are present in China. For example, celastrol is one of the active components extracted from tripterygium wilfordii Hook F, an ivy-like vine also known as “Thunder of God Vine,” which belongs to the family of celastraceae and has been used as a natural medicine in China for hundreds of years (Fig. 1).

Characteristics Biological Properties Celastrol has strong antifungal, anti-inflammatory and antioxidant effects. It has been shown that celastrol isolated from the roots of Celastrus hypoleucus (Oliv) Warb f argutior Loes exhibited inhibitory effects against diverse phytopathogenic fungi. Celastrol was also found to inhibit the mycelial growth of Rhizoctonia solani

CED-4 Definition Homologous of APAF-1 in C. elegans. CED-4 is one of three genes (Ced-3, -4 and -9) that control the process of programmed cell death in C. elegans. ▶APAF-1 Signaling ▶CED-3

Celastrol Q ING P ING D OU 1 , X IAO Y UAN 2 1

The Prevention Program, Barbara Ann Karmanos Cancer Institute and Department of Pathology, School of Medicine, Wayne State University, Detroit, MI, USA 2 Research and Development Center, Wuhan Botanical Garden, Chinese Academy of Science, Wuhan, Hubei, People’s Republic of China

Synonyms Tripterine; Quione methide friedelane tripterene (2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,

Celastrol. Figure 1 The chemical structure and nucleophilic susceptibility of celastrol. (a) The chemical structure of celastrol is shown. (b) Nucleophilic susceptibility of celastrol analyzed using CAChe software. Higher susceptibility was shown at the C2 and C6 positions of celastrol.

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Kuhn and Glomerella cingulata (Stonem) Spauld and Schrenk in vitro. Furthermore, celastrol has good preventive effect and curative effect against wheat powdery mildew in vivo. Celastrol in low nanomolar concentrations suppresses the production of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) by human monocytes and macrophages. Celastrol also decreases the induction of class II major histocompatibility complex (▶MHC) expression by microglia. In macrophage lineage cells and endothelial cells, celastrol decreases induction of nitric oxide (NO) production. Celastrol also suppresses adjuvant arthritis in the rat, demonstrating in vivo anti-inflammatory activity. Low doses of celastrol administered to rats could significantly improve the performance of these animals in memory, learning and psychomotor activity. In an isolated rat liver assay of lipid peroxidation, the antioxidant potency of celastrol (IC50 7 µM) is 15 times stronger than that of α-tocopherol or vitamine E. Under in vitro conditions, celastrol was found to inhibit ▶cancer cell proliferation and induce programmed cell death (or ▶apoptosis) in a broad range of tumor cell lines, including 60 National Cancer Institute (NCI) human cancer cell lines. As a ▶topoisomerase II inhibitor, celastrol was 5-fold more potent than the wellknown topoisomerase inhibitor etoposide to induce apoptosis in HL-60 leukemia cells. Celastrol was also found to be a tumor ▶angiogenesis inhibitor. In a sharp comparison, celastrol can block neuronal cell death in cultured cells and in animal models. These unique features of celastrol suggest potential use for treatment of cancer and neurodegenerative diseases accompanied by inflammation, such as Alzheimer’s disease. Potential Molecular Targets Celastrol is a naturally occurring potent inhibitor of the ▶proteasome and nuclear factor kappa B (NFκB). Proteasome, or 26S proteasome, is a multicatalytic protease complex consisted of a 20S catalytic particle capped by two 19S regulatory particles. The ubiquitinproteasome pathway is responsible for the degradation of most endogenous proteins involved in gene transcription, cell cycle progression, differentiation, senescence and apoptosis. Inhibition of the proteasomal chymotrypsin-like, but not trypsin-like activity is associated with induction of apoptosis in tumor cells. Both computational and experimental data support the hypothesis that celastrol is a natural proteasome inhibitor. Atomic orbital energy analysis demonstrates high susceptibility of C2 on A-ring and C6 on B-ring of celastrol toward a nucleophilic attack. Computational modeling shows that celastrol binds to the proteasomal chymotrypsin site (β5 subunit) in an orientation and conformation that is suitable for a nucleophilic attack

Celastrol. Figure 2 Docking solution of celastrol. Celastrol was docked to S1 pocket of β5 subunit of 20S proteasome. Celastrol was shown in pink while β5 subunit was shown in purple. The selected conformation with 92% possibility showed the distances to the OH group of N-Thr from C6 and C2 were 2.96 Å and 4.16 Å, respectively.

by the hydroxyl (OH) group of N-terminal threonine of β5 subunit. The distances to the OH of N-terminal threonine of β5 from the electrophilic C6 and C2 of celastrol are measured as 2.96 Å and 4.16 Å, respectively. Both carbons, more probably C6, of celastrol potentially interact with N-terminal threonine of β5 subunit and inhibit the proteasomal chymotrypsin-like activity (Fig. 2). Celastrol potently and preferentially inhibits the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value 2.5 μM. Celastrol at 1–5 μM inhibits the proteasomal activity in intact human prostate cancer cells. The inhibition of the cellular proteasome activity by celastrol results in accumulation of ubiquitinated proteins and three natural proteasome substrates, ▶IκB-α, Bax and p27, leading to induction of apoptosis in ▶androgen receptor (AR)-negative PC-3 cells. In AR-positive LNCaP cells, celastrol-mediated proteasome inhibition was accompanied by suppression of AR protein, probably by inhibiting ATP-binding activity of heat shock protein 90 (Hsp90) that is responsible for AR folding. Treatment of PC-3 tumor-bearing nude mice with celastrol (1–3 mg/kg/day, i.p., for 1–31 days) resulted in significant inhibition (65–93%) of the tumor growth. Multiple assays using the animal tumor tissue samples from both early and end time-points demonstrated in vivo inhibition of the proteasomal activity and induction of apoptosis after celastrol treatment. Antitumor activity of celastrol was also observed in a breast cancer mouse model. Celastrol inhibited 60% tumor growth in breast cancer xenograft through

Celecoxib

NFκB inhibition. NFκB inhibition by celastrol includes inhibition of its DNA-binding activity and inhibition of IκBα degradation induced by TNF-α or phorbol myristyl acetate. Further investigation showed that the cysteine-179 in the IκBα kinase was a potential target of celastrol-suppressed IκBα degradation. Since the proteasome is required for the activation of NFκB by degrading IκBα, the proteasome inhibition may also contribute to the NFκB inhibition by celastrol. TNF could send both anti-apoptotic and pro-apoptotic signals. The effects of celastrol on cellular responses activated by the potent proinflammatory cytokine TNF have also been investigated. Celastrol was able to potentiate the apoptosis induced by TNF and chemotherapeutic agents and inhibited invasion, both regulated by NFκB activation. TNF induced the expression of gene products involved in anti-apoptosis (IAP1, IAP2, ▶Bcl-2, Bcl-XL, c-FLIP, and survivin), proliferation (cyclin D1 and COX-2), invasion (MMP-9), and angiogenesis (VEGF), and celastrol treatment suppressed the expression of these genes. Celastrol also suppressed both inducible and constitutive NFκB activation. Furthermore, celastrol was found to inhibit the TNF-induced activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 nuclear translocation and phosphorylation, and NFκB-mediated reporter gene expression. Therefore, celastrol potentiates TNF-induced apoptosis and inhibits invasion through suppression of the NFκB pathway.

Clinical Relevance Due to its antioxidant or anti-inflammatory effects, celastrol has been effectively used in the treatment of autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus), asthma, chronic inflammation, and neurodegenerative diseases. As a bioactive component in Chinese traditional medicinal products from the extract of the roots of Tripterygium wilfordii Hook F, celastrol has been used since 1960s in China for autoimmune diseases, but has showed some side effects such as nausea, vomit, etc. Celastrol has not been used solely as a medication product. Celastrol has anti-tumor activities via inhibition of the proteasome and NFκB activation, indicating that celastrol has a great potential to be used for cancer prevention and treatment. This finding can be applied to various human cancers and diseases in which the proteasome is involved and on which celastrol has an effect.

References 1. Setty AR, Sigal LH (2005) Herbal medications commonly used in the practice of rheumatology: mechanisms of action, efficacy, and side effects. Semin Arthritis Rheum 34:773–784

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2. Sassa H, Takaishi Y, Terada H (1990) The triterpene celastrol as a very potent inhibitor of lipid peroxidation in mitochondria. Biochem Biophys Res Commun 172:890–897 3. Yang HJ, Chen D, Cui QZC et al. (2006) Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine,” is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res 66:4758–4765 4. Hieronymus H, Lamb J, Ross KN et al. (2006) Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators. Cancer Cell 10:321–330 5. Sethi G, Ahn KS, Pandey MK et al. (2006) Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NFκB-regulated gene products and TAK1-mediated NF-κB activation. Blood 109:2727–2735

Celebra ▶Celecoxib

Celebrex ▶Celecoxib

Celecoxib N UMSEN H AIL 1 , R EUBEN LOTAN 2 1

Department of Pharmaceutical Sciences, The University of Colorado at Denver and Health Sciences Center, Denver, CO, USA 2 Department of Thoracic Head and Neck Medical Oncology, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA

Synonyms Celebrex; Celebra; 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide

Characteristics Celecoxib, a diaryl-substituted pyrazole drug, was developed by G. D. Searle & Company, and is currently

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marketed by Pfizer Incorporated under the brand names Celebrex and Celebra. Celecoxib is a member of the class of agents known as ▶non-steroidal antiinflammatory drugs (▶NSAIDs). NSAIDs are the most commonly used therapeutic agents for the treatment of acute pain, fever, menstrual symptoms, osteoarthritis, and rheumatoid arthritis. Because of their ability to reduce tissue ▶inflammation, which is often associated with ▶tumorigenesis at various sites in the body (e.g., gastrointestinal tract and lung), celecoxib and certain other NSAIDs are also considered to have a potential in ▶cancer chemoprevention as exemplified by their ability to prevent the formation and decrease the size of polyps in ▶familial adenomatous polyposis (FAP) patients. Orally administered celecoxib exhibits good systemic bioavailability and tissue distribution with an estimated plasma half-life of approximately 11 h. Celecoxib binds to plasma albumin, and is metabolized primarily by hepatic enzymes prior to excretion. In humans, long-term exposures to celecoxib taken for arthritis pain relief at 100 mg twice daily caused no biologically significant adverse reactions. However, higher doses of 400 mg twice daily recommended for patients with FAP resulted in threefold increased risk of cardiovascular events (Fig. 1). ▶Cyclooxygenase Dependent Mechanisms for Cancer Chemoprevention by Celecoxib. Cyclooxygenases are enzymes that are indispensable for the synthesis of ▶prostaglandins. Prostaglandins are ▶hormones generated from ▶arachidonic acid, and they are found in virtually all tissues and organs. Prostaglandins typically act as short-lived local cell signaling intermediates that regulate processes associated with inflammation. In the early 1990s, cyclooxygenases were demonstrated to exist as two isoforms, cyclooxygenase-1 (COX-1), and cyclooxygenase-2 (COX-2). COX-1 is characterized as a constitutively expressed housekeeping enzyme that mediates physiological responses like platelet aggregation, gastric cytoprotection, and the regulation of renal

Celecoxib. Figure 1 The chemical structure of celecoxib.

blood flow. In contrast, COX-2 is recognized as the inducible cyclooxygenase isoform that is primarily responsible for the synthesis of the prostaglandins that are involved in pathological processes (e.g., chronic inflammation) in cells that mediate inflammation (e.g., macrophages and monocytes). COX-2 is inducible by ▶oncogenes (e.g., ▶RAS and ▶SRC), interleukin-1, ▶hypoxia, benzo[a]pyrene, ultraviolet light, epidermal growth factor, ▶transforming growth factor β, tumor necrosis factor α. Many of these inducers activate nuclear factor kappa B (NF-κB), which controls COX-2 expression and has been associated with tumorigenesis in various cell types. The COX-2 isoenzyme is frequently unregulated in cancer cells, as well as cells that constitute premalignant lesions, which are important targets for ▶cancer chemoprevention. The expression of the inducible COX-2 is enhanced in 50% of colon adenomas and in the majority of human ▶colorectal cancers, as opposed to COX-1, which typically remains unchanged. Thus, the increase in COX-2 expression, which is an early event in colon carcinogenesis, is believed to be necessary for tumor promotion. Aberrant COX2 expression has also been implicated in tumorigenesis in the lung, ▶prostate, esophagus, ▶breast, ▶liver, ▶pancreas, and ▶skin. The activity of COX-2 to produce arachidonic acid metabolites appears to enhance the proliferation of transformed cells and/or increases their survival through the suppression of ▶apoptosis. Furthermore, COX-2 expression by tumor cells can stimulate ▶angiogenesis at the tumor site and alter tumor cell adhesion to promote ▶metastasis. Celecoxib is a highly selective inhibitor of COX-2. Traditional NSAIDs (e.g., aspirin) inhibit both COX-1 and COX-2 isozymes. In contrast, celecoxib is approximately 20 times more selective for COX-2 inhibition compared to its inhibition of COX-1. This specificity allows celecoxib, and other selective COX2 inhibitors, to reduce inflammation while minimizing adverse drug reactions (e.g., stomach ulcers and reduced platelet aggregation) that are common with nonselective NSAIDs. This selectivity for COX-2 is also intimately associated with the putative cancer chemopreventive activity of celecoxib, which has been demonstrated in ▶colorectal cancer prevention. Epidemiological studies have shown that persons who regularly take aspirin have about a 50% lower risk of developing colorectal cancer. Celecoxib was the most effective ▶NSAID in reducing the incidence and multiplicity of colon tumors in a rat colon carcinogenesis model. Moreover, in a clinical setting celecoxib has been used effectively to suppress the development and/ or reduce the number of colorectal polyps in patients with FAP. This inflammatory disease often predisposes individuals to the development of ▶colorectal cancers. The anti-inflammatory mediated anticancer effects of

Celecoxib

celecoxib may be tissue-specific considering that celecoxib reduced lung inflammation in mice, but failed to inhibit the formation of chemically induced lung tumors in these animals. Cyclooxygenase Independent Mechanisms for Cancer Chemoprevention by Celecoxib. The results of several in vitro and animal studies suggest the celecoxib may suppress tumorigenesis through several COX-2independent mechanisms, which may account, at least in part, for celecoxib’s anti-cancer effects in humans. For example, celecoxib inhibited the proliferation of various cancer cell types in vitro irrespective of their expression of COX-2, including transformed haematopoietic cells and immortalized and transformed human bronchial epithelial cells that were deficient in COX2 expression. Celecoxib also inhibited the growth of human COX-2-deficient ▶colon cancer cells that were transplanted as xenografts in nude mice. Thus, the chemopreventive effect of COX-2-specific inhibitors like celecoxib may be due to their effect on COX-2 as well as targets other than COX-2. One putative COX-2 independent target for celecoxib is the ▶phosphatidylinositol 3-kinase (PI3K) pathway, which is often deregulated in tumor cells. Celecoxib appears to directly inhibit the phosphoinositide-dependent kinase-1 (PDK1), and its downstream substrate protein kinase B/AKT, in the ▶PI3K pathway. Protein kinase B/AKT inhibits apoptosis through the ▶phosphorylation, and thus inactivation, of the proapoptotic ▶BCL-2 family protein BAD. During apoptotic stimuli, BAD antagonizes BCL-2 and BCL-XL activity, which can promote ▶mitochondrial membrane permeabilization and cell death. The inhibition of the PI3K pathway by celecoxib is believed to be specific in its ability to promote apoptosis in transformed cells. For example, rofecoxib, another specific COX-2 inhibitor, had only marginal protein kinase B/AKT inhibitory activity in tumor cells during apoptosis induction. Another presumed COX-2 independent target of celecoxib in tumor cells is ▶sphingolipid metabolism. Celecoxib treatment increases the level of the ▶sphingolipid ceramide in murine mammary tumor cells irrespective of COX-2 expression. This increase in ▶ceramide was considered essential to apoptosis induction in these cells. Ceramide has been shown to mediate apoptosis in response to inflammatory cytokines like Fas and tumor necrosis factor α, and/or conditions associated with ▶oxidative stress. During conditions of cell stress, the deregulation of ceramide generating and/or utilizing processes are believed to cause a net increase in cellular ceramide that is sufficient to trigger apoptosis induction via a mitochondrial membrane permeabilization mechanism. Celecoxib treatment has also been shown to suppress the activity of the ▶Ca2± ATPase located in the

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endoplasmic reticulum of human ▶prostate cancer cells. The inhibition of the Ca2± ATPase by celecoxib disrupted Ca2+ homeostasis in the prostate cancer cells. This activity was highly specific for celecoxib, and was not associated with the exposure to other COX2 inhibitors, including rofecoxib. Microsome and plasma membrane preparations from the human prostate cancer cells showed that only the Ca2± ATPases located in the endoplasmic reticulum were the direct targets of celecoxib. The disruption of Ca2+ homeostasis played a central role in apoptosis induction in the prostate cancer cells because it was required for the activation of Ca2+-dependet hydrolyses that carried out cellular degradation. Moreover, mitochondrial membrane permeabilization, which releases cytochrome c to activate cell death, is sensitive to elevations in intracellular free Ca2+. Consequently, the celecoxibinduced inhibition Ca2± ATPases located in the endoplasmic reticulum may provide a link to mitochondrial membrane permeabilization for apoptosis induction much in the same way that Celecoxib inhibition of the PI3K pathway can regulate BAD phosphorylation to trigger mitochondrial-mediated cell death. It is apparent that the central hypothesis of a dominant role for COX-2 inhibition in cancer prevention by celecoxib may need re-examination. Furthermore, the COX-2 dependent and independent action of celecoxib in cancer prevention may be tissue specific. Since the aberrant expression of COX-2 is implicated in the pathogenesis of various types of human cancers, perhaps this inducible enzyme may be a useful surrogate ▶biomarker of the anticancer activity of celecoxib when evaluating the chemoprevention of cancer at various sites in the body. Although the precise molecular mechanism for its chemopreventive effects are still fairly unknown, celecoxib may be still useful as a chemopreventive agent for a variety of malignancies, especially since it triggers less toxicity and adverse side effects during long-tern use when compared to traditional NSAIDs. Celecoxib may be useful when combined with other cancer chemopreventive/ therapeutic agents to control the process of tumorigenesis.

References 1. Chun KS, Surh JY (2006) Signal transduction pathways regulating cyclooxygenase-2 expression: potential molecular targets for chemoprevention. Biochem Pharmacol 68:1089–1100 2. Grosch S, Maier TJ, Schiffmann S et al. (2006) Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 98:736–747 3. Kismet K, Akay MT, Abbasoglu O et al. (2004) Celecoxib: a potent cyclooxygenase-2 inhibitor in cancer prevention. Cancer detect Prev 28:127–142

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4. Schroeder CP, Kadara H, Lotan D et al. (2006) Involvement of mitochondrial and akt signaling pathways in augmented apoptosis induced by a combination of low doses of celecoxib and N-(4-hydroxyphenyl) retinamide in premalignant human bronchial epithelial cells. Cancer Res 66:9762–9770 5. Psaty BM, Potter JD (2006) Risks and benefits of celecoxib to prevent recurrent adenomas. N Engl J Med 355:950–952

Cell Adhesion Molecules K RIS V LEMINCKX Department of Molecular Biology, Ghent University, Department of Molecular Biomedical Research, VIB, Ghent, Belgium

Synonyms Cell adhesion receptors; Adhesion molecules; CAMs

Definition

Cell ▶adhesion molecules are transmembrane or membrane-linked glycoproteins that mediate the connections between cells or the attachment of cells to substrate (such as stroma or basement membrane). Dynamic cell-cell and cell-substrate adhesion is a major morphogenetic factor in developing multicellular organisms. In adult animals, adhesive mechanisms underlie the maintenance of tissue architecture, allow the generation of force and movement, and guarantee the functionality of the organs (e.g. to create barriers in secreting organs, intestines and blood vessels) as well as the generation and maintenance of neuronal connections. Cell adhesion is also an integrated component of the immune system and wound healing. At the cellular level, cell adhesion molecules do not function just as molecular glue. Several signaling functions have been attributed to adhesion molecules, and cell adhesion is involved in processes such as ▶contact inhibition, growth and ▶apoptosis. Deficiencies in the function of cell adhesion molecules underlie a wide variety of human diseases including cancer. By their adhesive activities and their dialogue with the ▶cytoskeleton, adhesion molecules directly influence the invasive and metastatic behavior of tumor cells, and by their signaling function they can be involved in the initiation of tumorigenesis.

Characteristics At the molecular level, cell adhesion is mediated by molecules that are exposed on the external surface of the cell and are somehow physically linked to the cell membrane. In essence, there are three possible

mechanisms by which such membrane-attached adhesion molecules link cells to each other (Fig. 1a). First, molecules on one cell bind directly to similar molecules on the other cell (▶homophilic adhesion). Secondly, adhesion molecules on one cell bind to other adhesion receptors on the other cell (▶heterophilic adhesion). Finally, two different adhesion molecules on two cells may both bind to a shared secreted multivalent ligand in the extracellular space. Also, cell-cell adhesion between two identical cells is called ▶homotypic (cell) adhesion, while ▶heterotypic (cell) adhesion takes place between two different cell types. In the case of cellsubstrate adhesion the adhesion molecules bind to the ▶extracellular matrix (ECM). Cell Adhesion Molecules and the Cytoskeleton Adhesion molecules can be associated with the cell membrane either by a glycosylphosphatidyl-inositol (GPI) anchor or by a membrane-spanning region. In the latter case the cytoplasmic part of the molecule often associates indirectly with components of the cytoskeleton (e.g. actin, intermediate filaments or submembranous cortex). This implies that adhesion molecules, which by themselves establish extracellular contacts, can be structurally integrated with the intracellular cytoskeleton, and they are often clustered in specific restricted areas in the membrane, the so-called ▶junctional complex (Fig. 1b). This combined behavior of linkage to the cytoskeleton and clustering, considerably strengthens the adhesive force of the adhesion molecules. In some cases, exposed adhesion molecules can be in a conformational configuration that does not support binding to its adhesion receptor. A signal within the cell can induce a conformational change that activates the adhesion molecule. Dynamic adhesion can also be mediated via regulated endocytosis of the adhesion molecules. These mechanisms of regulation allow for a dynamic process of cell adhesion that, amongst others, is required for morphogenesis during development and for efficient immunological defense. Classification of Cell Adhesion Molecules Based on their molecular structure and mode of interaction, five classes of adhesion molecules are generally distinguished; the ▶cadherins, ▶integrins, immunoglobulin (Ig) superfamily, selectins and ▶proteoglycans (Fig. 2). Cadherins Cadherins and proto-cadherins form a large and diverse group of adhesion receptors. They are Ca2+-dependent adhesion molecules, involved in a variety of adhesive interactions both in the embryo and the adult. Cadherins play a fundamental role in metazoan embryos, from the earliest gross morphogenetic events (e.g. separation of germ layers during gastrulation) to the most delicate


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Cell Adhesion Molecules. Figure 1 Different modes of cell-cell and cell-substrate adhesion and the mechanism of cytoskeletal strengthening. (a) Three possible mechanisms by which cell adhesion molecules mediate intercellular adhesion. A cell surface molecule can bind to an identical molecule (homophilic adhesion) on the opposing cell or can interact with another adhesion receptor (heterophilic adhesion). Alternatively, cell adhesion receptors on two neighboring cells can bind to the same multivalent, secreted ligand (linkermediated adhesion). Intercellular adhesion can take place between identical cell types (▶homotypic adhesion) or between cells of different origin (▶heterotypic adhesion), independently of the involved adhesion molecules. Cell-substrate adhesion molecules attach cells to specific compounds of the extracellular matrix. Cell-cell and cell-substrate adhesion can occur simultaneously. (b) Intercellular and cell-substrate adhesion can be strengthened by indirect intracellular linkage of the cytoplasmic tail of the adhesion molecules to the cytoskeleton and by lateral clustering in the membrane.

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Cell Adhesion Molecules. Figure 2 The five major classes of cell adhesion molecules and their binding partners. Cadherins are Ca2+-dependent adhesion molecules that consist of a varying number of cadherin repeats (five in case of the classical cadherins). The conformation and activity of cadherins is highly dependent on the presence of Ca2+-ions. In general, cadherin binding is homophilic. Integrins are functional as heterodimers and consist of an a- and b-subunit. They interact with members of the immunoglobulin superfamily or with compounds of the extracellular matrix (e.g. fibronectin, laminin). Members of the immunoglobulin superfamily (Ig-like proteins) are characterized by a various number of immunoglobulin-like domains (open circles). Membrane-proximal, fibronectin type III repeats are often observed (gray boxes). They can either bind to other members of the Ig-family (homophilic) or to integrins. Selectins contain an N-terminal Ca2+-dependent lectin domain (circle) that binds carbohydrates, a single EGF-like repeat (gray box) and a number of repeats that are related to those present in complement-binding proteins (ovals). Proteoglycans are huge molecules that consist of a relatively small protein core to which long side chains of negatively charged glycosaminoglycans are covalently attached. They bind various molecules, including components of the extracellular matrix.

tunings later in development (e.g. molecular wiring of the neural network). The extracellular part of vertebrate classical cadherins consists of a number of cadherin repeats whose conformation is highly dependent on the presence or absence of calcium ions. Homophilic interactions can only be realized in the presence of calcium, usually by the most distal cadherin repeat. Classical cadherins are generally exposed as homodimers, and their cytoplasmic domain can be structurally or functionally associated with the actin

cytoskeleton. Cadherins are the major adhesion molecules in tissues that are subject to high mechanical stress such as epithelia (▶E-cadherin) and endothelia (VEcadherin). However, finer and more elegant intercellular interactions, such as synaptic contacts, also involve cadherins. Integrins Integrins are another group of major players in the field of cell adhesion. They are involved in various processes


such as morphogenesis and tissue integrity, homeostasis, immune response and inflammation. Integrins are a special class of adhesion molecules, not only because they mediate both cell-cell and cell-substrate interactions (with components in the ECM such as laminin, fibronectin and collagen) but also because they function as heterodimers consisting of an α- and β-subunit. To date, at least 16 α-subunits and 8 β-subunits have been indentified. Of the theoretical 128 heterodimeric pairings, at least 21 are known to exist. While most integrin heterodimers bind to ECM components, some of them, more particularly those expressed on leukocytes, are heterophilic adhesion molecules binding to members of the Ig superfamily. The α-subunit mostly contains a ligand-binding domain and requires the binding of divalent cations (Mg2+, Ca2+ and Mn2+, depending on the integrin) for its function. Interestingly, integrins may be present on the cell-surface in a non-functional and a functional configuration. The cytoplasmic domain appears to be responsible for the conformational change that activates the integrin. The Ig Superfamily Among the classes of adhesion molecules discussed here, the Ig superfamily is probably the most diverse. The main representatives are the neural cell adhesion molecules (NCAMs) and V(ascular)CAMs. As the name suggests, the members of this family all contain an extracellular domain consisting of different immunoglobulin-like domains. NCAMs sustain homophilic and heterophilic interactions that play a central role in regulation and organization of neural networks, specifically in neuron-target interactions and fasciculation. The basic extracellular structure consists of a number of Ig domains, which are responsible for homophilic interaction, followed by a discrete number of fibronectin type III repeats. This structure is linked to the membrane either by a GPI anchor or a transmembrane domain. The VCAM subgroup, including I(ntercellular) CAMs and the mucosal vascular addressin adhesion molecule (MAdCAM), are involved in leukocyte trafficking (or homing) and extravasation. They consist of membrane-linked Ig domains that make heterophilic contacts with integrins. Other members of this family that are associated with cancer are carcinoembryonic antigen (▶CEA), “deleted in colon cancer” (DCC) and platelet endothelial (PE)CAM-1. Selectins These types of adhesion molecules depend on carbohydrate structures for their adhesive interactions. Selectins have a C-type ▶lectin domain, that specifically binds to discrete carbohydrate structures present on cell-surface proteins. Intercellular interactions mediated by selectins

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are of particular interest in the immune system, where they play a fundamental role in trafficking and homing of leukocytes. Proteoglycans Proteoglycans are large extracellular proteins consisting of a relatively small protein core to which long chains of glycosaminoglycans are attached. Although poorly documented, proteoglycans may bind to each other or may be the attachment site for other adhesion molecules. Role of Adhesion Molecules in Cancer The Metastatic Cascade Cell adhesion molecules play an important role during the progression of tumors, more particularly in the metastatic cascade (Fig. 3). When a benign tumor becomes malignant, cells at the periphery of the tumor will lose cell-cell contact (step I) and invade the surrounding stroma (step II) (see also ▶invasion). Cells then extravasate and enter the vasculature or lymphatic system, where they are further transported. A fraction of the circulating tumor cells survives and is arrested at a distant site, attaches to the endothelium (step III) and extravasates through the blood vessel wall and into the surrounding tissue (step IV). Here the tumor cells grow, attract blood vessels and develop to a secondary tumor (▶metastasis). Adhesive Events in Metastasis All the classes of cell adhesion molecules play a role in the metastatic cascade. During the first step, tumor cells need to disrupt intercellular junctions in order to detach from the primary tumor. This step often involves suppression of cadherin function. The second step of ▶migration through the stroma and into the blood or lymphatic vessels requires dynamic cell-substrate adhesion, mostly mediated by integrins. In the third step, where cells arrest in the circulation by aggregation with each other or attachment to platelets, leukocytes and endothelial cells, critical roles have been attributed to cell adhesion molecules of the Ig superfamily, selectins, integrins and specific membrane-associated carbohydrates. The fourth step is similar to step II and mostly involves integrins. Details on the adhesive events associated with metastasis are outlined below. . In benign epithelial tumors, cells maintain firm intercellular adhesive contacts, mostly by formation of a junctional complex (including tight junctions, ▶adherens junctions and desmosomes). Establishment and maintenance of such a strong junctional complex requires expression and function of cadherins (more particularly E-cadherin). Loss of E-cadherin expression or function appears to be a hallmark of progression of a benign epithelial tumor (adenoma)

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Cell Adhesion Molecules. Figure 3 Cell adhesion processes involved in the metastatic cascade. A subset of cells (gray) growing in a primary tumor will reduce cell-cell contacts (Step I) and migrate in the surrounding stroma by increasing specific cell-substrate adhesion (Step II). These invasive tumor cells can extravasate into the circulation and, at distant sites, attach to the endothelial blood vessel wall through specific cell-cell interactions (Step III). Once these cells have extravasated through the vessel wall they use cell-substrate adhesion molecules to invade the surrounding stroma (Step IV). See text for details.

to a malignant one (carcinoma). Epithelial tumor cells often acquire invasive properties by mutational inactivation of E-cadherin or one of its cytoplasmic binding partners (catenins). It is important to keep in mind that cadherin-mediated adhesion is a dynamic process and that E-cadherin can be temporarily inactivated at the functional level, for example by phosphorylation or other posttranslational modifications. E-cadherin and other molecules of the junctional complex are very often suppressed or functionally modulated in the epithelial-mesenchymal transitions (EMT), a hallmark of malignant tumor progression. EMT can be a tumorintrinsic feature or can be induced by their microenvironment. Paracrine factors such as scatter factor or juxtacrine signaling via Ephrin/Eph receptor or via Semaphorins/plexins can affect adhesion via direct activity on the cell adhesion molecules or via regulation of the cytoskeleton. . Dynamic cell-substrate adhesion is a critical factor in the migration of invasive tumor cells into the surrounding stroma. Integrins are instrumental in this process. Several studies have correlated the migratory behavior of tumor cells either with an increased or decreased expression of particular integrins. This apparent paradox may be explained by the fact that firm but temporary cell-substrate contacts are required for cells to migrate on a substrate. In order to crawl directionally through the stroma, a cell needs to “grab” the ECM, release after pulling itself forward and then has to establish the next contact. Both inhibiting adhesion and preventing

release of the substrate contacts “locks” the cell in its position and prevents migration. It should be remembered that integrins may exist in two functional states and that signals passed through the cytoplasm determine whether membrane-exposed integrins are functional or not. . In the third step of the metastatic cascade, cell-cell interactions are again the most determining. Homotypic interactions between circulating tumor cells promote formation of aggregates that are preferentially retained in the capillary network. PECAM-1 is a cell adhesion molecule potentially involved in this process. It should be pointed out that (re)expression of the invasion-suppressor molecule E-cadherin would actually promote metastasis formation. Besides these homotypic interactions, heterotypic interactions are also of major importance in the metastatic process. Tumor cells can attach to the blood-vessel wall either directly or indirectly through platelets and leukocytes. The adhesion molecules involved in this process are similar to those involved in the “multistep adhesion cascade” observed during homing and extravasation of leukocytes or trafficking of lymphocytes. Cell adhesion events include interactions of tumor-associated lectins with selectins expressed on platelets, leukocytes and endothelium (P-, L- and ▶E-selectins, respectively). These adhesion molecules are also involved in the initial transient low-affinity interactions (rolling) of circulating leukocytes (and probably tumor cells) with the endothelium. Other and

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more stringent heterotypic heterophilic interactions in this metastatic stage include the binding of integrins on tumor cells to ICAMs expressed on the surface of the endothelial cells. . The fourth step in the metastatic cascade is extravasation and invasion at a distant site. This process is very similar to step 2 and the same adhesion molecules are likely to be involved. Specific interactions of the tumor cells with molecules present on the endothelial cells (e.g. N-cadherin) will facilitate the extravasation process.

Other Cancer-Related Functions of Cell Adhesion Molecules Recently, it has become clear that some cell adhesion molecules are involved in signaling processes that are relevant to cancer. Germline mutations in E-cadherin predispose patients to the development of diffuse gastric carcinomas, and in lobular breast carcinoma E-cadherin seems to act as a tumor suppressor. Interestingly, β-catenin, a protein cytoplasmically linked to cadherins, has a central role in ▶Wnt signaling and has oncogenic properties that are counteracted by the adenomatous polyposis coli (▶APC) gene product. Signaling by integrins can also be an important factor that prevents cells from undergoing apoptosis (apoptosis upon loss of cell adhesion is called ▶anoikis), which might be critical when tumor cells are traveling in the circulation. Interdisciplinary research has revealed new unexpected functions for known cell adhesion molecules. The suspected tumor suppressor DCC, a member of the Ig superfamily of adhesion molecules, turned out to be the receptor for netrin-1, an axonal chemoattractant crucial in neuronal development. Other molecules known to have adhesive or repulsive activities in the axonal growth cone or in migrating neural crest cells, turn out to have similar activities in tumor cells (see also the chapters on ▶EPH receptors, ▶Ephrin signaling in cancer, ▶Semaphorins and ▶Plexins).

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Cell-Adhesion Molecules (CAM) Definition Are cell-surface proteins that are involved in binding cells together in tissues and also in less permanent cell– cell interactions. ▶Adhesion

Cell Adhesion Receptors ▶Cell Adhesion Molecules

Cell Biology F ILIPPO A CCONCIA 1,2 , R AKESH K UMAR 1,3 1

Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA 2 IFOM, The FIRC institute for Molecular Oncology, Milan, Italy 3 Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

Definition Cell biology deals with all aspects of the normal and of the tumor cell, their normal and abnormal multiplication, their differentiation, their stem origins, and their regulated cell death.

Characteristics References 1. Cavallaro U, Christofori G (2004) Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4:118–132 2. Chothia C, Jones EY (1997) The molecular structure of cell adhesion molecules. Annu Rev Biochem 66:823–862 3. Hynes RO (2000) Cell adhesion: old and new questions. Trends Cell Biol 9:M33–M37 4. Mizejewski GJ (1999) Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 222:124–138 5. Sanderson RD (2001) Heparan sulfate proteoglycans in invasion and metastasis. Semin Cell Dev Biol 12:89–98

The Cell The intracellular environment is separated from the external environment by a lipid bilayer called plasma membrane. The plasma membrane controls the movement of substances in and out of the cell and it is important for the cell to sense the surrounding environment. Within the cell the nucleus occupies most of the space. The cell nucleus contains genes, which drive all cellular activities and processes. Genes are organized in chromosomes (i.e., genome) and are made of DNA. The genetic information is used to produce proteins, which are the critical effectors required for all cellular processes. The nucleus is separated from the rest of the cellular content by the nuclear membrane,

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which remains in contact with the cytoplasm as well as the nucleoplasm. In the cytoplasm, proteins are organized into specific functional structures and also connected with the structural network referred to as cytoskeleton network, which physically sustains the cell. Moreover several intracellular organelles are located in the cytoplasm (e.g., mitochondria, Golgi apparatus) and allow the cells to self sustain. To continuously adjust the intracellular processes and to promptly respond to the demands of the extracellular environment, cells need to exchange matter, energy, and information with the external milieu. Cell Division and Reproduction One of the unique features of cell is its ability to divide and produce two daughter cells that are an exact copy of their parental cell, by a process called “mitosis.” However, some differentiated cells undergo the process of meiosis. For simplicity, meiotic division can be considered as the sum of two successive mitotic divisions, which result in four daughter cells with half the number of chromosomes and rearranged genes. These specialized cells (i.e., gametes) serve as reproductive cells. The fusion of the female and male gametes (eggs and spermatozoa, respectively) results in a new cell called zygote. The zygote, by definition, is a stem cell. Following mitotic division, it becomes an embryo and, at the end of the embryonic development, results in a new organism. Cell Proliferation The physiological functions of an organ require maintenance of homeostasis, a process of regulated balance between cell proliferation and cell death (also known as ▶apoptosis), in the differentiated tissue. Indeed, a variety of extracellular stimuli activate specific ▶signal transduction pathways that affect the expression and activity of molecules involved in the control of cell proliferation or cell death. Thus, the balance between ▶cell cycle progression and apoptosis defines the cell fate, and this process depends on genetic factors as well as the kinetics of signal transduction pathways in exponentially growing cells. Cell Cycle In mammalian cells, one cell cycle takes about 24 h in most cell types and can be schematically divided into two stages: mitosis and interphase. Mitosis (M phase) consists of a series of molecular processes that result in cell division. On the other hand, the interphase can be subdivided into three major gaps (G1, S, and G2 phase). The G1 phase of the cell cycle separates the M and S phases. In G1 phase, cells express a specific pattern of gene products required for the DNA synthesis; the G2 phase of the cell cycle resides in between the S and M phases and is important for the

completion of processes that are necessary for mitosis. The G0 phase of the cell cycle is entered by the cells from the G1. In the G0 phase, cells are out of the cell cycle and into a quiescent state where they do not proliferate. Regulation of Cell Cycle Progression Cell cycle progression is achieved through a series of coordinated molecular events that allow the cells to transit across the restriction points, also known as cell cycle checkpoints. There are three main restriction points in the cell cycle (G2/M, M/G1, and G1/S, respectively). Broadly, these checkpoints are defined as points after which the cell is committed to progress to the next phase in a nonreversible manner. Therefore, the transition between the phases of the cell cycle is strictly regulated by a specific set of proteins. ▶Cyclindependent kinases (CDK) act in various phases of the cell cycle by binding to its activating proteins called cyclins. For example, both ▶cyclin D/CDK4 and cyclin E/CDK2 complexes regulate transition of the cells through G1/S phase whereas cyclin A/CDK1, cyclin A/ CDK2, and cyclin B/CDK1 complexes are active during the rest of the cell cycle. On the other hand, another class of regulatory proteins, the cyclin-dependent kinase inhibitors (CKI) (e.g., p21Cip/Kip; p19Ink4d) antagonizes the activation of CDK activity, thus impeding the progression of the cell cycle. Programmed Cell Death Programmed cell death (PCD) is a physiological process of eliminating a living cell. The PCD involves activation of specific intracellular programs that commit cells to a “suicidal route.” The process of PCD plays an important role in a variety of biological events, including morphogenesis, maintenance of tissue homeostasis, and elimination of harmful cells. To date, different forms of PCD have been described among which apoptosis, necrosis, and ▶autophagy are the most common. Apoptosis One of the critical events in apoptosis is the activation of cystein proteases, called caspases, upon a given signal. The initiator caspases (▶Caspase 8 and 9) are the first enzymes involved in the activation of the apoptotic cascade. Caspase 8 and 9 activate the downstream effector caspases (caspase 3, 6, and 7) by proteolytic cleavage which in turn results in the hydrolysis and inactivation of the enzymes involved in the processes of DNA repair such as by poly-ADPribose polymerase (PARP). Upon stimulation of apoptotic cascade, cells display a specific set of characters, which constitute the hallmark of apoptosis (DNA fragmentation, cell shrinkage, cytoplasmic budding, and fragmentation). The activation of caspases

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is achieved through two principle pathways – an extrinsic pathway that transduces signals from the plasma membrane directly to the caspases, and an intrinsic pathway that involves activation of caspases through a series of biochemical events leading to permeabilization of the mitochondrial membrane and release of cytochrome c (▶Cytochrome P450) in the cytoplasm. Apoptotic cells are eventually eliminated by the immune system without the activation of inflammatory reactions (▶Inflammation). Necrosis Necrosis results from a severe physical, mechanical, or metabolic cellular damage. The necrotic phenotype is very different from those of an apoptotic cells. Overall, the cell switches off its metabolic pathways and the DNA condenses at the margins of the nucleus and the cellular constituents start to degrade. In general, necrosis consists in a general swelling of the cell before it disintegrates. Furthermore, upon leakage of the intracellular content, necrotic cells stimulate an inflammatory response that usually damages the surrounding tissue. Autophagy Autophagy, i.e., autophagic cell death, occurs by sequestration of intracellular organelles in a double membrane structure termed autophagosome. Subsequently, the autophagosomes are delivered to the lysosomes and degraded. Autophagy is responsible for the turnover of dysfunctional organelles and cytoplasmic proteins and thus, contributes to cytosolic homeostasis. Autophagy can occur either in the absence of detectable signs of apoptosis or concomitantly with apoptosis. Indeed, autophagy is activated by signaling pathways that also control apoptosis. Signal Transduction Extracellular signals are transduced by the activation of a series of phosphorylation-dependent intracellular pathways initiated by cell surface receptors. Eventually, such signals feed into the nucleus, stimulate transcription factors, and regulate gene transcription. Signaling Targets Signaling pathways regulate gene transcription by triggering the promoter activity of the target gene. For example, regulation of cyclin D is critical for cell cycle progression. The extracellular signal-mediated activation of specific signal transduction pathways stimulates the activity of transcription factors such as AP-1, SP-1, and NF-κB, which coordinate the activation of the cyclin D1 promoter and thus lead to cyclin D1 expression. On the other hand, signaling molecules can also change the activity of a preexisting protein. For example, activation of p21-activated kinase (PAK) induces the

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phosphorylation of phosphoglucomutase (PGM) that stimulates its enzyme activity and the phosphorylation of ▶estrogen receptor alpha (ERα) thus inducing its transcriptional activity. One of the most studied signaling pathways is the extracellular-regulated kinase (ERK) (▶MAP kinase) cascade. It consists of three steps of sequential phosphorylations that impact on diverse cellular effectors. The ERK cascade is activated by mitogenic stimuli (e.g., growth factors (▶Fibroblast growth factors)) and plays a critical role both in cell proliferation and cell survival. Indeed, activation of ERK induces the activation of AP-1 transcription factor, which, in turn, regulates cyclin D1 expression in addition to many of other proliferative molecules. Further, ERK activity leads to an increased expression of the antiapoptotic protein ▶Bcl-2 and inactivation of the proapoptotic protein ▶Bad. Conversely, the JNK/SAPK (▶JNK subfamily and cancer) and the p38/MAPK (MAP kinase) pathways mediate stress and apoptotic stimuli (e.g., UV, ischemic-reperfusion damage). Activation of JNK/SAPK and p38/MAPK often results in an increased expression of proapoptotic proteins (e.g., Bax), and in the activation of the caspase cascade and cytochrome c release from the mitochondria. Systems Biology Systems biology represents a new analytical tool that has begun to emerge for balanced comprehensive analyses of cellular pathways at the level of genes and proteins. Signal transduction pathways often cross-talk and influence each other, and the functionality of the effector molecule is influenced by the overall outcome of a set of signaling pathways. Thus, cells form a web of intracellular interactions that are critical for a timely and dynamic response. The intracellular signaling network is considered a complex system rapidly adapting to extracellular challenges. Therefore, an additional level of complication is the evaluation of the network as a whole, rather than the individual pathway. Cell Motility and Migration ▶Motility and ▶migration are important components for the functionality of a variety of cell types, and are involved in physiologic processes such as embryonic development, immune response, as well as in pathologic processes such as ▶invasion and ▶metastasis. Cell motility and migration are coordinated physiological processes that allow the cells to move or to invade the surrounding tissues, respectively. They occur as a result of a complex interplay between the focal ▶adhesion sites (cell-to-substrate contacts) and the ▶extracellular matrix (ECM) (substrate). Phenotypically, migratory cells develop motile structures such as pseudopodia, lamellipodia, and filopodia. An ordered sequence of events (protrusion of motile structures, formation and disruption of focal contacts) generate the traction forces

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that drive the cell movement. Moreover, when migration is required, cells secrete specific proteolytic enzymes (matrix metalloproteinases, MMPs) that digest the ECM, thus opening a passage across the substrate. Cytoskeleton is critical for the correct occurrence of cell motility and migration. Cytoskeleton Cytoskeleton is a network of cytoplasmic proteins, which define the cell “bones.” Many different protein filaments are important for cytoskeleton functions. In particular, microtubules, built from different types of tubulin, originate from specific intracellular structures called microtubules organizing centers (MTOC). Dynamic changes in the polymerization and depolymerization of tubulin maintain microtubule integrity and resulting functions. Furthermore, actin microfilaments form a network of cytoskeleton-associated proteins and connect the focal adhesion with the intracellular cytoskeleton. The dynamic remodeling of microtubules and microfilaments has an impact on cell motility, migration and cell–cell adhesion, ▶endocytosis, intracellular trafficking, organelle function, cell survival, gene expression, and cell division. Signaling Regulation At the focal adhesion sites, cells accumulate receptors (e.g., growth factor receptors), adaptors (e.g., vinculin), and signaling molecules, as well as structural and motor proteins (e.g., actin, myosin). Migration-specific stimuli (e.g., integrins engagement of ECM, growth factor stimulation, and mechanical stimuli) activate specific biochemical pathways. ▶Focal adhesion kinase (FAK), integrin-linked kinase (ILK), PAK, and ▶Src play key roles in modulating cell migration and invasion. The FAK/Src complex regulates the assembly and disassembly of focal contacts, F-actin cytoskeleton remodeling, and the formation of lamellipodia and filopodia through the activation of specific downstream cytoskeleton-associated signaling pathways. Further, ILK is also implicated in cell motility and migration by linking integrins with cytoskeleton dynamics through the ▶PI3K signaling pathway. Also, PAK1 dynamically regulates cytoskeletal changes by coordinating upstream signaling with multiple effectors. By acting on actin reorganization, PAK1 drives directional cell motility and migration. Tumor Biology Cancer is a progressive disease that arises from the clonal expansion of a single transformed cell into a mass of uncontrolled proliferating cells. Tumorigenesis is a multistep process and involves progressive conversion of a normal cell into a malignant cell, which subsequently invades the surrounding tissues. The process of tumorigenesis consists of major steps

(initiation, promotion, and progression), each involving specific molecular mechanisms, often interlaced with each other, that drive tumor development. Initiation and Promotion In general, initiation of tumorigenesis is referred to as the first oncogenic stimulus. However, such as initial event is not sufficient for tumor induction. In most cases, a second oncogenic stimulus must occur in a restricted time frame, thus promoting an irreversible effect. Chemical (e.g., aromatic compounds (▶Polycyclic aromatic hydrocarbons)), physical (e.g., ▶UV radiation), as well as biological (e.g., viruses as ▶human papillomavirus) stress have impact on the cells and can induce DNA mutations (e.g., point mutations). In addition, gene deletion or duplication also alters gene function and contributes to the process of tumorigenesis. These genomic changes result in the production of proteins with altered functions or in the overexpression or downregulation of specific proteins, which affects the associated cellular functions. Protooncogenes or oncogenes are genes that encode for proteins involved in the induction of cell proliferation (e.g., cyclin D1, CDK, EGFR, Src, Ras, etc.) and whose overexpression or hyperactivation leads to an uncontrolled cell proliferation. On the other hand, tumor suppressor genes are genes encoding for proteins that negatively regulate cell proliferation (e.g., p53, PARP, CKI, etc.). Inactivating mutations or downregulation of tumor suppressor genes are also critical for enhanced cell proliferation. In addition to DNA damage, oncogenes and tumor suppressor genes, abnormal changes in the epigenetic cellular information (e.g., DNA▶ methylation) can also participate in clonal evolution of human cancers. Progression The modified balance between the growth-inhibitory programs and proliferative networks allow the cell to escape the physiological growth restrains. These selective growth advantages produce a population of more aggressive or transformed cells that resist clearance by the immune system (i.e., immune defense escape), and in turn, contributes to the accumulation of additional mutations and eventually, in tumor growth. In this context, an in situ tumor develops, that is the uncontrolled mass of transformed cells stays within the limit of the tissue in which the first cell resided. During this phase, tumor volume increases in parallel with an increased dedifferentiation of the cells that also secrete angiogenic factors (▶Angiogenesis) to promote blood vessels formation in the tumor. Metastasis Metastasis is the process by which highly vascularized tumor cells acquire the ability to invade the

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blood-stream and seed in distant organs. Deregulation of cytoskeleton-associated proteins and secretion of protein factors play a critical role in the functionality of the metastatic cells. Stem Cell Biology In 1998, the group of Prof. James Thomson reported the isolation of a human embryonic stem cell line from the blastocyst stage of a human embryo. This cell line showed stability in a specifically developed culture medium and, upon transplantation in the nude mice, had the ability to form tumor-like structures made up of all the major human tissue types. This pioneer study opened the field of stem cell biology. Since then, enormous research efforts have been focused on the understanding of stem cell biology as well as their potential medical and therapeutic implications. Nonetheless, although the last 10 years witnessed an enormous progress, the field of stem cell research is in its infancy. The first controversy is the definition of stem cell itself. For simplicity, a stem cell is a clonal self-renewing entity that is multipotent and can generate several different cell types. This definition introduces three major characteristic of the stem cells: selfrenewal, clonality, and potency. Self-Renewal and Clonality Self-renewal is the process by which a stem cell undergoes an asymmetric mitotic division that produces, rather than two identical daughter cells, one cell that is completely identical to the parental stem cell and another cell that is already committed to a more restricted developmental path and more specialized abilities. Thus, stem cells have both the ability to selfmaintain their clonal cell population and to produce a population of clones with more differentiated characteristics. In this way, stem cells form a hierarchy of potency.

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stem cell population or mutation that can lead to tumorigenesis. One possibility is that the asymmetric division produces two daughter cells and, because of intrinsic factors, such cells follow different fates in spite of residing in the same ▶microenvironment. Alternatively, the two daughter cells become functionally different because they are exposed to different extrinsic factors. Most likely, both intrinsic and extrinsic factors are integrated in the milieu of the surrounding microenvironment, also known as the stem cell niche. Signals from the niche determine the type of gene regulation that allows the asymmetric division to take place. In this model, one daughter cell stays in the niche and the other one moves out. Indeed, the importance of the microenvironment in stem cell biology is highlighted by the ability of a particular stem cell to transdifferentiate or to dedifferentiate when put in a different niche. Although the concept of plasticity is debated in the literature, it is part of the “stemness” of a cell, which is the hallmark for a cell to be defined as a stem cell. Social Implications The ability to scientifically manipulate the human embryo or human adult stem cells has opened new perspectives for treatment of several human diseases. However, it has also initiated intense philosophical and political debates on the ethical issues associated with the use of such potential tools in medical practice.

References 1. Pestell RG, Albanese C, Reutens AT et al. (1999) The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation. Endocr Rev 20:501–534 2. Lowe SW, Cepero E, Evan G (2004) Intrinsic tumour suppression. Nature 432:307–315 3. Potten C, Wilson J (2004) Apoptosis – the life and death of cells. Cambridge University Press, New York 4. Gearhart J, Hogan B, Melton D et al. (2006) Essential of stem cell biology. Academic Press, London 5. Feinberg AP, Tycko B (2004) The history of cancer epigenetics. Nat Rev Cancer 4:143–153

Potency Stem cells have the ability to give rise to a population of daughter stem cells with a reduced differentiation. The totipotent cells are the first embryonic cells that can become any kind of cell type (e.g., zygote). These cells become pluripotent cells, which can differentiate in most but not all cell types (e.g., embryonic stem cells). Next, cells that are committed to produce only a certain lineage of cell types (e.g., ▶adult stem cells) are the multipotent cells. Some multipotent cells can only generate one specific kind of terminally differentiated cell type and thus, such cells are called unipotent cells.

Definition

Environmental Regulation The molecular mechanism by which regulatory processes occur in stem cells are not clear but are believed to be tightly regulated to avoid imbalance in

Consists of a paraffin block made from the cellular material of cytologic specimens (most commonly fine needle aspiration biopsies and body fluids) and is processed similar to histology. Can be a useful adjunct in cytology because it gives a better idea of tissue

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architecture and allows for multiple sections for ancillary stains. ▶Fine Needle Aspiration

Cell Cycle

completed. It is a regulatory mechanisms that monitors the progression of the cell cycle, so that one phase is not started before another has finished. The activation of checkpoints, for example by damaged DNA, arrests cell cycle progression. ▶Hypoxia ▶HSP90 ▶Decatenation G2 checkpoint

Definition The sequence of cellular transformations that accompany transition from one mitotic cell division to another. The cell cycle is composed of four phases known as G1, S, G2 and M. S is the period of DNA synthesis, M is mitosis when sister chromatids are condensed and segregated to two daughter cells. G1 lies between M and S and is a phase of preparation for DNA synthesis, G2 is between S and M and is a phase of preparation for mitosis. G0 refers to a quiescent state into which some cells in multicellular organisms enter from G1. Cells typically achieve full differentiation in the G0 phase. ▶Decatenation G2 Checkpoint ▶Cell-Cycle Targets for Cancer Therapy ▶Cyclin Dependent Kinases ▶Chelators as Anticancer Drugs

Cell Cycle Arrest Definition

The halt of the ▶cell cycle, often as a result of cellular stress with physical or chemical treatment as a mechanism of cellular defense. ▶Sulforaphane

Cell-Cycle Checkpoint Definition The cell-cycle checkpoint is a mechanism for stopping progression through the cell cycle when a key event, such as DNA replication, is not completed or when the genome is damaged. It is a restriction point during the cell cycle in which a cell monitors if preceding events required for cell division have been correctly

Cell-Cycle Targets for Cancer Therapy R OLF M U¨ LLER Institute of Molecular Biology and Tumor Research (IMT), Philipps-University Marburg, Marburg, Germany

Definition Knowledge of the molecular mechanisms governing the mammalian ▶cell cycle and their dysfunction in cancer cells has grown considerably in the last decade. It is now clear that the cell utilizes two distinct kinds of regulatory mechanisms to control cell-cycle progression: while progression past the ▶restriction point in late G1 is solely governed by extracellular signals, ▶checkpoints sense cellular damage or dysfunctions that are not compatible with a proper cell division, such as DNA damage. The detailed knowledge of the underlying molecular mechanisms, pathways and molecules provides the basis for a new approach to cancer therapy.

Characteristics Cell-cycle progression in mammalian cells is controlled through fundamentally different regulatory pathways. Progression through G1 across the restriction point (R-point) is controlled by external signals that are transmitted, for example, by mitogens or through cell adhesion processes. Beyond this point, cell-cycle progression is governed by a genetic program that is largely independent of extracellular signals but regulated by internally controlled checkpoints. These checkpoints ensure proper DNA replication, DNA integrity, progression through G2 and mitosis. A central role in cell-cycle progression is exerted by the ▶cyclindependent kinases (CDKs), which are composed of a regulatory cyclin subunit (e.g., cyclin A, B, D or E) and a catalytic kinase subunit (e.g., CDK1, 2, 4 or 6). The activity of CDKs is controlled by phosphorylation, phase-specific expression and proteolysis as well as the

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Cell-Cycle Targets for Cancer Therapy. Figure 1 The E2F pathway and its regulation by Rb and G1 CDKs.

association with CDK inhibitors (CDIs) belonging to the INK4 (p15, ▶p16, p18, p19) or KIP (p27, p57)/CIP (p21) families. Restriction Point Control The G1 CDK-cyclin complexes regulate progression across the restriction point through phosphorylation of the ▶retinoblastoma protein Rb and its kins p107 and p130. In early-mid G1 the transcription factor ▶E2F is found in complexes with Rb and ▶histone deacetylase (HDAC). These complexes actively repress transcription via E2F binding sites in the respective target genes. The phosphorylation of the E2F-RbHDAC complexes by ▶cyclin D-CDK4/6 and cyclin E-CDK in mid-late G1 leads to the disruption of these complexes and the generation of transcriptionally active “free” E2F, which results in the induction of numerous E2F target genes (Fig. 1). The relevance of R-point control for tumorigenesis is emphasized by the fact that the ▶INK4-cyclin D-CDK4-Rb pathway is defective in the vast majority of human tumors due to genetic alteration of its components (Fig. 2). Therefore, this pathway is of major interest with respect to therapeutic intervention. Checkpoint Control A major role in checkpoint control is exerted by the ▶p53 tumor suppressor pathway. In response to DNA damage (or other insults to the cell) p53 induces a number of genes that either invoke cell-cycle arrest (such as the CDI p21/CIP) or trigger apoptosis (Fig. 3). The activity and the steady-state level of p53 is regulated by ▶MDM-2, a oncoprotein that associates with p53, inhibits its transcriptional activity and targets

Cell-Cycle Targets for Cancer Therapy. Figure 2 Deregulation of E2F activity in cancer cells through impairment of the INK4-cyclin D-CDK 4–8211; Rb pathway.

p53 for degradation by the proteasome. MDM-2 itself is targeted for proteolysis by the tumor suppressor ▶p14ARF (or p19ARF in mice). The importance of this pathway is demonstrated by the fact that each of its components can be a target for genetic alterations in human tumors, and that a defective p53 pathway is found in more than 50% of all human malignancies. This emphasizes the relevance of p53 for the development of new anti-cancer therapies.

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Cell-Cycle Targets for Cancer Therapy. Figure 3 Loss of p53 function in human cancer cells.

Another checkpoint activated in G2 in response to DNA damage is governed by the checkpoint kinase-1 (▶CHK1; Fig. 4). CHK1 phosphorylates the CDC2 (CDK1) phosphatase CDC25C, which results in the association of CDC25C with a p53-induced specific isoform of 14–3–3. This renders CDC25C inactive, so that the cyclin B-CDC2 complex remains in its phosphorylated inactive form. As a consequence, progression into mitosis is prevented and DNA repair can occur. Since many anti-cancer drugs exert their function through DNA damage, this checkpoint may have a negative impact on their efficacy. An analogous checkpoint operating in G1 has recently been identified. This checkpoint is activated when the CDK2 phosphatase CDC25B is targeted for degradation in response to DNA-damage that will leave the cyclin E kinase in an inactive (phosphorylated) state. As a consequence, cell-cycle progression into S-phase is prevented. Clinical Relevance Cancer is clearly a proliferative disease resulting from deregulated cell-cycle progression. The inhibition of specific proteins driving the cell cycle is therefore an obvious strategy for the rational discovery of new anticancer drugs. In this context it is of particular interest that the interference with coordinated cell-cycle progression can result in apoptosis of tumor cells. This is exemplified by the observation that the deregulated expression of proteins, such as ▶Myc or E2F-1, in conjunction with a non-physiological cell-cycle block is

incompatible with cell survival. It has also been shown that the direct inhibition of CDKs, for example by CDIs or through ▶antisense nucleic acid, can trigger programmed cell death in tumor cells. These and other findings have laid the foundation for the definition of a new class of anti-tumor agents that function through a direct inhibition of proteins driving the cell cycle. One of the prototypes of this class of compounds is the synthetic flavone ▶Flavopiridol. Flavopiridol is a general inhibitor of CDKs, induces cell-cycle arrest and apoptosis, and is not influenced by many of the genetic alterations conferring resistance on human tumor cells. Accordingly, Flavopiridol has shown promising tumor responses in preclinical models and is currently undergoing clinical trials. Numerous other chemical CDK inhibitors have recently been identified and are currently being evaluated for their anti-tumor properties. It can be anticipated that CDK-inhibiting drugs will constitute a new class of powerful chemotherapeutics. Other interesting targets for therapeutic intervention are the proteins governing checkpoint control, for instance in response to DNA damage. Checkpoint control can invoke a transient cell cycle block, but can also trigger apoptosis. Both types of checkpoints are of relevance to tumor therapy. While the functionality of an apoptosis-inducing mechanism in response to drug- or radiation-induced cellular damage is desirable, checkpoint control leading to cell-cycle arrest is counterproductive for any therapy that relies on cell proliferation, such as radiation or conventional therapy. The p53 checkpoint is lost in many tumor cells, and thus the ability to undergo apoptosis in response to chemo- or radiotherapy. The restoration of this checkpoint could therefore sensitize many tumor cells to conventional therapies. Strategies along these lines involve the development of compounds that can reactivate mutant p53 or inhibit MDM-2, or the use of gene therapeutic approaches for the reintroduction of functional p53 genes. Other drug-based strategies aim to improve the efficacy of existing therapies that rely on DNA-damage, such as radiation or DNA-damaging chemotherapy. A prime candidate in this context is the kinase ▶CHK1 that regulates the G2 checkpoint (Fig. 4). First results obtained with an inhibitor of the G2 checkpoint, UCN01, suggest that this may indeed be the case. Numerous other mechanisms controlling cellcycle progression have been discovered, and approaches for therapeutic intervention are being developed, pointing to the great potential of targeting the cell cycle for the development of new anti-cancer drugs. It can be anticipated that this new class of anti-cancer drugs will lead to a clear advance in clinical oncology.

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Cell-Cycle Targets for Cancer Therapy. Figure 4 Regulation of the G2 checkpoint.

References 1. Jacks T, Weinberg RA (1998) The expanding role of cell cycle regulators. Science 280:1035–1036 2. Russell P (1998) Checkpoints on the road to mitosis. Trends Biochem Sci 23:399–402 3. Hueber AO, Evan GI (1998) Traps to catch unwary oncogenes. Trends Genet 14:364–367 4. Johnson DG, Walker CL (1999) Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol 39:295–312 5. Mailand N, Falck J, Lukas C et al. (2000) Rapid destruction of human Cdc25A in response to DNA damage. Science 288:1425–1429 6. Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512 7. Gray N, Detivaud L, Doerig C et al. (1999) ATP-site directed inhibitors of cyclin-dependent kinases. Curr Med Chem 6:859–875 8. Garrett MD, Fattaey A (1999) CDK inhibition and cancer therapy. Curr Opin Genet Dev 9:104–111

Cell Differentiation Definition This is a concept from developmental biology describing the process by which cells acquire a “type”. The morphology of a cell may change dramatically during differentiation, but the genetic material remains the same, with few exceptions. A cell that is able to differentiate into many cell types is known as pluripotent. These cells are called stem cells in animals and meristematic cells in higher plants. A

cell that is able to differentiate into all cell types is known as totipotent. In mammals, only the zygote and early embryonic cells are totipotent, while in plants, many differentiated cells can become totipotent with simple laboratory techniques. ▶Orphan Nuclear Receptors and Cancer

Cell Division Definition Synonym cell proliferation; Is the process of cell doubling by which a cell, called the parent cell, divides into two cells, called daughter cells. Cell division is a physiological process that occurs in almost all tissues. However, a process of pathological cell division can be seen in cancers. ▶Cell Cycle

Cell Fate Definition The ultimate differentiated state to which a cell has become committed. ▶Polycomb Group

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Cell-free Circulating Nucleic Acids

Cell-free Circulating Nucleic Acids ▶Circulating Nucleic Acids

Cell Migration A highly complex process, regulated by multiple gene pathways enabling the motility of cells through the adhesion and invasion of extracellular matrices. ▶Tissue Inhibitors Of Metalloproteinases (Timps)

Cell Lines Definition Are cell populations with the feature of dividing indefinitely when growing in culture. There are tumor and non-tumor cell lines from different organisms including humans.

Cell Motility ▶Migration ▶Motility

Cell Movement Cell Locomotion

▶Motility

▶Migration

Cell Polarity Cell-Mediated Immunity Definition Synonym Cell-mediated immune response describes any adaptive immune response in which antigenspecific T cells have the main role. It is defined operationally as adaptive immunity that cannot be transferred to a naïve recipient with serum antibody. ▶Sjögren Syndrome

Definition Cell direction or orientation to maintain the property of having two opposite poles, apical and basolateral domains. ▶Tight Junction

Cell Scattering Definition

Cell Membrane Definition

▶Plasma membrane

A common tissue culture assay used to monitor ▶Met receptor activation. When non-transformed dog kidney epithelial (MDKC) cells are grown in tissue culture, the cells spontaneously arrange themselves into tightly connected epithelial sheets. Following Met activation, these cell sheets breakdown and the individual cells migrate away from each other.

Cellular Senescence

Cell Signaling Definition

Synonym ▶signal transduction, refers to the process by which a cell converts one type of stimulus into another using ordered sequences of biochemical reactions.

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architecture, de-differentiation, loss of cellular adhesion, loss of cellular polarity, keratinization within the deeper areas of the epithelium. ▶Squamous Cell Carcinoma

Cellular Immortalization Cell-Surface Receptors Definition Are cell surface receptor is a cellular proteins embedded within the cell membrane that receive and respond to extracellular soluble ligands such as neurotransmitters, hormones, growth factors, or chemokines.

Definition The process by which cells cultured in vitro, or in the organism, escape from cellular senescence and grow forever. This can happen spontaneously, or can be caused by chemical carcinogens, oncogenic viruses, or radiations. ▶Chemically Induced Cell Transformation ▶Senescence and Immortalization

▶CXC Chemokines

Cellular Immunity b-Cell Tumor of the Islets ▶Insulinoma

Cellular Antigens ▶CD Antigens

Cellular Atypia

Definition Immune protection provided by the direct action of immune cells (as distinct from soluble molecules such as antibodies).

Cellular Self-Cannibalism ▶Autophagy

Cellular Senescence

Definition

Definition

Histological features associated with epithelial dysplasia, the degree of which is determined by the number of atypia present in the dysplastic lesion. Atypia include densely stained nuclei, pleomorphic nuclei, altered nucleus:cytoplasm ratio, aberrant mitosis, frequent mitosis, supra-basal mitosis, disorganized tissue

The process of programmed cell aging, by which cells die after a specific number of population doublings, usually 60 population doublings. ▶Chemically Induced Cell Transformation ▶Senescence and Immortalization

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Cellular Transformation Assay

Central Cleavage

Definition

Definition

Cell biological test to demonstrate oncogenic activity of a candidate gene. Typically the gene in question is cloned into a mammalian expression vector, transfected into appropriate recipient cells and expressed. The transforming activity is detected on the basis of morphological alterations such as the loss of the typical fibroblastic or epithelial cell shape, anchorage independent proliferation, as determined in semi-solid agar medium, or tumor formation following injection of transfected cells into nude mice. The classical cellular transformation assays were done with donor DNA prepared from tumors and pre-neoplastic mouse NIH/3T3 cells as recipients.

Central cleavage refers to the symmetric cleavage of ▶carotenoids at their central 15,15′ double bond by carotene 15,15′-oxygenase, a main pathway for vitamin A formation from provitamin A carotenoids.

▶RAS Transformation Targets

▶Brain Tumors

Central Nervous System Definition The brain and spinal cord.

Central Neurocytoma CENP-E ▶Neurocytoma

Definition Centromeric protein E; Is a kinetochore-associated kinesin-like motor protein that is responsible for chromosome movement and alignment in mitosis. In animal model, CENP-E deletion in mice causes early embryonic lethality, with embryos unable to implant or develop past implantation.

Central Neurofibromatosis ▶Neurofibromatosis 2

▶Mitotic Arrest-Deficient Protein 1 (MAD1)

Centrocytic (Mantle Cell) Lymphoma Censoring

▶Mantle Cell Lymphoma

Definition Censoring, particularly in survival studies, occurs when the outcome of interest is not measured fully as, for example, when a trial is ended after a specified period of time so that failure times are not precisely measured.

Definition

▶Kaplan–Meier Survival Analysis

Constricted portion of the chromosome. The centromere divides the chromosome into a short “p” and a

Centromere

Centrosome

long “q” arm. The centromere is a region of chromosomes with a special DNA sequence and structure. The centromere plays a role in cellular division and it is the region where sister chromatids join after doubling the chromosomes during prophase and metaphase of mitosis. ▶Micronucleus Assay

Centrosome K ENJI F UKASAWA Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute 12902 Magnolia Dr. Tampa, FL 33612-9416, OH, USA

Synonyms Major microtubule organizing center; MTOC; Spindle pole body; SPB, in yeast

Definition

The centrosome is a nonmembranous organelle (1–2 μm in diameter) normally localized at the periphery of nucleus, and its primary function is to nucleate and anchor microtubules.

Characteristics Structure and Function The centrosome in mammalian cells consists of a pair of centrioles and the surrounding protein aggregates consisting of a number of different proteins (known as pericentriolar material; PCM). The centrioles in the pair structurally differ from each other; one with a set of appendages at the distal ends (mother centriole) and another without appendages (daughter centriole).

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These appendages are believed to be important for nucleating and anchoring microtubules. The daughter centriole acquires the appendages in late G2-phase of the cell cycle. As the primary function of the centrosome is to nucleate and anchor microtubules, centrosomes organize the cytoplasmic microtubule network during interphase, which is involved in vesicle transport, proper distribution of small organelles, and establishment of cell shape and polarity. In mitosis, centrosomes become the core structures of spindle poles and direct the formation of mitotic spindles. (Fig. 1). Centrosome Duplication Upon cytokinesis, each daughter cell receives only one centrosome. Thus, the centrosome, like DNA, must duplicate once prior to the next mitosis. In other words, cells have either one unduplicated or two duplicated centrosomes at any given time point during the cell cycle. Since DNA and centrosome are the only two organelles that undergo semiconservative duplication once in a single cell cycle, cells are equipped with a mechanism that coordinates these two events, likely to ensure these two organelles to duplicate once, and only once. In late G1/early S-phase, the centrosome initiates duplication by physical separation of the paired centrioles, which is followed by the formation of a procentriole in the proximity of each preexisting centriole. During S and G2, the procentrioles elongate and two centrosomes continue to mature by recruiting PCM. By late G2, two mature centrosomes are generated. The coupling of the initiation of DNA and centrosome duplication is in part achieved by late G1-specific activation of cyclin-dependent kinase 2 (CDK2)/ cyclin E. CDK2/cyclin E triggers initiation of both DNA synthesis and centrosome duplication. The activation of CDK2/cyclin E is controlled by the late G1-specific expression of cyclin E as well as the basal level expression of p53 and its transactivation target p21Waf1/Cip1 (p21), a potent CDK inhibitor. Several

Centrosome. Figure 1 Structure and function of centrosomes. (a) The centrosome consists of a pair of centrioles and surrounding protein aggregates (PCM). (b and c) Mouse embryonic fibroblasts were immunostained for γ-tubulin (one of major centrosomal proteins, green – appearing in yellow) and α- and β-tubulin (primary constituents of microtubules, red). Cells were also counterstained for DNA with DAPI. Panel b: interphase cell, panel c: mitotic cell.

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potential targets of CDK2/cyclin E for centrosome duplication have been identified, including nucleophosmin, Mps1 kinase, and CP110. For instance, nucleophosmin localizes between the paired centrioles, likely functioning in the pairing of the centrioles. CDK2/ cyclin E-mediated phosphorylation promotes dissociation of nucleophosmin from the centriole pairs, leading to physical separation of the paired centrioles. (Fig. 2). Abnormal Amplification of Centrosomes and Chromosome Instability in Cancer The presence of two centrosomes at mitosis ensures the formation of bipolar mitotic spindles. Since chromosomes are pulled toward each spindle pole, the bipolarity of mitotic spindles is essential for the accurate chromosome segregation into two daughter cells during cytokinesis. Abrogation of the regulation underlying the numeral homeostasis of centrosomes (i.e., regulation of centrosome duplication) results in abnormal amplification of centrosomes (presence of >2 centrosomes), which in turn increases the frequency of mitotic defects (i.e., formation of >2 spindle poles) and chromosome segregation errors/chromosome instability (see [1] for the full description of the mechanisms for generation of centrosome amplification). Chromosome instability has been recognized as a hallmark of cancer, and contributes to multistep carcinogenesis by facilitating the accumulation of genetic lesions required for acquisition of various malignant phenotypes. To date, a

number of studies have shown that centrosome amplification is a frequent event in almost all types of solid tumors, including breast, bladder, brain, bone, liver, lung, colon, prostate, pancreas, ovary, testicle, cervix, gallbladder, bile duct, adrenal cortex, and head and neck squamous cell, to name a few. Centrosome amplification has also been observed in certain cases of leukemia and lymphoma. Many studies have also shown the strong association between the occurrence of centrosome amplification and a high degree of aneuploidy. Thus, centrosome amplification can be reasonably considered as a major contributing factor for chromosome instability in cancer. (Fig. 3). Loss of Tumor Suppressor Proteins and Centrosome Amplification In view of carcinogenesis, it is important to mention that loss or inactivating mutation of certain tumor suppressor proteins, most notably p53 and BRCA1, results in centrosome amplification. For both p53 and BRCA1, they were initially implicated in the control of centrosome duplication and numeral homeostasis of centrosomes by the observations that centrosome amplification and consequential mitotic aberrations were frequent in the embryonic fibroblasts (as well as various tissues) of p53-null mice as well as mice harboring BRCA1 mutation, which implies that destabilization of chromosomes due to centrosome amplification contributes to the cancer susceptibility

Centrosome. Figure 2 The centrosome/centriole duplication cycle. Late G1-specific activation of CDK2/cyclin E triggers initiation of both DNA and centrosome duplication. Centrosome duplication begins with the physical separation of the paired centrioles, which is followed by formation of procentrioles. During S- and G2-phases, procentrioles elongate, and two centrosomes progressively recruit PCM. In late G2, the daughter centriole of the parental pair acquires appendages (shown as red wedges), and two identical centrosomes are generated. During mitosis, two duplicated centrosomes form spindle poles, and direct the formation of bipolar mitotic spindles. Upon cytokinesis, each daughter cell receives one centrosome.

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Centrosome. Figure 3 Representative immunostaining images of centrosome amplification in human cancer. The touch preparations of G3 tumor grade bladder cancer specimens and the adjacent normal bladder epithelium samples were subjected to immunostaining for γ-tubulin (centrosome, green) and counterstained for DNA with DAPI (blue). No centrosome amplification can be seen in normal bladder epithelium (a), while a high frequency of centrosome amplification in the G3 tumors (b).

phenotype associated with loss or mutational inactivation of p53 as well as BRCA1. Centrosome Amplification and Cancer Chemotherapy In cells inhibited for DNA synthesis (i.e., by exposure to DNA synthesis inhibitors such as aphidicolin (Aph) or hydroxyurea (HU)), centrosomes undergo multiple rounds of duplication in the absence of DNA synthesis, resulting in abnormal amplification of centrosomes. However, this phenomenon preferentially occurs when p53 is either mutated or lost. In the presence of wildtype p53, centrosome duplication is also blocked by exposure to DNA synthesis inhibitors; p53 is upregulated upon prolonged exposure to Aph or HU, leading to transactivation of p21, which in turn blocks the initiation of centrosome duplication via continuous inhibition of CDK2/cyclin E. In contrast, in cells lacking p53, p21 fails to be upregulated in response to the cellular stress imposed by DNA synthesis inhibitors, allowing “accidental” activation of CDK2/ cyclin E, which triggers initiation of centrosome duplication. Considering the high frequency of p53 mutation in human cancer, it is important to address the effect of commonly used anticancer drugs targeting S-phase (DNA replication) on centrosomes. When p53null cells were exposed to subtoxic concentrations of the S-phase targeting chemotherapeutic agents (i.e., 5′-fluorouracil, arabinoside-C), centrosome amplification was efficiently induced. Moreover, after removal of drugs, these cells resumed cell cycling, and suffered dramatic destabilization of chromosomes. This finding may be significant in the context of cancer chemotherapy using the S-phase targeting drugs. During chemotherapy, not all cells in tumors receive a maximal dose of drugs – such cells may not be killed, but only arrested for cell cycling. If these cells harbor p53 mutations, centrosome amplification occurs during the drug-induced cell cycle-arrest. Upon cessation of chemotherapy, these cells resume cell cycling in the

presence of amplified centrosomes, and suffer significant mitotic aberrations and chromosome instability, which increases the risk of acquiring further malignant phenotypes. This may in part explain why the recurrent tumors after chemotherapy are often found to be more malignant than the original tumors. Many S-phase targeting anticancer drugs have been found to be effective, and there is no doubt that DNA duplication should be one of the major targets for future development of more effective anticancer drugs. However, the possibility that the S-phase targeting drugs may exacerbate a chromosome instability phenotype by inducing centrosome amplification should be taken into consideration. Another important issue to be addressed is the concept of centrosome duplication as a target of cancer chemotherapy. Like DNA replication, centrosome duplication occurs only in proliferating cells. Inhibition of centrosome duplication will not only suppress centrosome amplification and chromosome instability, but also block cell division and possibly induce cell death – cells with one centrosome fail to form bipolar mitotic spindles, and are often undergo cell death. Moreover, in contrast to genotoxic drugs which impose an increased rate of secondary mutations through interfering with DNA metabolisms, such side effects will likely be minimal in the protocol designed to block centrosome duplication. ▶Genomic Imbalance ▶Microtubule-Associated Proteins

References 1. Fukasawa K (2005) Centrosome amplification, chromosome instability and cancer development. Cancer lett 230:6–19 2. Hinchcliffe EH, Sluder G (2002) Two for two: Cdk2 and its role in centrosome doubling. Oncogene 21:6154–6160

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3. Tarapore P, Fukasawa K (2002) Loss of p53 and centrosome hyperamplification. Oncogene 21:6234–6240 4. Deng CX (2002) Roles of BRCA1 in centrosome duplication. Oncogene 21:6222–6227 5. Bennett RA, Izumi H, Fukasawa K (2004) Induction of centrosome amplification and chromosome instability in p53-null cells by transient exposure to sub-toxic levels of S-phase targeting anti-cancer drugs. Oncogene 23:6823–6829

Ceramide E RICH G ULBINS Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany

Definition Ceramide belongs to the group of sphingolipids and is constituted by the amide ester of the sphingoid base D-erythro-sphingosine and a fatty acid of C16 through C32 chain length. At present, the differential biological function of different ceramide species is unknown and, thus, the term ceramide is used collectively to represent all long chain ceramide molecules.

Characteristics Formation of Ceramide Ceramide molecules are very hydrophobic and exclusively present in membranes. Sphingomyelin, the choline-ester of ceramide is hydrolyzed by acid, neutral and alkaline sphingomyelinases to release ceramide. Ceramide is also de novo synthesized via a pathway involving the serine-palmitoyl-CoA transferase. Under some circumstances ceramide can be also formed from sphingosine by a reverse activity of the acid ceramidase. Ceramide-Induced Changes of Biological Membranes The formation of ceramide within biological membranes results in a dramatic change of the biophysical properties of the lipid bilayer. Ceramide molecules have the tendency to self-associate and to form small ceramideenriched membrane microdomains. These membrane microdomains spontaneously fuse to large ceramideenriched membrane macrodomains that constitute a very hydrophobic and stable membrane domain. Furthermore, ceramide molecules seem to compete with and displace cholesterol from membrane domains. Ceramide-enriched membrane platforms serve to re-organize and cluster/ aggregate receptor molecules in the membrane resulting in a very high density of receptors within a small area of the cell membrane. At least for some receptors the transmembranous domain of the receptor determines its

preferential partitioning in ceramide-enriched membrane platforms. Ceramide-enriched membrane macrodomains are also involved in the recruitment or exclusion, respectively, of intracellular signaling molecules that mediate transmission of signals into the cell via a particular receptor. In general, clustering of receptors in ceramide-enriched membrane domains serves to amplify a weak primary signal. For instance, it was shown that ceramide-enriched membrane platforms amplify CD95 signaling ~100-fold. Ceramide in Receptor-Mediated Signaling Death receptors, in particular CD95 or DR5, activate the acid sphingomyelinase and trigger the translocation of the enzyme onto the extracellular leaflet of the cell membrane. Translocation of the acid sphingomyelinase onto the extracellular leaflet of the cell membrane may occur by fusion of intracellular vesicles that are mobilized upon receptor stimulation with the cell membrane. Surface exposure and stimulation of the acid sphingomyelinase results in very rapid release of ceramide in the cell membrane. Ceramide forms membrane platforms and mediates clustering of the death receptors, which is required for the induction of cell death via these receptors (Fig. 1). However, ceramide is not only involved in the mediation of apoptotic stimuli, but also many other stimuli trigger the release of ceramide including CD40, CD20, FcγRII, CD5, LFA-1, CD28, TNFα, Interleukin-1 receptor, PAF-receptor, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, γ-irradiation, UV-light, doxorubicin, cisplatin, gemcitabine, disruption of integrin-signaling and some conditions of developmental death. Signaling Molecules Regulated by Ceramide Ceramide interacts with and activates phospholipase A2, kinase suppressor of Ras (KSR; identical to ceramide-activated protein kinase), ceramide-activated protein serine-threonine phosphatases, protein kinase C isoforms and c-Raf-1. Furthermore, ceramide inhibits the potassium channel Kv1.3 and calcium release activated calcium (CRAC) channels. Lysosomal ceramide specifically binds to and activates cathepsin D resulting in translocation of cathepsin D into the cytoplasm and induction of cell death via the pro-apoptotic proteins Bid, Bax and Bak. Ceramide in Mitochondria and Cell Death Besides a function of ceramide in the plasma membrane and lysosomes for the induction of cell death, ceramide is also generated in mitochondria via the de novo synthesis pathway, a reverse activity of the ceramidase and/or activity of the acid sphingomyelinase. Although at present the function of ceramide in the mediation of mitochondrial pro-apoptotic events is poorly defined,

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Ceramide. Figure 1 Receptors cluster in ceramide-enriched membrane domain to transmit signals into cells. The interaction of a ligand with its receptor results in translocation of the acid sphingomyelinase onto the extracellular leaflet and a concomitant release of ceramide. Ceramide spontaneously forms ceramide-enriched microdomains that fuse to large ceramide-enriched macrodomains. These domains trap activated receptor molecules finally resulting in clustering of many receptor molecules within a small area of the cell membrane. The high density of receptor molecules and associated intracellular molecules amplifies the primarily weak signal, permits the generation of a strong signal and, thus, efficient transmission of the signal into the cell. Modified from A. Carpinteiro et al. Cancer Letters.

it was suggested that C16-ceramide molecules form large channels in mitochondrial membranes that may permit the exit of cytochrome c from mitochondria to execute death. Genetic Evidence for a Function of Ceramide in Apoptosis The role of the acid sphingomyelinase and ceramide for CD95 and DR5-triggered apoptosis was evidenced by studies on acid sphingomyelinase-deficient cells or mice, respectively, that revealed a resistance of these cells to CD95- and DR5-triggered apoptosis, but also γ-irradiation- and UV-light- or P. aeruginosa-triggered cell death. Ceramide in g-Irradiation- and UV-A Light-Induced Apoptosis The acid sphingomyelinase and ceramide are critically involved in the response of cells to γ-irradiation. Animals or cells lacking the acid sphingomyelinase are resistant to γ-irradiation-induced cell death. In particular, endothelial cells in acid sphingomyelinasedeficient mice are resistant to γ-irradiation. Ceramide also plays a critical role for UV-light induced apoptosis. UV-A and UV-C light activate the acid sphingomyelinase, trigger the release of ceramide and the formation of large ceramide-enriched membrane domains in the cell membrane to initiate. Ceramide and Chemotherapy In addition to a central role of ceramide in γ-irradiationinduced cell death, ceramide is also critically involved in the induction of cell death by at least some chemotherapeutic drugs. Thus, doxorubicin-, cisplatin- und

gemcitabine-induced cell death of malignant and nonmalignant cells requires expression of the acid sphingomyelinase, release of ceramide and/or the formation of ceramide-enriched membrane platforms to trigger death. Rituximab, an anti-CD20 antibody, requires expression of the acid sphingomyelinase and the generation of ceramide to kill leukemic cells. Short Chain Ceramide Short chain ceramide molecules composed of a fatty acid chain with C2 through C12 length are water-soluble and, thus, very much differ from endogenous long ceramide molecules (C16-C32). However, they are very efficient reagents to kill tumor cells in vitro. Cationic pyridinium-ceramides seem to accumulate in mitochondria of tumor cells and may, thus, serve as a new class of anti-tumor reagents, although at present no convincing concepts are available to selectively target tumor cells in vivo by and to avoid effects of short chain ceramide on normal cells.

References 1. Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25:4798–4811 2. Kolesnick RN, Goni FM, Alonso A (2000) Compartmentalization of ceramide signaling: physical foundations and biological effects. J Cell Physiol 184:285–300 3. Gulbins E, Kolesnick RN (2003) Raft ceramide in molecular medicine. Oncogene 22:7070–7077 4. Jaffrezou JP, Laurent G (2004) Ceramide: A new target in anticancer research? Bull Cancer 91:E133–E161 5. Ogretmen B, Hannun YA (2004) Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 4:604–616

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Ceramide Kinase (CerK)

Ceramide Kinase (CerK) Definition Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes. ▶Ceramide

irreversibly binding to the active site cysteine thiol in the β-ketoacyl-synthase domain of fatty acid synthase. ▶Fatty Acid Synthase

Cervical Definition Pertaining to the neck.

Ceramide-1-Phosphate (C1P) Definition Ceramide-1-phosphate (C1P) is a phosphorylated form of ▶ceramide and possesses antitumor properties. ▶Lipid Mediators

Cervical Cancers J IRO F UJIMOTO Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu City, Japan

Definition

C-erb-B2 ▶HER-2/neu

Cerebral Edema Definition Is the accumulation of fluid in the brain, often as a result of a pathological condition. ▶Convection Enhanced Delivery (CED)

Cerulenin Definition An antifungal antibiotic isolated from several species, including Cephalosporium, Acrocylindrum, and Helicoceras. It inhibits the biosynthesis of fatty acid by

The regions of the uterus are the corpus and the cervix. Cancer originating from the cervix is defined as cancer of the cervix. When cancers are simultaneously detected in the cervix and corpus, squamous cell carcinoma (SCC) is designated as a cancer of the cervix and adenocarcinoma is designated as a cancer of the corpus. When cancer occupies both the cervix and vagina without the junctional area (the fornix), the cancer extending to the exocervix is recognized as a cancer of the cervix. Thus, cervical cancer is defined apart from cancer of the uterine corpus (cancer of the uterine endometrium) and cancer of the vagina.

Characteristics The main gynecological cancers originate from the cervix, endometrium, and ovary. Among them, cervical cancer is the most common malignancy in women. Main risk factors are . Young age at first intercourse, especially shortly after the menarche . High number of sexual partners . High number of sexual partners of the partner . High number of children . Excessive douching Smoking appears to increase the incidence of SCC, but not of adenocarcinoma or adenosquamous carcinoma. Immunosuppression by smoke-derived nicotine and its metabolite cotinine in the cervical mucus may enhance

Cervical Cancers

the effects of sexually transmitted disease (STD) including human papillomavirus (HPV) infection. Most epidemiological risk factors for cervical cancer are associated with STDs. HPV induces an STD, human venereal condyloma, which is associated with cervical, vaginal and vulvar dysplasia, and invasive carcinomas. HPV particles and DNA, especially HPV-16, HPV-18, and HPV-33, are detected in cervical and vulvar dysplasia and in invasive carcinomas. Additionally, it has been demonstrated that HPV transforms human cell lines. HPV infection of the cervix is a main etiology of cervical cancer. Symptoms Main symptoms of cervical cancer are . Vaginal bleeding, which may be recognized as postmenopausal bleeding, irregular menses, or postcoital bleeding . Abnormal vaginal (watery, purulent or mucoid) discharge In advanced cases, corresponding local symptoms occur. A Pap smear even in unsymptomatic cases is useful for the early detection of cervical dysplasia and cancers. Among women over the age of 18 who have had sexual intercourse, high-risk women should be screened at least yearly. Pathology Histopathological types in cervical cancers are mainly SCC and adenocarcinoma, which account for about 90% of all cervical cancers (adenosquamous carcinoma, glassy cell carcinoma, adenoid cystic carcinoma, adenoid basal carcinoma, carcinoid, small cell carcinoma, and undifferentiated carcinoma also occur). SCCs are keratinizing or nonkeratinizing in most cases and may be verrucous, condylomatous, papillary, or lymphoepithelioma-like carcinomas in a few cases. Adenocarcinomas are classified into mucinous, endometrioid, clear cell, serous, and mesonephric adenocarcinomas; mucinous adenocarcinomas are subclassified with endocervical type into adenoma malignum and villoglandular papillary adenocarcinoma, and intestinal type adenocarcinoma. Staging Clinical staging represents the degree of advancement of the tumor, and is defined by the FIGO classification established in 1994 and by the TNM classification of malignant tumors set by the UICC in 1997 as follows (classified by FIGO [TNM]): . Stage 0 (Tis): carcinoma in situ (preinvasive carcinoma). . Stage I (T1): cervical carcinoma confined to the uterus.

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. Stage II (T2): tumor invades beyond the uterus but not to the pelvic wall or to the lower third of the vagina. . Stage III (T3): tumor extends to the pelvic wall and/ or involves the lower third of the vagina and/or causes hydronephrosis or nonfunctioning kidney. . Stage IVA (T4): tumor invades the mucosa of the bladder or rectum and/or extends beyond the true pelvis. . Stage IVA (Ml): distant metastasis. Stage IA (TIa) has been further classified by microinvasive depth and width into stage IA1 (Tlal) (depth of stromal invasion ≤3 mm, horizontal spread ≤7 mm) and stage IA2 (Tla2) (depth of stromal invasion >3 mm, ≤5 mm; horizontal spread ≤7 mm). Stage IB (Tlb) has been further classified by tumor size into stage IB1 (Tlbl) (greatest dimension ≤4 cm) and stage IB2 (Tlb2) (greatest dimension >4 cm). In cases staged IA2 (Tla2) or less advanced, colposcopically directed biopsy in the transformation zone of the cervix, endocervical curettage or cervical conization are required. Prognosis Unfavorable prognostic factors include younger age, advanced clinical stage, certain histopathological types, vessel permeation, large tumor volume, parametrium involvement, and lymph node metastasis. Nodal metastasis is an especially critical prognostic factor after curative resection. Vascular endothelial growth factor (VEGF)-C and osteopontin contribute to the aggressive lymphangitic metastasis in uterine cervical cancers. Platelet-derived endothelial cell growth factor (PD-ECGF) contributes to the advancement of metastatic lesions as an agiogenic factors. PD-ECGF, VEGF-C, and osteopontin levels in metastatic lesions are prognostic indicators. Furthermore, serum PDECGF level reflects the status of advancement of cervical cancers and is recognized as a novel tumor marker for both SCC and adenocarcinoma of the cervix, while the tumor marker SCC is well known only as an indicator for SCC of the cervix. VEGF-C and osteopontin contribute to the aggressive lymphangitic metastasis in uterine cervical cancers. Therapy The treatment for cervical cancer consists mainly of surgery and radiation. Chemotherapy is performed in combination with surgery and/or radiation for advanced cases, and immunotherapy is an adjuvant treatment for surgery, radiation, and chemotherapy. The standard treatment for carcinoma in situ is cervical conization or total hysterectomy. The standard treatment for microinvasive carcinoma stage IA (Tla) is modified radical hysterectomy regardless of regional lymphadenectomy. The standard surgical treatment for

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invasive carcinoma is radical hysterectomy with regional lymphadenectomy. Although oophorectomy can be avoided in some cases during the reproductive period, ovarian metastasis must be considered especially in adenocarcinoma of the cervix. When ooporectomy is avoided, the ovary is better shifted out of radiation area. For patients who undergo oophorectomy, hormone replacement therapy can be useful. In more advanced cases, extended radical hysterectomy or pelvic exenteration is appropriate. After surgery external irradiation is followed in some cases. The standard radiotherapy without surgery for invasive carcinoma is intra-cavitary and/or external irradiation. Recently, neoadjuvant therapy (chemotherapy) has been tried in order to make surgery more successful, and concurrent radio-chemotherapy has been tested for the purpose of enhancing the effect of radiation.

References 1. Fujimoto J, Toyoki H, Sato E et al. (2004) Clinical implication of expression of vascular endothelial growth factor-C in metastatic lymph nodes of uterine cervical cancers. Br J Cancer 91:466–469 2. Fujimoto J, Sakaguchi H, Hirose R et al. (1999) Clinical implication of expression of platelet-derived endothelial cell growth factor (PD-ECGF) in metastatic lesions of uterine cervical cancers. Cancer Res 59:3041–3044 3. Fujimoto J, Sakaguchi H, Aoki I et al. (2000) The value of platelet-derived endothelial cell growth factor as a novel predictor of advancement of uterine cervical cancers. Cancer Res 60:3662–3665

Cetuximab

have been identified in several types of cancer, such as lung cancer and colorectal cancer. Cetuximab is approved for treatment of EGFRpositive, irinotecan-refractory ▶metastatic colon cancer. ▶Monoclonal Antibody Therapy ▶Epidermal Growth Factor Receptor Inhibitors

c-FLICE-like Inhibitory Protein Definition c-FLIP, also known as FLAME-1/I-FLICE/CASPER/ CASH/MRIT/CLARP/Usurpin, is a death-effectordomain (DED)-containing protein that exists in three different isoforms, FLIPL, FLIPS, and FLIPR. All cFLIP isoforms contain two N-terminal DEDs, whereas only FLIPL also harbors a C-terminal part of catalytically inactive caspase-like domains homologous to caspase-8. FLIPL, FLIPS, and FLIPR function as inhibitors of apoptosis by blocking caspase-8 activation at the death-inducing signaling complex (DISC). In addition, FLIPL may promote ▶apoptosis at low expression levels by facilitating autocatalytic activation of procaspase-8 and is also involved in nonapoptotic pathways, e.g., NF-κB or MAPK signaling. ▶c-FLIP ▶Usurpin ▶Caspase-8

Definition Is a chimeric IgG1κ monoclonal antibody specifically binding to epidermal growth factor receptor (EGFR). Epidermal growth factor receptor (EGFR) (EGFR; ErbB-1; HER1 in humans) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. Ligands which induce activation of EGFR are epidermal growth factor and transforming growth factor α, for example. Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer. EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity resulting in activation of several signal transduction cascades which lead to DNA-synthesis and cell proliferation. EGFR mutations can lead to EGFR overexpression or overactivity and consequently result in uncontrolled cell division. Mutations of EGFR

c-FLIP Definition

▶c-FLICE-like Inhibitory Protein

CG ▶Cancer Germline Antigens

Checkpoint

cGMP

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Chaperone

Definition

Definition

Cyclic guanosine monophosphate.

Molecular chaperones are proteins that aid the proper folding and assembly of proteins incompletely folded under conditions of cellular stress or protein synthesis. Molecular chaperones include small Hsp, Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, the calnexin/calreticulin families, and so forth.

▶Nitric Oxide

CGP57148

▶Calreticulin ▶Dioxin ▶Methylation-Controlled J Protein (MCJ) ▶Autophagy

▶STI-571 ▶Imatinib

Chaperonins z Chain Definition A transmembrane protein associated with key functional receptors of the immune system, the T cell antigen receptor (TCR) and NK killing receptors (NKP30, NKP46 and CD16). The ζ chain has a key role in receptor assembly, expression and signaling function. Down-regulation of ζ chain expression associated with immunosuppression has been shown in various chronic pathologies characterized by chronic inflammation, including cancer, autoimmune, and infectious diseases. ▶Inflammation

Channels

Definition

A family of conserved ▶chaperone proteins which have a characteristic multi-subunit ring structure. They function by enclosing a nascent protein and preventing its non-specific aggregation during assembly. ▶Molecular Chaperones

Charged Particle Therapy ▶Proton Beam Therapy

Checkpoint

Definition

Definition

Channels are pores in biological membranes that have a rather limited specificity. Substance flow through channels is controlled by the channels’ open probability so that high transport rates of 107–109 molecules per second can be achieved.

Checkpoints represent intrinsic mechanisms that are activated when cell-cycle progression would be detrimental to the cell, as in case of DNA-damage, incomplete DNA synthesis, metabolic dysfunctions or mitotic spindle damage. In such cases, cell-cycle progression is transiently halted until the respective problem is fixed, for instance by the DNA repair

▶Membrane Transporters

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machinery. Major checkpoints are governed by the tumor suppressor gene product p53 or the ▶check point kinases CHK1 and CHK2. Control mechanism that ensures that the next step in the cell cycle does not proceed until a series of preconditions have been fulfilled including the completion of all previous steps; this is particularly true for all pathways associated with DNA replication and chromosome formation; impaired chromosome checkpoints can result in chromosomal.

Chelators as Anti-Cancer Drugs DAVID B. LOVEJOY, Y U Y U, D ES R. R ICHARDSON Department of Pathology, University of Sydney, NSW, Australia

Synonyms Chelation therapy

▶Cell-Cycle Targets for Cancer Therapy ▶Fragile Histidine Triad ▶Chromosomal instability ▶Aneuploidy

Checkpoint Kinases Definition Checkpoint kinases are a group of at least four protein kinases (ATM, ATR, CHK1 and CHK2) and their relatives, which play an important role in the mechanisms that sense and signal DNA damage, culminating in the activation of cell-cycle checkpoints and DNA repair. ▶Checkpoint ▶Cell-Cycle Checkpoint

Chelation Therapy ▶Chelators as Anti-Cancer Drugs

Chelator Definition A chemical or drug capable of binding metal ions. ▶Chelators as Anticancer Drugs

Definition Iron is an element fundamental for life. Many vital cellular processes such as energy metabolism and DNA synthesis consist of reactions that require catalysis by iron-containing proteins. These proteins include ▶cytochromes, and ▶ribonucleotide reductase (RR). The latter is more significant in the context of cellular proliferation due to its role in catalyzing the rate-limiting step of DNA synthesis. Ultimately, the importance of iron is highlighted by the fact that iron-deprivation leads to G1/S ▶cell cycle arrest and ▶apoptosis. Cancer cells in particular, have a higher iron requirement because of their rapid rate of proliferation. In order to satisfy their iron requirement, some cancer cells have altered iron metabolism. In addition, iron ▶chelators also demonstrate the ability to inhibit growth of aggressive tumors such as ▶neuroblastoma. For these reasons, iron-deprivation through iron chelation is seen as an exploitable therapeutic strategy for the treatment of cancer.

Characteristics Iron Metabolism in Cancer Cells In order to attain more iron, cancer cells have higher numbers of the transferrin receptor-1 molecule (TfR1) on their cell surface. The TfR1 binds the serum iron transport protein, transferrin (Tf). Hence, cancer cells are able to bind more Tf, and thus, take up iron at a greater rate than their normal counterparts. This is reflected by the ability of tumors to be radiolocalized using a radioisotope of gallium, 67Ga, which binds to the iron-binding site on Tf for delivery via TfR1. 67 Ga can bind to iron-binding sites of Tf due to the similar atomic properties between gallium(III) and iron (III). Additionally, gene therapy by administration of anti-sense TfR1 targeted to the sequences of TfR1 mRNA also showed selective anti-cancer activity, further demonstrating the importance of TfR1 in mediating cancer cell growth. Apart from TfR1 up-regulation, the expression of the iron-storage protein ferritin is also often altered in neoplastic cells, especially neuroblastoma (NB) and breast carcinoma. In childhood NB, serum ferritin levels are elevated at stages III and IV of the disease.

Chelators as Anti-Cancer Drugs

In a longitudinal study, it was found that the elevated level was associated with a markedly poorer prognosis of the disease. In addition, serum ferritin levels also exceeded the normal limit in ▶hepatocellular carcinoma and were found to be directly related to axillary lymph node status, presence of metastatic disease (▶metastasis) and clinical stages of breast cancer. Desferrioxamine, an Iron Chelator with Some Anti-Cancer Activity Desferrioxamine (DFO) is a natural ligand secreted by the bacterium Streptomyces pilosus to selectively sequester iron for biological use (Fig. 1). DFO is used clinically for the treatment of iron overload disorders such as the transfusion-related iron overload in β-thalassemia. DFO is active against aggressive tumors including NB and leukemia in cell culture and clinical trials. The cytotoxicity of DFO in vitro was prevented by coincubation of the cells with iron or iron saturated DFO, indicating that its anti-proliferative activity was due to depletion of cellular iron. Furthermore, DFO induces a block in cell cycle progression. Therefore, it was proposed that the mechanism of action of DFO involved the depletion of cellular iron, leading to the inhibition of ribonucleotide reductase for DNA synthesis and cell cycle arrest. In human NB cells, 5 days of exposure to DFO resulted in approximately 90% cell death. In contrast, the effect of DFO was minimal on non-NB cells, suggesting that it had selective anti-NB activity. A clinical trial showed that seven of nine NB patients

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had up to 50% reduction in bone marrow infiltration after a course of DFO administered for 5 days. Other clinical trials using DFO as a single agent and in combination with other chemotherapeutic drugs confirmed the anti-cancer potential of this chelator. However, in some animal studies and clinical trials, DFO was found to exhibit limited or no activity. DFO also suffers a number of limitations as a result of its highly hydrophilic nature. It has poor gastrointestinal absorption and a short plasma half-life of about 12 min due to rapid metabolism. As a result, DFO is not orally active and needs to be administered via subcutaneous infusion for prolonged periods ranging from 8 to 12 h for five to seven times per week. The prolonged infusion results in pain and swelling, which consequently leads to poor patient compliance. DFO is also expensive to produce. Despite these limitations and mixed results in clinical trials, DFO nonetheless provides “proof of principle” that iron chelation therapy may be specific and useful for cancer treatment. Other Chelators with Anti-Cancer Potential The limitations of DFO as an anti-cancer agent have encouraged the search for other active iron-chelating drugs against cancer. Other experimental iron chelators include Triapine® (3-AP; Fig. 1), an iron-binding thiosemicarbazonebased drug currently in clinical trials for cancer therapy. Triapine® is a chelator that binds iron via a sulfur and two nitrogen donor atoms, and is suggested to be one

Chelators as Anti-Cancer Drugs. Figure 1 Chemical structures of the iron chelators desferrioxamine (DFO), N,N′,N″-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (tachpyridine or tachpyr), 3-aminopyridine2-carboxaldehyde-thiosemicarbazone (3-AP or Triapine®), 2-hydroxy-1-napthaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) showing coordination to iron (Fe) through pyridyl nitrogen, aldimine nitrogen and thionyl sulfur donor atoms.

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of the most potent inhibitors of RR yet identified. In clinical trials, high doses of Triapine® (160 mg/m2/day) resulted in dose-limiting toxicities, including reduction in white blood cells, jaundice, nausea and vomiting. Lower doses of Triapine® administered as a 96-h iv infusion at 120 mg/m2/day every 2 weeks was found to be well tolerated. In clinical trials with patients with advanced cancer, Triapine® was combined with the cytotoxic cancer drug gemcitabine, which also targets DNA synthesis. Of the 22 patients examined after treatment with gemcitabine and Triapine®, three were observed to have an objective response, and one patient had evidence of tumor reduction. In this trial, Triapine® was suggested to cause oxidation of hemoglobin to methemoglobin. This may have led to or contributed to the hypoxia, acute hypotension, and electrocardiogram changes in patients receiving this chelator. An asymptomatic myocardial infarction was also observed in one individual administered Triapine® and this may also be related to its oxidative effects. Triapine® continues to be examined in clinical trials, particularly in combination with standard chemotherapy drugs. However, these deleterious effects must be considered when designing future studies with compounds of this class. Tachpyridine, (or tachpyr; Fig. 1) is a novel chelator based upon the framework of the triamine cis,cis1,3,5-triaminocyclohexane. Tachpyridine is cytotoxic to cultured bladder cancer cells with an activity approximately fifteen times greater than that of DFO. Although tachpyridine has the potential to chelate a number of metals, including calcium(II), magnesium (II), manganese(II), copper(II) and zinc(II), toxicity studies on tachypyridine complexes suggest that iron and zinc depletion mediates its cytotoxic effects. Similar to Triapine®, Tachpyridine induces apoptotic cell death independent of functional p53 (see Iron Chelation and Cell Cycle Control Molecules, below) (▶p53 gene family). In addition, tachpyridine-iron complexes produce toxic free-radicals (▶reactive oxygen species), which was also thought to contribute to its anti-tumor activity. Tachpyridine arrests cells at the G2 phase, whereas the majority of iron chelators arrest cells at the G1/S phase due to inhibition of ribonucleotide reductase. The G2 phase stage of the cell cycle is particularly sensitive to the effects of radiation. Ionizing radiation increases the sensitivity of tumor cells to the action of tachpyridine. Currently, tachpyridine is in preclinical development with the National Cancer Institute, USA. PIH Chelators The most comprehensively assessed alternate chelators for cancer treatment are the pyridoxal isonicotinoyl hydrazone (PIH) analogues. This class of chelators bind iron through the carbonyl oxygen, imine nitrogen, and phenolic oxygen (Fig. 1).

Originally conceived for the treatment of iron overload disorders, several chelators of the PIH class were found to inhibit the growth of cancer cells. In fact, the chelator 311 (Fig. 1) was found to be highly active against a range of cancer cells. These compounds also showed marked ability to remove Fe from cells and prevent cellular Fe uptake from transferrin. The marked anti-cancer activity of chelator 311 was attributed to its relatively high ▶lipophilicity, which facilitates entry into the cell. Indeed, a general trend observed with the PIH analogues was that anti-cancer activity increased as the chelator became more lipophilic. Mechanistically, PIH analogues have multiple modes of anti-cancer activity, aside from chelation of iron and inhibition of ribonucleotide reductase. Some members of the PIH class of chelators (e.g. DpT chelators, see below) increase the generation of toxic free-radicals (reactive oxygen species) in cancer cells and affect the expression of cell-cycle control molecules (see Iron Chelation and Cell Cycle Control Molecules, below). Additional studies with 311 have also shown that it can markedly induce the expression of the metastasis suppressor protein, ▶Drg-1 in tumor cells. The Drg-1 protein is known to play a critical role in suppressing tumor growth and metastasis. Hence, induction of Drg-1 by potent iron chelators such as 311 may significantly contribute to the anti-cancer activity of these analogues. The DpT Chelators: Dp44mT The DpT class of chelators are structurally-related to PIH analogues, but feature a sulfur donor atom instead of the hydrazone oxygen donor atom (Fig. 1). The chelator Dp44mT has recently been shown to be the most effective of the DpT series of ligands in terms of anti-cancer activity. It acts with selectivity against tumor cells and has much less effect on the growth of normal cells. Dp44mT also showed high iron chelation efficacy and prevented cellular uptake of iron from iron-labeled Tf. Another mechanism of its action involves the generation of toxic free-radicals (reactive oxygen species) when Dp44mT interacts with cellular iron pools. Initially, in vivo studies of Dp44mT in mice bearing chemotherapy-resistant M109 lung carcinoma showed a reduction in the size of the tumor by 53% after 5-days of treatment. A later investigation also found marked inhibition of the growth of human lung, neuroepithelioma and melanoma xenografts growing in mice. In fact, a 7-week administration of Dp44mT in mice bearing human melanoma xenografts resulted in the decrease of tumor growth to 8% of that in untreated control mice. At the dose given, no hematological abnormalities were detected, although at a higher dose, myocardial fibrosis was identified. This side effect at a high dose may be due to the marked redox activity of the Dp44mT-iron complex. However, at a lower dose


Dp44mT was well tolerated with no hematological abnormalities and less cardiotoxicity. Other studies with Dp44mT showed that it also markedly increased the expression of the metastasis suppressor protein, Drg-1 in tumor cells. Induction of Drg-1 could potentially be a very significant component of the anti-cancer mechanism of Dp44mT. Further development of DpT series chelators is currently underway. Iron Chelation and Cell Cycle Control Molecules Iron-deprivation generally leads to G1/S phase cell cycle arrest as a result of inhibition of ribonucleotide reductase. This has prompted many studies assessing the effect of iron chelation by DFO and chelator 311 on the expression of many cell cycle control molecules, namely, cyclins, ▶cyclin dependent kinases (cdks), cdk inhibitors and p53 (p53 gene family). Consistently, these studies found that iron chelation markedly decreased the expression of ▶cyclin D (D1, D2 and D3), and to a lesser extent cyclin A and B. The expression of cdk2 and cdk1, but not cdk4, were also decreased upon iron chelation. These effects were dependent on iron-deprivation, as iron-chelator complexes were unable to induce such effects. Cyclins D, E, A and cdks 2, 4 and 6 are involved in progression through the G1 phase, although cyclin E, A and cdk2 are also involved in S phase progression. The formation of the cyclin A-cdk2 complex is essential for G1/S progression. Cyclin B and cdk1 on the other hand, are important for mitosis. During the G1 phase, cyclin D and E bind to cdk4 and cdk2 respectively to phosphorylate (▶phosphorylation) the ▶retinoblastoma protein (pRb) (▶Biological and Clinical Functions). This results in the release of molecules such as the ▶E2F transcription factor from pRb that promotes the expression of genes for S phase. The decrease in the expression of these cyclins upon iron chelation causes hypophosphorylation of pRb, which in turn leads to the G1/S phase arrest. In addition to cyclins and cdks, iron chelation also affects the expression of cell cycle modulatory molecules. In particular, iron chelators caused a marked increase in the expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (▶p21(WAF1/CIP1/ SDI1)) at the mRNA level. p21WAF1/CIP1 mediates G1/S phase arrest by directly binding the cyclin-cdk complexes. It was speculated that the increased level of p21WAF1/CIP1 upon iron chelation was consistent with its potential role in the G1/S phase arrest. However, an increase of p21WAF1/CIP1 expression only occurred at the mRNA level, with either no change or a decrease in p21WAF1/CIP1 protein expression being observed. This was unexpected and it was subsequently demonstrated that p21WAF1/CIP1 protein level could be controlled by proteasomal (▶proteasome) degradation after iron chelation.

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In contrast, investigations examining p53 showed that its protein expression and DNA-binding activity were increased after chelation. p53 is a tumor suppressor and acts as a transcription factor that is involved in the transcription of a variety of genes involved in cell cycle arrest, differentiation, apoptosis and DNA repair. An increase in p53 after iron chelation may be the result of a decrease in deoxyribonucleotide levels due to inhibition of RR activity or changes in intracellular redox status. Despite the fact that p21WAF1/ CIP1 is a down-stream effector of p53, elevated expression of p21WAF1/CIP1 upon iron chelation occurs through a p53-independent pathway. The ability of chelators to potentially inhibit tumor cell growth by a p53-independent pathway is significant, since p53 is the most frequently mutated gene in cancer. This also explains why cells with wild-type or mutant p53 are similarly sensitive to the growth inhibitory effects of iron chelators. However, the function of increased p53 expression after chelation remains a subject for further investigation. Conclusions The demonstration that some iron chelators may be clinically useful for cancer treatment followed on from initial observations that rapid cancer cell proliferation requires iron. Currently, the iron chelator, Triapine®, is being examined in a variety of clinical trials, with focus on a potential role in combination chemotherapy. The search for more effective anti-cancer Fe chelators than DFO has also led to the development of other potent Fe chelators, including Dp44mT and tachpyridine, and significant progress has been made towards understanding their molecular targets. However, further in vivo experiments and pre-clinical studies will be necessary to build upon the promise of these agents.

References 1. Yu Y, Wong J, Lovejoy DB et al. (2006) Chelators at the cancer coalface: desferrioxamine to triapine and beyond. Clin Cancer Res 12:6876–6883 2. Buss JL, Greene BT, Turner J et al. (2004) Iron chelators in cancer chemotherapy. Curr Top Med Chem 4:1623–1635 3. Kalinowski D, Richardson DR (2005) Evolution of iron chelators for the treatment of iron overload disease and cancer. Pharmacol Rev 57(4):1–37 4. Le NTV, Richardson DR (2004) Iron chelators with high anti-proliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene: a novel link between iron metabolism and proliferation. Blood 104:2967–2975 5. Whitnall M, Howard J, Ponka P et al. (2006) A class of iron chelators with a wide spectrum of potent anti-tumor activity that overcome resistance to chemotherapeutics. Proc Natl Acad Sci USA 103:14901–14906

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Chemical Biology

Chemical Biology Definition The application of chemical tools and ideas to biological problems. ▶Small Molecule Screens

Chemical Biology Screen ▶Small Molecule Screens

Chemical Carcinogenesis J OSEPH R ICHARD L ANDOLPH , J R . Department of Molecular Microbiology and Immunology, and Dept. of Pathology; Cancer research Laboratory, USC/ Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences, School of Pharmacy, Health Sciences Campus, University of Southern California, Los Angeles, CA, USA

Definition

▶Chemical carcinogenesis (▶carcinogenesis) is the process of the genesis of a ▶tumor (carcinoma), and the series of sequential steps that occur when lower animals or humans are treated with ▶chemical carcinogens that leads to tumor development. After all these steps are accomplished, the physiological mechanisms regulating control of growth in the normal cells are degraded, and the normal cells are degraded and converted into tumor cells. The tumor cells then grow in an unregulated fashion and evade the host immune system, leading to development of visible tumors.

Characteristics Normal Cell Types in Animals and the Tumors They Give Rise to During embryogenesis in mammals (warm-blooded animals), there are three primary germ layers of the early embryo which develop into all the basic cell types, tissues, and organs in the body. These are the ectoderm, the endoderm, and the mesoderm. The ectoderm and endoderm are epithelial layers. Most of the epithelial

organs in the body are derived from the endodermal and the ectodermal germ layers. The epidermis of the skin, the corneal epithelium, and mammary glands develop from the ectoderm. The endoderm layer develops into the liver, pancreas, stomach, and intestines. The mesoderm develops into the kidney and linings of male and female reproductive tracts. Three types of cells are important in chemical carcinogenesis. These cell types are (i) ▶epithelial cells, which form the coverings and internal parts of organs, (ii) ▶fibroblasts, which are connective tissue cells derived from primitive mesenchymal cells, and (iii) cells of the hematolymphopoietic series, which are derived from the blood-forming elements. These cell types all have special, specific characteristics. In humans, 92% of the tumors that arise are derived from epithelial cells (▶Epithelial cell tumors). These tumors are called carcinomas. The remaining 8% of the tumors are derived from a combination of tumors derived from fibroblasts, called sarcomas, and tumors derived from white blood cells, called leukemias (▶Leukemia diagnostics) and lymphomas. Carcinogens There are a group of molecules and radiations referred to as “carcinogens.” A ▶carcinogen (▶Carcinogen macromolecular adducts) is any molecule, or group of molecules, such as viruses (▶Virology), or radiation (▶Radiation carcinogenesis; ▶radiation oncology) that can cause tumors in lower animals and humans, when they are exposed to this agent. This happens when carcinogens cause normal cells to transform, or convert into transformed cells and tumor cells during experiments in vitro, called chemical transformation experiments. Chemicals referred to as chemical carcinogens (chemical carcinogenesis) can cause tumors in lower animals in humans exposed to them. Examples of chemical carcinogens are vinyl chloride, aflatoxin B1 (a metabolite and biocide of the fungus, Aspergillus flavus) (▶Aflatoxins), benzo(a)pyrene (a polycyclic aromatic hydrocarbon formed when organic matter is pyrolyzed in the absence of oxygen) (▶Polycyclic aromatic hydrocarbons), and beta-naphthylamine (an aromatic amine used to manufacture dyestuffs that causes bladder cancer in animals and humans) (▶Aromatic amines). Nitrosamines are another class of chemical carcinogens. An example is dimethylnitrosamine (DMN). Many nitrosamines are synthetic compounds. Some are believed to form in the stomach of humans when amines (derived from fish in the diet) contact nitrous acid (formed from the nitrate from fertilizer that is used to grow foodstuffs) in the acidic conditions (acid pH) of the stomach. Chemicals in all these classes of carcinogens can cause tumors in humans and in lower mammals.

Chemical Carcinogenesis

There are also a number of radiations that cause tumors in humans and lower animals. These include ionizing radiations, such as alpha particles (charged helium nuclei), beta particles (naked electrons), and gamma particles. There are also tumor viruses, consisting of RNA (RNA tumor viruses) and DNA (DNA tumor viruses). When animals are treated with these viruses, tumors are formed. Examples of RNA tumor viruses are the Rous sarcoma virus, the Abelson leukemia virus, and the Kirsten Ras virus. Examples of DNA tumor viruses are the polyoma virus, the SV40 (simian virus 40) (▶SV40) virus, the ▶Epstein Barr virus, and the human papilloma viruses 16 and 18 (▶Human papilloma viruses). Mechanisms of Chemical Carcinogenesis There are two broad mechanisms of chemical carcinogenesis. In the first type, which we refer to here as “▶complete carcinogenesis,” a mammal is treated with a large dose of a chemical carcinogen, such as 7,12-dimethylbenz(a)anthracene, and the animals treated eventually develop tumors. Carcinogenesis with complete carcinogens is usually dose-dependent, such that the higher doses of carcinogens that the animals are treated with, the high the yield of tumors per animal and in the percentage of animals with tumors. The second mechanism of chemical carcinogenesis, discovered by Dr. Isaac Berenblum of the Weizmann Institute in Israel, is referred to as ▶“two-step carcinogenesis,” or “initiation and promotion”. In initiation and promotion experiments, Berenblum treated mice on the skin of their shaved backs with chemical carcinogens at low doses and also with ▶tumor promoters. Berenblum was testing the hypothesis that carcinogenesis was due to irritation and inflammation. Hence, he used croton oil, a product of the plant, Euphorbia lathyris, which the plant uses as a biocide against insects. Croton oil is a very irritating substance, which is important in the plant’s use of it as a biocide against insects. When mice were treated with low doses of 7,12-dimethyl-benz(a)anthracene (DMBA, a carcinogenic PAH), one time, they exhibited no tumors. A second group of animals was treated with the tumor promoter, croton oil, once per week, and the animals also exhibited no tumors. When the mice were treated with a low dose of DMBA, and then once weekly with croton oil, they developed many tumors. If the latter treatment was reversed, i.e. the animals were treated first with croton oil once per week, and then later treated with a low dose of DMBA, the animals showed no tumors. If the animals were treated with a low dose of DMBA, then no treatment was performed for a significant amount of time, then the animals were treated with croton oil once per week, the animals also developed a high yield of tumors. In this system, treatment of the animals with the low dose of DMBA is referred to as the “initiation step,” and later

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treatment with croton oil is called the “promotion” step. Initiation is believed to be a ▶genotoxic event, likely a ▶mutation, and is an irreversible step. Initiated cells can be promoted to tumors cells if they are treated with croton oil long enough. The promotion step is believed to be due to the binding of tetradecanoyl-phorbol acetate (TPA, the most active constituent of the mixture of phorbol esters in croton oil), to protein kinase C, triggering signal transduction and cell division in cells bearing mutations in ▶proto-oncogenes. If promotion is interrupted, then tumorigenesis is reversible, i.e. the cellular death rate will equal the cellular growth rate, and the tumor will regress. If promotion is continued long enough, the tumor becomes fixed and will not regress. Eric Hecker of the German Cancer Research Center (Deutsch Krebs Forschung Zentrum) in Heidelberg, Germany, fractionated croton oil used by Berenblum, by high pressure liquid chromatography, and found that TPA was the most active tumor promoter in it. From experiments with high doses of chemical carcinogens, and experiments with initiation and promotion, we now have evidence that chemical carcinogens such as DMBA cause mutations in proto-oncogenes, such as ras genes, converting them into activated ▶oncogenes. In complete carcinogenesis experiments, further mutations in other proto-oncogenes can also occur, leading to activation of additional oncogenes. In addition, activated metabolites of the carcinogens (formed in the animals/ mammals by cytochrome P450 or other enzymes of metabolic activation), also cause mutational inactivation of ▶tumor suppressor genes, or breakage of chromosomes bearing them, leading to loss of these tumor suppressor genes. Together, activation of oncogenes and inactivation of tumor suppressor genes is believed to lead to the genesis of tumors in mammals. Insights into Mechanisms of Chemical Carcinogenesis from Studies of Chemically Induced Neoplastic Transformation Studies of the abilities of chemical carcinogens to convert normal cells into tumor cells in cell culture dishes have given us substantial insight into the molecular mechanisms of chemical carcinogenesis. In cell culture, normal fibroblasts and normal epithelial cells grow if they are fed properly, until they eventually fill the culture dish, and touch each other. Growth then ceases. This process is called contact inhibition of cell division. Cells can then be removed from the cell culture dish with a protease called trypsin, diluted, and replated into new cell culture dishes. This process can be repeated many times, until the population of total cells has undergone sixty population doublings. At this point, the cells senesce (▶Senescence and immortalization), or die. This is due to progressive shortening of telomeres (▶Telomerase), structures at the end of chromosomes, with each successive DNA replication

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and cell division. Telomere shortening acts as a cellular and molecular “clock,” to mark the lifetime of the cell. This process aids in the control of the normal physiology of the organism, by removing old cells which accumulated many mutations, which could eventually lead to cancer. ▶Chemically induced cell transformation is the process by which normal cells are treated with chemical carcinogens in vitro in a cell culture dish or flask, and then their growth control mechanisms degrade, converting or transforming them into transformed cells. There are two mechanisms by which cells can be converted by chemical carcinogens into transformed cells. Firstly, cells can be treated with genotoxic (DNAdamaging) (▶Genetic toxicology) chemical carcinogens. Many of these genotoxic carcinogens are ▶mutagens (▶Mutation rate). These carcinogens either already are direct mutagens (rare), or more commonly they are pre-carcinogens, and can be converted into mutagenic proximate carcinogens by ▶cytochrome P450 enzymes or other enzyme systems that activate the pre-carcinogens into mutagens. The carinogens (benzo(a)pyrene, aflatoxin B1, and nitrosamines are all examples of pre-carcinogens that are metabolically activated into mutagens by various types of cytochrome P450 enzymes. Most pre-carcinogens are hydrophobic (fat-loving) compounds that would bioaccumulate in the body, and cause alterations in the properties of enzymes and membranes in cells. Mammals must therefore derive strategies to eliminate hydrophobic pre-carcinogens. The cytochrome P450 enzyme system, and other enzyme systems, have evolved in order to metabolize these pre-carcinogens, to make them water-soluble, so they can be excreted in the urine and removed from the body. Since these compounds are inherently chemically inert, a necessary first chemical reaction step has evolved, in which cytochrome P450 enzymes attack pre-carcinogens like benzo(a)pyrene (BaP) with molecular oxygen and reducing equivalents (NADPH and NADH) to generate epoxides and diol epoxides from it. These metabolites are mutagens, and this step results in “metabolic activation.” In a second step, which is closely coupled to the first step, these active metabolites are reacted with and conjugated to, molecules of water by the enzyme, epoxide hydrolase, converting them to trans-dihydrodiols and tetraols, which are highly water-soluble, so they are excreted in the urine. The small amount of epoxides and diol epoxides derived from BaP then bind covalently to DNA bases, resulting in mutations in proto-oncogenes, activating them into oncogenes, and mutations in tumor suppressor genes, inactivating them. In a second mechanism of ▶carcinogenesis, chemicals called “non-genotoxic carcinogens,” transform normal cells into tumor cells in a different way, by non-mutagenic mechanisms. One example is the

chemical, 5-azacytidine, a chemical analog of a normal base. 5-azacytidine binds to DNA methyltransferases (▶Methylation), inhibiting them. This results in a loss of methylation of the cytidine in DNA. If this occurs in quiescent proto-oncogenes, then these can become transcriptionally activated, leading to cell transformation. Other examples of non-genotoxic carcinogens include hormones, such as testosterone and estrogen. Higher steady-state levels of testosterone and estrogen are believed to lead to aberrantly high numbers of cell divisions in prostate and breast tissue. The resultant spontaneous mutations that occur are believed to lead to prostate cancer and breast cancer, respectively. The process by which a normal cell is converted into a tumor cells, or chemically induced ▶neoplastic transformation (▶Neoplastic cell transformation), occurs in four steps. In the first step, when cells are treated with mutagenic chemical carcinogens, there occur mutations in proto-oncogenes, activating them to oncogenes, and mutations in tumor suppressor genes, inactivating them. The cells then develop the ability to grow in multi-layers, and form foci. This is particularly true for fibroblastic cells, less so for epithelial cells. This first step in cell transformation is called morphological cell transformation, or focus formation. Further genetic changes occur in the transformed cells. The second step that occurs is that the cells become immortal, and do not die or senesce. Some activated oncogenes (v-myc) can cause cells to become immortal. This step would be called transformation to cellular immortality. In the third step, cells develop the ability to grow in soft agar, in three dimensional suspension. This step is called anchorage-independent cell transformation, or transformation to anchorage independence. A final step that develops after further genetic change is that cells develop the ability to form tumors when injected into athymic (nude) mice. This step is called neoplastic transformation, or the ability of cells to be transformed so that they form neoplasms, or new growths, which we call tumors. Often, a number of activated oncogenes, two or more, may cooperate together to perturb normal cellular physiology, to cause neoplastic transformation of normal rodent or human cells in culture. It is now believed by scientists that activation of proto-oncogenes into oncogenes, and inactivation of tumor suppressor genes, such that approximately eight total genes are genetically altered, leads to the aberrant expression of approximately 150 genes or more in the tumor cells. This then leads to neoplastic transformation of cells in culture, hence to chemical cacinogenesis in the animal. We believe that chemically induced neoplastic transformation is a good model for how cells in the animal become converted (transformed) into tumor cells when the animal is treated with chemical carcinogens.

Chemical Tool

Significance of Chemical Carcinogenesis The significance of the process of chemical carcinogenesis is two-fold. Firstly, the assay for chemical carcinogenesis in lower animals, usually mice and rats, can be used to test chemicals to determine whether they are carcinogens by virtue of their ability to induce tumors in mice and rats. Those chemicals that are able to cause a reproducible, dose-dependent induction of tumors in mice and/or rats, are presumed to be human carcinogens. This presumption is due first to the relationship that rodents and humans are both warm-blooded animals, or mammals. As such, their biochemistry and physiology is similar. In addition, many chemical carcinogens were first found to be carcinogenic in rodent carcinogenesis bioassays, and later found to be carcinogens in humans. Almost all carcinogens that have been shown to be carcinogenic in humans are also carcinogenic in rodents (aflatoxin B1, vinyl chloride, asbestos, cigarette smoke, asbestos, polycyclic aromatic hydrocarbons). Secondly, the process of chemical carcinogenesis as studied in rodents has led to unique insights into the mechanisms of carcinogenesis. Investigators frequently use whole animal carcinogenesis bioassays to study how proto-oncogenes are activated into oncogenes, how tumor suppressor genes are inactivated by chemical carcinogens, and how oncogene activation and tumor suppressor gene inactivation leads to induction of tumors in mammals. Studying the mechanisms of carcinogenesis in rodents has also led to the identification of agents that interfere with this process, and may eventually be used to prevent the induction of cancer in humans. ▶Toxicological Carcinogenesis ▶Genetic Toxicology

References 1. Landolph JR Jr, Xue W, Warshawsky D (2006) Whole animal carcinogenicity bioassays, Chapter 2. In: Warshawsky D, Landolph JR Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/ Taylor and Francis Group, Boca Raton, FL, pp 25–44 2. Verma R, Ramnath J, Clemens F et al. (2005) Molecular biology of nickel carcinogenesis: identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by specific insoluble nickel compounds. Mol Cell Biochem 255:203–216 3. Warshawsky D (2006) Carcinogens and mutagens, Chapter 1. In: Warshawsky D, Landolph JR Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, FA, pp 1–24 4. Warshawsky D Landolph JR Jr (2006) Overview of human cancer induction and human exposure to carcinogens, Chapter 13. In: Warshawsky D, Landolph JR Jr (eds)

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Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, FA, pp 289–302 5. Weinberg RW (2007) Multi-step tumorigenesis, Chapter 11. In: The biology of cancer. Garland Science, Taylor and Francis Group, LLC, New York, NY, pp 399–462

Chemical Castration Definition Removal of the gonads (ovary and testis) is required to ablate serum levels of sex steroids (progesterone, estrogen, and testosterone). As continuous administration of GnRH analogs removes the influence of the pituitary to regulate gonadal function, this inhibitory effect became known as “Chemical castration.” ▶Gonadotropin-Releasing Hormone

Chemical Genetic Screen ▶Small Molecule Screens

Chemical Mutagenesis ▶Genetic Toxicology

Chemical Tool Definition A chemical tool is a small, drug-like molecule that can be used to identify new targets in a ▶signal transduction pathway. The chemical tool is usually identified through the screening of a cell-based or in vivo assay, and then used as an affinity probe to identify its molecular target. The chemical tool provides the link between the target and the desired phenotype in the assay. ▶Luciferase Reporter Gene Assays

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Chemically Induced Cell Transformation

Chemically Induced Cell Transformation J OSEPH R ICHARD L ANDOLPH J R . Department of Molecular Microbiology and Immunology, and Pathology; Cancer Resaerach Laboratory, USC/ Norris Comprehensive Cancer Center, Keck School of Medicine; Department of Molecular Pharmacology and Pharmaceutical Sciences; Health Sciences Campus, University of Southern California, Los Angeles, CA, USA

Definition

▶Chemically induced cell transformation is the series of sequential steps that occur when mammalian cells are treated with ▶chemical carcinogens, and converted into tumor cells. The intermediate cell phenotypes (cell properties) are acquired one at a time, including first morphological ▶transformation (change in cell shape, leading to crisscrossing of cells in abnormal patterns), then anchorage independence (growth of cells as colonies or balls of cells in three dimensional suspension of agar, without attachment to the plastic dishes cells are usually grown on), and finally ▶neoplastic transformation (▶Neoplastic cell transformation), or the ability of cells to form tumors when injected into nude (athymic) mice.

Characteristics Normal Growth of Normal Cells In the mammalian organism (warm-blooded animals), there are many types of cells. In general, these cell types are divided into (i) ▶epithelial cells, which form the coverings of organs, (ii) ▶fibroblasts, which are connective tissue cells, and (iii) cells of the hematolymphopoietic series, which are derived from the bloodforming elements. These cell types all have special, specific characteristics. These three general cell types can be grown outside the body in an artificial situation, in cell culture medium in plastic cell culture dishes. This constitutes a model system in which the physiology of cells can be studied outside of the complicated conditions of the body. When grown in cell culture, epithelial cells and fibroblastic cells attach to the cell culture dish, by virtue of the surface charge of the cell relative to that of the plastic of the cell culture dish. These normal fibroblastic and epithelial cells must anchor to the bottom inside of the cell culture dish in order to be able to replicate their DNA and divide. This is called anchorage dependence of cell growth. These cells continue to grow if fed properly with cell culture medium, containing 5–10% fetal calf serum and cell culture medium. Cell culture

medium consists of sugars, amino acids, salts, and buffers, along with an indicator to detect the acidity of the culture medium (pH indicator), all dissolved in water. In cell culture, the normal fibroblasts and normal epithelial cells continue to grow if they are fed properly, until they eventually fill the culture dish, and touch each other. Growth then ceases. This process is called ▶contact inhibition of cell division. These cells can then be removed from the cell culture dish with a protease called trypsin, diluted, and replated into new cell culture dishes. This process can be repeated many times, until the population of total cells has undergone approximately 60 population doublings. This is called the “Hayflick Limit,” after Dr. Leonard Hayflick, who discovered it. At this point, the cells undergo ▶cellular senescence (▶Senescence and cellular immortalization), or die. This is due to progressive shortening of telomeres (▶Telomerase), structures at the end of chromosomes that are progressively shortened with each successive DNA replication and cell division. Hence, telomere shortening acts as a cellular and molecular “clock,” to mark the lifetime of the cell. This process is believed to aid in the control of the normal physiology of the organism, and to rid it of old cells which have many ▶mutations, which could eventually lead to cancer. If these normal cells are injected into mice lacking an immune system (athymic or “nude” mice), they will not grow and will not form tumors. In contrast, cells of the hemato-lymphopoietic series grow in three-dimensional suspension (the blood) in vivo. Hence, when grown in vitro (outside the body), these cells must also be grown in three-dimensional suspension. A common practice is to grow the cells in varying concentrations of agar. When injected into athymic or “nude” mice, these normal cells, whether cells of the hematopoietic (red blood cell) or lymphoid (white blood cell) lineages, will not form tumors. Carcinogens There are a group of molecules and radiations referred to as “carcinogens.” A ▶carcinogen (▶Carcinogen macromolecular adducts) is any molecule or group of molecules, such as viruses (▶Virology) or radiation (▶Radiation carcinogenesis; ▶radiation oncology) that can cause tumors in lower animals when they are treated with this agent. These agents can also cause normal cells to transform (convert) into transformed cells and tumor cells. There are a group of chemicals referred to as chemical carcinogens (▶Chemical carcinogenesis). These are specific chemicals that can cause tumors in animals treated with them. Examples of these are vinyl chloride, aflatoxin B1 (a metabolite and biocide of the fungus, Aspergillus flavus) (▶Aflatoxins), benzo(a)pyrene (a polycyclic aromatic hydrocarbon formed when organic matter is burned in the absence of

Chemically Induced Cell Transformation

oxygen) (▶Polycyclic aromatic hydrocarbons), and beta-naphthylamine (an aromatic amine used to manufacture dyestuffs that causes bladder cancer in animals and humans) (▶Aromatic amines). Another class of chemical carcinogens is called nitrosamines. An example is dimethylnitrosamine (DMN). Many nitrosamines are synthetic compounds. Some are believed to form in the stomach of humans when amines (derived from fish in the diet) contact nitrous acid (formed from the nitrate from fertilizer that is used to grow foodstuffs) in the acidic conditions (acid pH) of the stomach. Chemicals in all these classes of carcinogens can cause tumors in humans and in lower mammals. There are also a number of radiations (Radiation carcinogenesis) that can cause tumors in humans and lower animals. These include ionizing radiations, such as alpha particles (charged helium nuclei), beta particles (naked electrons), and gamma particles. In addition, there are also tumor viruses, consisting of RNA (RNA tumor viruses) and DNA (DNA tumor viruses). When animals are treated with these viruses, tumors are formed. Examples of RNA tumor viruses are the Rous sarcoma virus, the Abelson leukemia virus, and the Kirsten Ras virus. Examples of DNA tumor viruses are the polyoma virus, the SV40 (simian virus 40) (▶SV40) virus, the ▶Epstein Barr virus, and the human papilloma viruses 16 and 18 (▶Human papilloma viruses). Chemically Induced Cell Transformation – Description and Mechanisms Chemically induced cell transformation is the process by which normal cells are treated with chemical carcinogens in vitro in a cell culture dish or flask, and they then convert or transform into transformed cells. There are two mechanisms by which cells can be converted by chemical carcinogens into transformed cells. Firstly, cells can be treated with ▶genotoxic (DNA-damaging) (▶Genetic toxicology) chemical carcinogens. Many of these genotoxic carcinogens are ▶mutagens (▶Mutation rate). These carcinogens either already are direct mutagens (rare), or more commonly they are pre-carcinogens, and can be converted into mutagenic proximate carcinogens by ▶cytochrome P450 enzymes or other enzyme systems that activate the pre-carcinogens into mutagens. The Pre-carinogens benzo(a)pyrene, aflatoxin B1, and nitrosamines are all examples of pre-carcinogens that are metabolically activated into mutagens by various types of cytochrome P450 enzymes. The perspective for this process is that most pre-carcinogens are hydrophobic (fat-loving) compounds that would bioaccumulate in the body, and cause alterations in the properties of enzymes and membranes in cells. Hence, the organism must derive a strategy to eliminate these hydrophobic precarcinogens. Therefore, the cytochrome P450 enzyme system, and other enzyme systems, have evolved in order

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to metabolize these pre-carcinogens, to make them water-soluble, so they can be excreted in the urine and removed from the body. Since these compounds are inherently chemically inert, a necessary first chemical reaction step has evolved, in which cytochrome P450 enzymes first attack pre-carcinogens like benzo(a)pyrene (BaP) with molecular oxygen and reducing equivalents (NADPH and NADH) to generate epoxides and diol epoxides from it. These metabolites are mutagens, and this step results in “metabolic activation.” In a second step, which is closely coupled to the first step, these active metabolites are reacted with and conjugated to, molecules of water by the enzyme, epoxide hydrolase, converting them to trans-dihydrodiols and tetraols, which are highly water-soluble, so they are excreted in the urine. The small amount of epoxides and diol epoxides derived from BaP then go on to bind covalently to DNA bases, resulting in mutations in proto-oncogenes, activating them into ▶oncogenes, and mutations in ▶tumor suppressor genes, inactivating them. In a second mechanism of ▶carcinogenesis, chemicals called “non-genotoxic carcinogens,” transform normal cells into tumor cells in a different way, by non-mutagenic mechanisms. One example is the chemical, 5-azacytidine, a chemical analog of a normal base. 5-azacytidine binds to DNA methyltransferases (▶Methylation), inhibiting them. This results in a loss of methylation of the cytidine in DNA. If this occurs in quiescent proto-oncogenes, then these can become transcriptionally activated, leading to cell transformation. Other examples of non-genotoxic carcinogens include hormones, such as testosterone and estrogen. Higher steady-state levels of testosterone and estrogen are believed to lead to aberrantly high numbers of cell divisions in prostate and breast tissue. The resultant spontaneous mutations that occur are believed to lead to prostate cancer and breast cancer, respectively. The process of chemically induced neoplastic transformation, or the process of generating a tumor cell, falls into at least four steps. In the first step, when cells are treated with mutagenic chemical carcinogens, there occur mutations in proto-oncogenes, activating them to oncogenes, and mutations in tumor suppressor genes, inactivating them. The cells then develop the ability to grow in multi-layers, and form foci. This is particularly true for fibroblastic cells, less so for epithelial cells. This first step in cell transformation is called ▶morphological cell transformation, or focus formation. Further genetic changes occur in the transformed cells. The second step that occurs is that the cells become immortal, and do not die or senesce. Some activated oncogenes (v-myc) can cause cells to be come immortal. This step would be called transformation to cellular immortality. A third step that occurs is that the cells develop the ability to grow in soft agar,

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in three-dimensional suspension. This step is called ▶anchorage-independent cell transformation, or transformation to anchorage independence. A final step that develops after further genetic change is that the cells develop the ability to form tumors when injected into athymic (nude) mice. This step is called neoplastic transformation, or the ability of the cell to be transformed so that it forms neoplasms, or new growths, which we call tumors. Often, a number of activated oncogenes, two or more, may cooperate together to perturb normal cellular physiology, to cause neoplastic transformation of normal rodent or human cells in culture.

4. Verma R, Ramnath J, Clemens F et al. (2005) Molecular biology of nickel carcinogenesis: identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by specific insoluble nickel compounds. Mol Cell Biochem 255:203–216 5. Weinberg RW (2007) Multi-step tumorigenesis, Chapter 11. In: The biology of cancer. Garland Science, Taylor and Francis Group, LLC, New York, NY, pp 399–462

Significance of Chemically Induced Neoplastic Transformation The significance of the process of chemically induced neoplastic transformation is twofold. Firstly, the assay for chemically induced morphological cell transformation can be used an assay to detect chemical carcinogens. Those chemicals that have the ability to induce foci of morphologically transformed cells are highly likely to be able to induce tumors in animals. Hence, this assay can detect chemical carcinogens by virtue of their ability to induce foci of morphologically transformed cells. Secondly, the study of chemically induced morphological, anchorage-independent, and neoplastic transformation in vitro is frequently used as a model system to study the process of chemical carcinogenesis. Investigators frequently use these assays to study how proto-oncogenes are activated into oncogenes, and how tumor suppressor genes are inactivated by chemical carcinogens, and how oncogene activation and tumor suppressor gene inactivation leads to induction of morphological transformation, cellular immortality, anchorage-independent transformation, and neoplastic transformation.

Definition

References

Synonyms

1. Kumar V, Abbas AK, Fausto N (2005) Neoplasia, Chapter 7. In: Robbins and Cotran’s pathologic basis of disease, 7th edn. Elsevier Saunders, Philadelphia, PA, pp 269–342 2. Landolph JR Jr (2006) Chemically induced morphological and neoplastic transformation in C3H/10T1/2 mouse embryo cells, Chapter 9. In: Warshawsky D Landolph JR Jr (eds) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, FL, pp 199–220 3. Pitot HC, Dragan YP (2001) Chemical carcinogenesis, Chapter 8. In: Klaassen CD (ed) Casarett and Doull’s toxicology, the basic science of poisons, 6th edn. McGraw-Hill Medical Publishing Division, New York, NY, pp 239–320

Directed migration; Directed motility

Chemoattractant

A molecule that is capable of promoting cell movement by inducing ▶chemotaxis. ▶Chemokine Receptor CXCR4

Chemoattractant Cytokine ▶Chemokine

Chemoattraction J OSE LUIS R ODRI´ GUEZ -F ERNA´ NDEZ Departamento de Fisiología Celular y Molecular, Centro de Investigaciones Biológicas, Madrid, Spain

Definition Chemoattraction is the process whereby a cell detects a chemical gradient of a ligand called chemoattractant and, as a consequence, gets oriented and subsequently moves in the direction from a low to a high concentration of the chemoattractant. Chemoattraction is controlled by specific chemoattractant receptors that are able to detect selectively these ligands. Chemoattraction is called ▶chemotaxis or ▶haptotaxis when the chemical gradient of the chemoattractant is presented to the cell either in a soluble or bound to a substrate form,

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respectively. As it is not clear which one of these two types of motile processes takes place in vivo, it is more appropriate to refer to these directional motile processes with the more general term of chemoattraction.

Characteristics

Chemoattraction. Figure 1 Classical and new names of chemokines are included. Red identifies “inducible” or “inflammatory” chemokines, green “homeostatic” agonists and yellow ligands belonging to both realms. BCA, B cell activating chemokine; BRAK, breast and kidney chemokine; CTACK, cutaneous T-cell attracting chemokine; ELC, Epstein–Barr virus-induced receptor ligand chemokine; ENA-78, epithelial cell-derived neutrophil-activating factor (78 amino acids); GCP, granulocyte chemoattractant protein; GRO, growth-related oncogene; HCC, human CC chemokine; IP, IFN-inducible protein; I-TAC, IFN-inducible T-cell α chemoattractant; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; Mig, monokine induced by gamma interferon; MIP, macrophage inflammatory protein; MPIF, myeloid progenitor inhibitory factor; NAP, neutrophil-activating protein; PARC, pulmonary and activation-regulated

Chemoattractants use specific chemoattractant receptors to guide different migratory cell types towards specific sites in the organism. These receptors, upon binding to the chemoattractant, transform the information of this ligand in intracellular signals that result in the movement of the migratory cell towards the positions where chemoattractant is present at high concentration. Therefore, the analysis, in a specific context, in one hand, of the type of chemoattractant receptors expressed by a certain migratory cell and, on the other hand, the position in the organism of the chemoattractants recognized by these receptors, allow to make predictions on the potential tissues where this cell can be attracted. Upon arrival to the position where the chemoattractant is at a high concentration, adhesive receptors may contribute to slow down (function largely performed by selectin adhesive receptors for cells in blood vessels) and eventually attach (cells use ▶integrin receptors for this function in most cell types) the cells to these sites. Chemoattractants can be conveniently classified according to the type of receptor that they bind. In this regard, the first and the largest group include chemoattractants that bind members of the ▶G-protein coupled receptor (GPCR) superfamily. In this first group is included the family of ▶chemokines. A second group is formed by chemoattractants that bind tyrosine kinase receptors (e.g. Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF)). A third group includes ligands that bind receptors different of the two aforementioned families (e.g. laminin and fibronectin, which bind integrin receptors). This article deals mainly with the chemokines because they have been the chemoattractant family most studied in relation to ▶cancer and ▶metastasis. Chemokines Chemokines (chemotactic chemokines) are a family of peptides (60–100 amino acid (aa)) that includes some 50 members (Fig. 1). Based on the number and spacing

chemokine; RANTES, regulated upon activation normal T cell expressed and secreted; SCM, single C motif; SDF, stromal cell-derived factor; SLC, secondary lymphoid tissue chemokine; TARC, thymus and activation-related chemokine; TECK, thymus expressed chemokine.

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of the conserved Cystein (C) residue in the N-terminus of the protein, chemokines are subdivided in four families (C, CC, ▶CXC, CX3C), where X is any intervening amino acid between the cysteins. Chemokines receptors transmit intracellular signals that can control either chemoattraction or other functions (Fig. 1). The chemokine receptors (some 20 members) are included in the G-protein coupled receptor (GPCR) superfamily. They are classified based on the class of chemokines that they bind, i.e. receptors that bind C, CC, CXC, CX3C chemokines are called respectively to CR, CCR, CXCR and CX3CR receptors. Based largely on studies performed in the immune system, chemokines have been classified in three functional groups, homeostatic, inducible and dual function (Fig. 1). The first group, which includes chemokines constitutively produced by “resting cells” in specific organs or in tissues inside these organs, controls homeostatic migratory processes that determinate the correct location of different cell types in the organism under normal conditions. Inducible or inflammatory chemokines are secreted in different tissues in emergency situations and serve to attract to these places specialized cell types that contribute to the resolution of the emergency situation. A third group is formed by dual function chemokines, which can be either homeostatic or inducible depending on the context (Fig. 1). Although chemoattraction is the function most commonly regulated by chemokines, however, studies performed mainly on leukocytes have demonstrated that these peptides, acting through specific chemokine receptors, may control additional cellular functions, including proliferation, ▶adhesion, motility, survival or protease secretion, among other functions. By controlling these activities, chemokines may contribute to modulate the functions of leukocytes and other cell types. Chemokines and Cancer Cancer is a disease where cells have disrupted the mechanisms that regulate their normal growth and, consequently, proliferate without control. This affliction becomes life threatening when cancer cells become metastatic, that is, they acquire the ability to leave their original sites of growth (primary tumor) and invade other tissues or organs where the uncontrolled growing cells can form new colonies (▶metastases) that can interfere with vital functions. The process leading to metastasis formation has been divided into several steps. In a first step, the cancer cells detach from the substrate and from the neighboring cells and escape from the primary tumors. A second step involves the penetration of the cancer cells into the blood or lymphatic vessels and their ▶migration through these vessels. In the case of cells that migrate through the

afferent lymphatics, they migrate first to the lymph nodes from where they can exit through the efferent lymphatics, eventually ending up in the blood vessels. In a third stage, cancer cells extravasate from blood vessels and home into new sites in the organism where new metastatic colonies can be formed. During these migratory processes the cells undergo changes in their adhesive properties that are regulated by modulation of the activities and/or levels of integrin receptors. Moreover, cancer cells and/or associated stromal cells secrete proteases which, by degrading extracellular matrix (ECM) proteins of connective tissues facilitate the moving of the cells and the ▶invasion of other tissues. Finally, at the metastatic sites, the cancer cells attach and grow as secondary colonies. In addition, they may secrete chemokines and other soluble factors that induce new vascular vessel formation (▶angiogenesis) and contribute to maintain the growth of the metastatic cells. Although millions of cells may be shed into the blood from primary tumors, however only a reduced percentage of these cells are able form metastases, suggesting that metastatic cells develop mechanisms that increase their survival in the face of a hostile environment. Chemoattraction: A Key Process to Attract Cancer Cells to New Biological Niches Since the work of Stephen Paget in the second half of the nineteenth century, it is known that metastatic cells do not move randomly, displaying in contrast a marked tropism toward specific organs (Table 1). A variety of experimental data indicates that chemokines may play an important role in determining this bias of the metastatic cells. Analysis of the phenotype of multiple metastatic cell types shows that these cells express specific sets of chemokine receptors (Table 1). Furthermore, a clear correlation has been observed between the expression of a specific chemokine receptor by a metastatic cell and the presence of its respective ligands in the metastatic sites, suggesting the involvement of these receptors in the homing processes (Table 1). Finally, a direct role for chemokines and their receptors in the control of the tropism of metastatic cells is corroborated in studies that show that interference with the binding to the chemokine receptors impairs the ability to metastasis to specific organs. For instance, antibody neutralization of ▶CXCR4 in breast cancer cells reduced the ability of these cells to form metastases in the lung, both upon intravenous injection and after ▶orthotopic implantation of the cells. Conversely, over-expression of CCR7 in B16 ▶melanoma resulted in a dramatic enhancement in the ability of these cells to form metastases in the draining lymph nodes upon intravenous injection of the cells in mice. From these studies it has also emerged that CCR7 and

Chemoattraction Chemoattraction. Table 1

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Chemokine receptors involved in cancer metastases

Chemokine/s receptor/s/ligand/s

Site/s of metastases

CXCR3/CXCL9, -10, -11 CXCR4/CXCL12

Lung, bone, lymph node Lung, bone, lymph node

Cancer cell types

Acute lymphoblastic leukemia, Chronic myelogenous leukemia, colon, melanoma Breast, ovarian, prostate, glioma, pancreas, melanoma, esophageal, lung (small cell lung cancer), head and neck, bladder, colorectal, renal, stomach, astrocytoma, cervical cancer, squamous cell cancer, osteosarcoma, multiple myeloma, intraocular lymphoma, follicular center lymphoma, rhabdomyosarcoma, neuroblastoma, B-lineage acute lymphocytic leukemia, B-chronic lymphocytic leukemia, non-Hodgkin lymphoma, acute myeloid leukemia, thyroid cancer, acute lymphoblastic leukemia, chronic myelogenous leukemia CXCR5/CXCL13 Lymph node Head and neck, chronic myelogenous leukemia CXCR7/CXCL11, -12 Lymph node Breast, cervical carcinoma, glioma, lymphoma, lung carcinoma CCR4/CCL17, -22 Skin Cutaneous T-cell lymphoma CCR7/CCL19, -21 Lymph node Breast, Melanoma, lung (non-small cell lung cancer), head and neck, colorectal, stomach, chronic lymphocytic leukemia CCR9/CCL25 Small intes- Melanoma, prostate tine CCR10/CCL27 Skin Melanoma, cutaneous T-cell lymphoma

CXCR4 are the chemokine receptors most commonly expressed by metastatic cells. This finding contributes to explain the ability of multiple metastatic cell types that express these receptors to colonize the lymph node and other organs where CXCL12 (ligand for CXCR4 and CXCR7) and CCL19 and CCL21 (both ligands of CCR7) are expressed (Table 1). Premetastatic niche is the name given to the specific regions, whose formation is induced by soluble factors released by primary tumor cells, which eventually become colonized by distant metastatic cells from the primary tumors. It has been shown that chemokines expression may confer premetastatic niches the ability to attract metastatic cells from the distant primary tumor. In this regard, it has been shown that chemokines S100A8 and S100A9, expressed by myeloid and endothelial in premetastatic niches in the lung, are responsible of attracting incoming Lewis Lung carcinoma metastatic cells to these niches because neutralization of the chemokines with antibodies reduced the metastases in these areas. In sum, chemokine/chemokine receptor pairs are important factors that control the colonization of cancer cells to specific sites in the organism.

Function/s regulated by chemokine receptor Chemoattraction Chemoattraction, angiogenesis, survival, growth

Chemoattraction Adhesion, survival, growth Chemoattraction Chemoattraction

Chemoattraction Chemoattraction, growth, survival

Other Biological Effects of Chemokines on Cancer Cells Apart from Chemoattraction Chemokines may affect cancer not only by regulating chemoattraction, but also by regulating other functions that control cancer progression. Chemokines can Contribute to Regulate the Growth of Cancer Cells Uncontrolled growth is a hallmark of cancer cells. Considering that chemokines may control cell growth in different cell types, the effect of chemokines on the proliferation of cancer cells is not unexpected. The growth of tumor cells may be affected by chemokines that can be either released in an ▶autocrine signaling fashion by the cancer cells or secreted by the stromal tissues associated to the cancer cells. As an example of the first case, it is known that CXCL1, -2, -3, and -8, secreted as autocrine growth factors by melanoma, pancreatic and liver cancer cells, regulate the proliferation of all these cell types. As an example of the second case, it has been reported that CXCL12, which is secreted in lung and lymph nodes, leads to the increase in the growth of ▶glioma, ovarian, small cell lung, basal cell carcinoma and renal cancer, all cancer cells

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types that colonize the aforementioned organs. The effects of chemokines on growth can be complex because, for instance interference with CCR5 seems to increase the proliferation of ▶xenografts of human breast cancer, suggesting that CCR5 inhibits the growth of this cancer cells. Chemokines can Contribute to Regulate the Survival of Cancer Cells A reduced susceptibility to ▶apoptosis, leading to a concomitant extended survival, is also an important factor to explain the uncontrolled growth and the ability of cancer cells to form metastases. Chemokines have been involved in regulating survival in leukocytes and other cells, therefore these ligands may potentially contribute to regulate the carcinogenic phenotype by modulating this function. Stimulation of melanoma B16 cells expressing CCR10 with its ligand CCL27 enhances the resistance of these cells to the apoptosis induced by stimulation of the death receptor CD95. These in vitro results are consistent with in vivo experiments that show that the neutralization of CCL27 ligand with antibodies results in the blocking of tumor cell formation. Also, stimulation of glioma cells with CXCL12 protects these cells from the apoptosis induced by serum deprivation. Recently, it has been shown that, CXCR7, a novel second receptor for CXCL12, is expressed in a variety of cancer cells. It has been indicated that CXCR7 may regulate survival, growth and adhesion. Thus, it is possible that CXCR7 may also contribute to control all these functions in cancer cells. Chemokines can Contribute to Regulate the Adhesion to New Sites in Cancer Cells Migratory cancer cells experience changes in adhesion, including processes of attachment and detachment, as they move through the organism. Enhanced adhesion is particularly crucial at the final stages of cancer progression where these cells require attaching to the new metastatic sites. Stimulation of cancer cells with chemokines may change the adhesion of these cells either by increasing the activity of ▶integrins or by inducing changes in the expression levels on the membrane of these receptors. As an example of the first case, it has been observed that stimulation of B16 melanoma cells with CXCL12 leads to an increase in the affinity of the β1 integrin by the ligand VCAM-1 both in in vitro and in in vivo experiments. As an example of the second case, stimulation of prostate tumor cells with CXCL12 induces enhanced expression of the integrins α3 and β5. Chemokines can Contribute to Control Protease Secretion in Cancer Cells Metalloproteins are largely responsible for ECM remodeling and play key roles in solid tumor cell invasion. In

this regard, it has been shown that chemokines enhance in protease secretion in some cancer cell types. For instance, stimulation of myeloma cells with CXCL12 induces metalloproteinase secretion. Chemokines can Contribute to Control Angiogenesis in Cancer Cells At metastatic sites cancer cells induce formation of new vessels (angiogenesis), which allow the nourishment of the metastatic colonies. Angiogenesis is a finely orchestrated process where endothelial cells proliferate, secrete proteases, change their adhesive properties, migrate and, finally, differentiate into new vessels. Chemokines can act as positive or negative regulators of the angiogenesis in the tumor microenvironment. In this regard, the members of the ▶CXC chemokine family play an important role during this process. The CXC family has been divided into two groups. A first group that includes members that present the triplet Glutamic-Leucine-Arginine (ELR) before the first Cys (ELR+ CXC chemokines), and a second group that include the members that lack this three amino acids (ELR− CXC chemokines). Although there are exceptions, by and large, ELR+ CXC chemokines (including CXCL1, -2, -3, -5, -6, -7 and -8) play proangiogenic roles, promoting vessel formation through the stimulation of the CXCR2 receptor. For instance, in human ovarian carcinoma CXCL8 induces both angiogenesis and tumorigenesis. Furthermore, treatment of mice that bear CXCL8-producing non-small cell ▶lung cancer cells with anti-CXCL8 antibodies blunted the growth of these tumors in the mice. Exceptions to the rule ELR+ CXC=angiogenic chemokines are the ELR+ CXC members CXCL1 and 2, which are angiostatic i.e. they inhibit angiogenesis. ELR− CXC chemokines, including CXCL9, -10, -11, are generally angiostatic. For instance, CXCL9 and CXCL10 inhibit Burkitt’s lymphoma tumor formation probably by blocking blood vessel formation. An exception to the rule ELR− CXC = angiostatic chemokine is CXCL12 that is angiogenic, as suggested by CXCL12 and CXCR4 KO mice that display cardiovascular development defects. It is believed that the angiogenic effects of CXCL12 are mediated by the vascular endothelial growth factor (VEGF) that is secreted by endothelial cells upon stimulation with CXCL12. The latter chemokine can be secreted in the tumor microenvironment by both the cancer cells and associated stromal cells. Finally, apart from CXC chemokines, other chemokines families may also regulate angiogenesis. In this regard, the CC chemokine CCL21 is angiostatic. In contrast, three CC family members (CCL1, -2, -11) and one CX3C family member (CX3CL1) can induce angiogenesis. All these chemokines, secreted inside the tumor, may potentially regulate the growth of the metastatic cells.

Chemokine

Therapeutical Aspects The multiple points at which chemokines may regulate cancer progression make them attractive targets to develop anti-cancer drugs. Several strategies have been adopted to harness the power of chemokines against cancer, including the use of antibodies against the overexpressed chemokine receptors in the target cancer cells to induce apoptosis of these cells. One common strategy has been the development of inhibitors to block the binding of the chemokines to the receptors and consequently the function of these receptors. The fact that chemokine receptors are on the membrane and that much information is available on the sequences, both on the ligands and on the receptors, necessary for receptor-ligand binding have enabled the development of numerous peptide or small molecule inhibitors that interfere with chemokine function. Some of these inhibitors have been developed against CCR1, CCR5, CXCR7 and CXCR4. Most of these inhibitors relay on their ability to inhibit survival or angiogenesis in the target cells. As CXCR4 is one of the most broadly expressed chemokine receptor in cancer cells, at least six peptides or small molecule inhibitors of the function of CXCR4 have been developed and used in preclinical cancer models. CXCR4 is particularly interesting due to its pro-angiogenic functions. A variety of data indicate that the growth and persistence of tumors and their metastases depend on an active angiogenesis at the tumor sites. In this regard, interference with this process is a powerful strategy to inhibit tumor growth. Interference with CXCR4 has been used in several cancer models, including many of the cancers indicated in Table 1. Although peptide inhibitors of chemokine receptors may not have by itself ▶tumoricidal affects, however, along with other strategies may be a powerful therapy against tumors. Summary and Final Conclusions Upon becoming carcinogenic and metastatic, a variety of cancer cells up-regulate the expression of chemokine receptors. In this regard, the microenvironment conditions inside the tumors are also known to induce chemokine receptor expression in some cases. For instance, the low oxygen concentration (▶hypoxia) inside a tumor induces CXCR4 expression which concomitantly leads to a more aggressive metastatic phenotype in cancer cells. Chemokine receptors endow cancer cells with “postal codes” that determine their migration to tissues where the ligands of these receptors are expressed and therefore are important for the metastatic ability of these cells. In addition, these receptors may confer or modulate cancer cells functions that, by regulating different steps in cancer progression, may contribute to the carcinogenic and metastatic phenotype of these cells. The case of the Kaposi’s sarcoma herpesvirus (KSHV), which induces cancer lesions similar to that of Kaposi sarcoma, is a dramatic

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example that shows the important role that chemokines and their receptors may play in cancer. Interestingly, this virus encodes a constitutively active receptor that displays a high degree of sequence similarity to chemokine receptors CXCR1 and CXCR2 and which can even be further activated by the CXCR2 ligands CXCL1 and/or CXCL8. KSHV is also pro-angiogenic and induces survival effects in the cancer cells where is expressed. Further supporting a causative role of CXCR2 in cancer, a constitutive form of CXCR2 can induce cell transformation in susceptible cell types.

References 1. Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4:540–550 2. Zlotnik A (2006) Chemokines and cancer. Int J Cancer 119:2026–2029 3. Sánchez-Sánchez N, Riol-Blanco L, Rodríguez-Fernández JL (2006) The multiples personalities of the chemokine receptor CCR7 in dendritic cells. J Immunol 176:5153–5159 4. Kakinuma T, Hwang ST (2006) Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol 79:639–651 5. Ben-Baruch A (2006) The multifaceted roles of chemokines in malignancy. Cancer Metastasis Rev 25:357–371

Chemoembolization Definition Chemoembolization is a procedure in which the blood supply to a tumor is interrupted through mechanical or surgical interventions (embolization) and cytotoxic drugs are administered directly into the tumor. This technique is used in hepatocellular carcinoma and neuroendocrine carcinomas, among other cancers. ▶Neuroendocrine Carcinoma

Chemokine L EI FANG , S AM T. H WANG Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Synonyms Chemotactic cytokine; Chemoattractant cytokine

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Definition Chemokines are a large group of small proteins that play multiple biological roles, including stimulating directional migration (▶chemotaxis) of leukocytes and tumor cells via their membrane-bound receptors.

Characteristics Chemokines are divided into four subgroups (C, CC, CXC, and CX3C) based on the spacing of the key cysteine residues near the N terminus of these proteins. The CC and CXC families represent the majority of known chemokines. Chemokines signal through seventransmembrane-domain receptors, which are coupled to heterotrimeric Gi-proteins. Activation of phospholipase C (PLC) and ▶phosphatidylinositol-3-kinase γ (PI3Kγ) by βγ subunits of ▶G-proteins is well established. So far, approximately 50 chemokines and 18 chemokine receptors have been identified. Some chemokine receptors bind to multiple chemokines and vice versa, suggesting possible redundancies in chemokine functions. Chemokine receptors permit diverse cells to sense small changes in the gradient of soluble and extracellular matrix-bound chemokines, thus facilitating the directional migration of these cells toward higher relative concentrations of chemokines. While soluble chemoattractants can induce directional migration, chemokines (due to their net positive charges) will often be bound to and presented by negatively charged macromolecules such as endothelial cell-derived proteoglycans in vivo. Chemokine gradients bound to solid surfaces are capable of mediating ▶haptotaxis of leukocytes and other cells. Chemokine receptor activation can also trigger conformational changes in membrane ▶integrins, permitting strong cell–cell adhesion in the presence of appropriate integrin receptors. This signaling pathway is particularly

relevant in triggering cellular integrins found on leukocytes and cancer cells to bind to their respective receptors (e.g. ICAM-1) on vascular endothelial cells, facilitating stable binding and spreading of cells to endothelium. The stable binding of metastatic tumor cells to vascular endothelial cells at distant sites of metastasis is likely to be a crucial early step in the process of ▶metastasis. Circumstantial evidence supports the idea that tumor cells use chemokines to promote their own survival and metastasis through multiple mechanisms. For example, certain chemokines secreted by tumor cells contribute to tumor growth and ▶angiogenesis. Members of chemokines that contain an ELR motif (Glu–Leu–Arg) act as angiogenic factors, which are chemotatic for endothelial cells in vitro and can stimulate in vivo. In contrast, members without an ELR motif inhibit angiogenesis. Chemokine-mediated tumor cell activation through cellular kinases such as PI3K, ▶Akt kinase, and other downstream mediators (Fig. 1) influences tumor cell resistance to apoptotic death. For example, activation of the chemokine receptor CCR10 prevents ▶Fasmediated tumor cell death induced by cytolytic antigenspecific T cells. Selected chemokine receptors are upregulated in a large numbers of common human cancers, including breast, lung, prostate, colon, and melanoma. Chemokine receptors expressed on tumor cells coupled with chemokines preferentially expressed in a variety of organs are believed to play critical roles in cancer metastasis to vital organs as well as draining lymph nodes. CXCR4 is by far the most common chemokine receptor expressed on most cancers. In addition, CXCL12, the ligand for CXCR4, is highly expressed in lung, liver, bone marrow, and lymph nodes, which represent the common sites of metastasis of many

Chemokine. Figure 1 ▶Chemokine receptor signaling. Upon stimulation by chemokine, βγ subunits of G-protein are dissociated from Gαi subunit. βγ subunits activate phospholipase C (PLC) and phosphatidylinositol 3 kinase γ (PI3Kγ), Whereas Gαi subunit directly activates ▶Src-like kinase.

Chemokine Receptor CXCR4

cancers. Chemokine receptor expression on cancer cells may influence the conversion of small, clinically insignificant foci of cancer cells at metastatic sites to rapidly growing, clinically serious secondary tumors. Cancers that upregulate CCR7 expression also facilitate their entry into lymphatic vessels, which strongly express the CCR7 ligand (CCL21), and subsequent retention within CCL21-rich secondary lymphoid organs. Upregulation of chemokine receptors such as CCR7 may be a major reason for efficient lymph node metastasis observed in many epithelial cancers. Chemokines released by tumor cells have been shown to attract ▶regulatory T cells, thus suppressing host responses to invasive tumors. Moreover, chemokine and their receptors are involved in ▶dendritic cell maturation, B and T cell development, and ▶Th1 and ▶Th2 polarization of the T cell response. These actions suggest the possibility that chemokines may play a role in altering the magnitude and polarity of host immune responses to cancer cells. Although individual chemokine and chemokine receptor appear to affect many aspects of cancer cell survival, migration, angiogenesis, and the host response to cancer cells, it is still unclear which of these functions predominate in the multistep establishment of primary tumors and secondary metastases.

References 1. Thelen M (2001) Dancing to the tune of chemokines. Nature Immunol 2:129–134 2. Murphy PM (2002) International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacol Rev 54:227–229 3. Rossi D, Zlotnik A (2000) The biology of chemokines and their receptors. Annu Rev Immunol 18:217–242 4. Kakinuma T, Hwang ST (2006) Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol 79: 639–651 5. Müller A, Homey B, Soto H et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410:50–56

Chemokine Receptor CXCR4 J ONATHAN B LAY Department of Pharmacology, Dalhousie University, Halifax, NS, Canada

Synonyms Receptor for CXCL12; Receptor for stromal cellderived factor-1 alpha (SDF-1α); CD184; Fusin

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Definition CXCR4 is a cell-surface protein that acts as a receptor for the molecule CXCL12 (stromal cell-derived factor-1 alpha, SDF-1α). CXCL12 is one of a class of signaling molecules called chemokines that regulate the movement and other activities of cells throughout the body. Although CXCL12 and CXCR4 play major roles in regulating stem cells and cells of the immune system, CXCR4 is also found on many cancer cells and plays a part in metastasis, spread of the cancer cells being influenced by tissue levels of CXCL12.

Characteristics Chemokines are a class of peptide mediators that play important roles in controlling cellular homing and migration both in embryonic development and in the regulation of cell populations in the adult. There are at least forty different chemokines that fall into four classes depending upon their peptide structure. The different classes are “C”, “CC”, “CXC” and “CX3C” chemokines, for which characteristic sequence motifs involve residues of the amino acid cysteine (C) either in sequence or separated by one or three other amino acids (X or X3). The chemokines themselves are peptides that can exist freely in solution in biological fluids and act by binding to corresponding ▶receptors. In the language of molecular interactions a chemokine is therefore known as a ▶ligand. Chemokines are denoted by the letter L within their name. CXCL12 is thus a ligand, and a chemokine of the CXC class of chemokine mediators. The chemokine receptors are named according to the chemokine class of their binding partner (or ligand), with the letter “R” to designate their receptor status. CXCR4 is therefore a receptor. As for chemokines, the numbers serve to distinguish individual members of the overall family. The partnership between chemokine receptors and the chemokines is not monogamous, and some chemokine receptors may bind as many as ten different chemokines. However, most receptors have between one and three distinct partners. With very few exceptions, these partnerships are within a particular chemokine class (e.g., CXCL chemokines bind selectively to certain CXCR receptors). At this point, the only chemokine factor known to bind to CXCR4 is CXCL12, although CXCL12 itself is able to bind to an alternate receptor (CXCR7, previously known as RDC-1) as well as to CXCR4. Chemokine receptors such as CXCR4 are seventransmembrane, ▶G-protein-coupled receptors. The protein chain of CXCR4 therefore winds back and forth across the outer membrane of the cell so that it crosses the membrane a total of seven times. One end of the protein chain (the amino terminus) protrudes from the outside of the cell. This region of the protein,

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together with certain parts of the three extracellular loops, forms the binding domain for CXCL12. The part of the receptor that protrudes from the inner face of the membrane (composed of the carboxy terminus and three intracellular loops) contains the characteristics that allow it to provoke a cascade of events within the cell (Fig. 1). These steps are initiated firstly by a linkage to one or more of a small family of proteins that interact directly with the receptor, called ▶G proteins (in this case primarily Gαi and Gαq). G protein involvement leads to the activation of three major signaling pathways: (i) the phospholipase C-diacylglycerol/IP3 pathway, (ii) the ras-raf-MAP kinase pathway, and (iii) the PI 3-kinase pathway. CXCR4 is a crucially important member of the chemokine receptor family. If CXCR4 or CXCL12 is absent during embryonic development, the organism is unable to survive. The key dependence on CXCL12 and CXCR4 reflects the importance of this signal/receptor pair in marshalling the correct formation of cells as tissues are formed from their more rudimentary cellular precursors in the embryo. The CXCL12:CXCR4 axis, as it is often called, is a central part of the normal development of the central nervous system (the brain itself) and the exquisitely organized tissue that replenishes the different cells of the blood through adult life (the ▶hematopoietic system). In addition, CXCR4 and CXCL12 seem to play a particular role in

the development of the gut, and their participation is important for the proper development of the blood vessel system that is required for efficient intestinal function in the adult. In adult organisms, CXCR4 and CXCL12 partly reprise their developmental role during tissue damage by participating in repair processes. Once the organism is fully formed, the most evident role for CXCR4 and CXCL12 in a normal individual is that of continued regulation of the hematopoietic system. This takes place mainly in the bone marrow, which acts as a reservoir for the ancestral cells (stem cells and other progenitor cells) that are needed for the continued production of various white cells (leukocytes) and other progeny that are required to ensure a proper defense against infection or injury, or to deal with replacement and remodeling of damaged tissues. These stem cells – which need to be maintained safely by the body until required to respond – are located within the protected environment of the bone marrow and are supported and nourished by a specialized grouping of cells that together are referred to as the “▶microenvironmental niche.” These supporting cells or “▶stromal cells” secrete a number of factors that serve to nourish the stem cells and to keep them within a safe environment in their primitive and “resting” state. Notable amongst these factors is CXCL12 (the “stromal cell-derived factor”), which can bind to CXCR4 on the stem cells. The binding of CXCL12

Chemokine Receptor CXCR4. Figure 1 The cellular signaling pathways of CXCR4. When the chemokine ligand CXCL12 binds to its receptor CXCR4, one or more of several pathways can be activated through initial links involving G proteins that associate with the receptor. These pathways, which are shown only in outline, involve a further network of interactions that eventually lead to a cellular response that may ensure cell growth, migration or survival.


to its receptor has several effects on cell behavior, but the principal outcome is to attract cells toward the source of CXCL12. In the case of stem cells in the bone marrow, this results in retention within the microenvironmental niche, or directs migrant stem cells back to this location. This ability of the CXCL12:CXCR4 axis to direct cell movement is what underlies its key role in orchestrating tissue development and repair. The phenomenon can be demonstrated in experiments using isolated cells, such that cells that have the CXCR4 receptor can be induced to migrate through pores in an artificial filter in response to an upward concentration gradient of CXCL12 in the fluid. This is a cellular response known as ▶chemotaxis, and CXCL12 is referred to as a ▶chemoattractant. Unfortunately, this normal and very important process by which CXCL12 and CXCR4 assist directed cell movement has been subverted by cancer cells to assist the spread of a cancer, or metastasis. Normal tissues that are not subject to inflammation or repair processes typically have very low levels of CXCR4. However, when cancers are formed the affected cells frequently experience a dramatic increase (“upregulation”) of CXCR4. This has been shown for the common adult cancers (carcinomas of the breast, colon, lung, prostate, cervix etc), which arise in the membranous linings (epithelia) of certain organs; but CXCR4 levels are also elevated in cancers arising in bone (e.g., osteosarcoma), muscle (e.g., rhabdomyosarcoma), nervous tissue (e.g., glioblastoma) or white cells (various leukemias). This is such a consistent finding that in many cancers the level, or “expression,” of CXCR4 can be used as cancer ▶biomarker. The levels of CXCR4 that are present on the cells give an indication of how the cancer is likely to behave in the future, and what therapeutic steps might need to be considered. Levels are assessed using a technique called immunohistochemistry. In this approach very thin slices or “sections” – no more than 0.005 mm thick – are taken from the suspect tissue onto glass slides. Special protein reagents called ▶antibodies are used that recognize any molecules of CXCR4 in the tissue, and additional steps in the process generate color wherever the antibody has bound. The resulting picture under a microscope tells the pathologist not only about the architecture of the tissue and the characteristics of the cells, but whether or not they have high levels of CXCR4. High levels (expression) of CXCR4 are associated with cancer aggressiveness, a likelihood that the cancer will spread or metastasize, and means that the outlook for the patient is likely to be poorer. The link between cancer aggressiveness/metastasis exists because the CXCL12:CXCR4 axis has a similar role of “directing traffic” in cancer as it does in normal circumstances. In this situation it is the cancer cells that possess the receptor – CXCR4 – and have levels at

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the cell surface that are much greater than are found on their normal counterparts. The exact reasons for these elevated levels of the chemokine receptor are not fully understood. Undoubtably the genetic changes that are characteristic of cancer cells lead to alterations in ▶transcription of the CXCR4 gene that may provide certain subpopulations with greater amounts of the CXCR4 protein, and these cells have a selective advantage. However, there are also indications that factors within the environment of the tumor can make the situation worse by stimulating the cell to make even more CXCR4. The hypoxic nature of tumor tissue causes an increase in CXCR4 gene transcription through a pathway involving ▶hypoxia-inducible factor-1 alpha (HIF-1α). Various small-molecularweight and polypeptide mediators have also been shown to enhance the cellular expression of this chemokine receptor. The cancer cells are therefore equipped to be attracted toward sources of CXCL12 and to be captured within environments that are high in concentrations of CXCL12. Thus, it is no coincidence that the tissues that are high in CXCL12 are also those in which cancers form secondary tumors or metastases. Such tissues include the lymph nodes – central filters in the system that drains fluid from all tissues – as well as the liver, lung and bone marrow. CXCL12 is believed to be one of the major factors driving metastasis (Fig. 2). As a colorectal cancer develops in the large intestine, for example, and small groups of tumor cells are shed into the blood circulation and the lymphatic drainage, circulating cells will find an attractive home as they encounter lymph nodes in the mesenteric fat around the intestinal wall, when they are delivered to the liver through the portal circulation, or as they lodge in the capillary beds of the lung after traversing the systemic circulation. Conversely, they have a much reduced probability of taking up residence in sites such as the heart or skeletal (voluntary) muscle, which are low in CXCL12. In addition to being attracted and retained in tissues that have high concentrations of CXCL12, the CXCR4-bearing cancer cells may respond in other ways. Although this may not be the case for all cancers, in some types (carcinomas of the colon and prostate, for example) there is evidence that once the cells have settled in to their new location, the presence of CXCL12 acting through CXCR4 also enhances their ability to grow and colonize the tissue. In this way, CXCL12 can also be regarded as a growth factor, alongside other polypeptide growth stimulators that participate in tumor expansion. One additional factor that makes CXCR4 of interest for many different clinicians and researchers is that it is one of the two major coreceptors by which the AIDS virus infects human cells. One of the proteins that is

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Chemokine Receptor CXCR4. Figure 2 How CXCR4 and CXCL12 work together to facilitate metastasis. Tumor cells have increased levels of the receptor at their cell surface. When the tumor grows sufficiently for the cancer cells to find their way into the bloodstream, some cells lodge in tissues (e.g., lungs, liver and bone marrow) that have high concentrations of CXCL12, the molecule for which CXCR4 is the receptor. CXCL12 both encourages the entry of cells into the tissue and promotes growth of the cell population, facilitating metastatic spread. Tissues that have low levels of CXCL12 are much less likely to accept metastases.

present within the outer surface of the HIV-1 virus, called gp120, binds to CXCR4, although at a slightly different site to CXCL12. When the virus binds to its major target (the CD4 protein) on susceptible cells, it requires a coreceptor in order to complete its cellular attack. This allows it to complete the molecular changes that allow it to infect the cell. Depending on the exact cell and viral type, the coreceptor may be CXCR4 or another chemokine receptor, CCR5. While the link with AIDS has limited direct relevance to most cancers, the two fields of research have synergized to extend our present understanding of CXCR4.

well as other cells, and the more widely expressed CXCR4. The tropism of specific chemokine receptors is associated with HIV clinical effects, with CCR5 linked to infection and CXCR4 tropism linked to progression to AIDS. ▶TAT Protein of HIV

Chemokinesis Chemokines

▶Motility

Definition

The name comes from “Chemotactic cytokines,” these small cytokines induce migration of diverse immune cells. The family of the chemokines is quite numerous, as are the chemokine receptors, and often there is “promiscuity,” in that a single chemokine can active multiple receptors and multiple chemokines can activate a single receptor. These molecules direct trafficking of leucocytes. Two chemokine receptors are also the principal co-receptors for HIV involved in viral entry: CCR5, expressed on monocytes and macrophages as

Chemoprevention Definition Chemoprevention involves the use, in healthy people, of natural or laboratory made substances to prevent cancer or reduce cancer risk both in high-risk individuals as well as in the general population. The

Chemoprotectants

aim is to reduce the cancer burden in humans. Most work is being done to reduce the risk for ▶oral cancer, ▶prostate cancer, ▶cervical cancer, ▶lung cancer, ▶colorectal cancer, and ▶breast cancer. The first chemopreventive agent to reach the clinic – and possibly the best known – was ▶tamoxifen, which has been shown to cut breast cancer incidence in highrisk women by 50%. It was followed by ▶finasteride, found to reduce ▶prostate cancer incidence by 25% in men at high risk for the disease. However, the largescale trials that confirmed these benefits brought to light a troublesome issue: the drugs caused serious side effects in some patients. This is an issue of particular concern when considering long-term administration of a drug to healthy people who may or may not develop cancer. Obviously, this is raising a number of ethical issues. An effective chemopreventive agent should not significantly alter quality of life, and should be ideally inexpensive, safe, well tolerated, and effective in preventing more than one cancer. Experience with ▶celecoxib (Celebrex) and other ▶COX-2 inhibitors illustrates the importance of an assessment of the risk/benefit ratio for patients. COX-2 inhibitors have shown impressive efficacy in the prevention of colon cancer and several other forms of cancer, but they also increase the risk of serious cardiovascular side effects. Attention has focused on ▶nutraceuticals and ▶phytochemicals as chemopreventive agents. ▶Curcumin (found in the curry spice turmeric), has shown dramatic anticancer results in preclinical studies owing to its significant anti-▶inflammation properties. Curcumin has been used for thousands of years in the diets of people in the Middle and Far East and therefore is believed to have a low probability of serious side effects. Under investigation for their potential in breast cancer chemoprevention are ▶aromatase inhibitors, a class of ▶estrogen blockers, which are approved to treat metastatic breast cancer in post-menopausal women. While the idea of cancer chemoprevention is extremely attractive, much research remains to be done to make this a generally applicable option for reducing the human cancer burden. An important element will be to identify informative ▶biomarkers to assess individual cancer risk and to possibly provide information of patients tolerance towards individual chemopreventive agents. ▶Celecoxib ▶Chemoprotectants ▶COX-2 ▶Cyclooxygenase 2 ▶Detoxification ▶Photochemoprevention ▶Phytochemicals and Cancer Prevention

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Chemoprotectants D EBASIS B AGCHI Department of Pharmacy Sciences, Creighton University Medical Center, Omaha, NE, USA

Synonyms Chemoprotection; Chemoprevention

Definition

▶Chemoprotectants are natural or synthetic chemical compounds which exhibit the ability to ameliorate, mimic, or inhibit the toxic or adverse effects of structurally different chemotherapeutic agents, ▶radiation therapy, cytotoxic drugs, or naturally occurring toxins, without compromising the anticancer or antitumor potential of the chemotherapeutic drugs. Chemoprotectants shouldn’t affect the ▶therapeutic efficacy of the chemotherapeutic agents, radiation or drugs, disrupt the serum enzyme levels, or induce significant injury to the tissues/organs. These chemoprotectants include anticancer, antitumor, anti▶angiogenic, and antioxidant compounds and used as an adjuvant in cancer ▶chemotherapy.

Characteristics According to the World Health Organization (WHO), cancer accounts for 7.6 million (or 13%) of all deaths in 2005, and the incidence of cancer is expected to rise with an estimated 9 and 11.4 million deaths from cancer in 2015 and 2030, respectively. Cancer chemotherapy and radiation therapy are the most promising choice available for the cancer patients. The global outlook of cancer therapy has made dramatic improvement since the discovery of various ▶synthetic and ▶natural chemoprotectants which slows down the progress of this deadly disease and enhances the life span of the cancer patients. Chemoprotectants may exert toxic effects. Thus, it is very important to determine the right dosage and exposure scenario for each chemoprotectant prior to the exposure to demonstrate adequate safety. Synthetic Chemoprotectants Amifostine. A white powder, water-soluble organic thiophosphate compound, chemically known as 2[(3-aminopropyl)amino]-ethanethiol dihydrogen phosphate (ester) or 2-(3-aminopropylamino)ethylsulfanyl phosphonic acid or aminopropylaminoethyl thiophosphate (Fig. 1a), and used as a ▶cytoprotective adjuvant in cancer chemotherapy to reduce the incidence of ▶neutropenia-related fever and infection caused by

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Chemoprotectants. Figure 1 Structures and IUPAC nomenclature of (a) Amifostine, (b) Dexazoxane, (c) Glutathione, (d) Mesna, and (e) N-acetylcysteine.

DNA-binding chemotherapeutic agents including cyclophosphamide and cisplatin. ▶Amifostine (empirical formula C5H15N2O3PS; molecular weight 214.22; trade name Ethyol, synonyms: Ethiofos, Ethanethiol, Gammaphos, WR2721, NSC-296961) is used to decrease the cumulative nephrotoxicity caused by cisplatin in patients with ovarian or lung cancer, as well as to reduce the incidence of moderate to severe xerostomia (dry mouth) in patients undergoing radiotherapy for head and neck cancer. Amifostine is dephosphorylated by alkaline phosphatase in tissues to a pharmacologically active free thiol metabolite, which readily scavenge noxious reactive oxygen species (ROS) generated by exposure to either cisplatin or radiation, as well as detoxify reactive metabolites of platinum and other alkylating agents. Pharmacokinetic studies show that amifostine is rapidly cleared from the plasma with a distribution half-life of 3 g/day) are required for an adrenolytic effect. Although responses to mitotane alone may occur in 20–30% of cases, most responses are transient, and the prospect for long-term survival is uncertain. The antitumor effect of mitotane is influenced by its pharmacokinetics and by the duration of its therapeutic exposure. Serum concentration plateaus after 8–12 weeks of treatment, and antitumor responses occur only when a serum concentration of at least 14 μg/mL is maintained for a prolonged period. The severe gastrointestinal (nausea, vomiting, diarrhea, and

abdominal pain) and neurologic (somnolence, lethargy, ataxia, depression, and vertigo) toxic effects of mitotane reduce patient adherence. Because mitotane is adrenolytic, all patients receiving this agent should be considered to have severe adrenal insufficiency and treated accordingly. ▶Cisplatin-based regimens, usually including etoposide and doxorubicin, are used in combination with mitotane, although less than 40% of patients respond. The use of radiotherapy in pediatric ACT has not been consistently investigated, although ACT is generally considered to be radioresistant. Furthermore, because many children with ACT carry germline TP53 mutations that predispose them to cancer, radiation may increase the incidence of secondary tumors. For most patients with metastatic or recurrent disease that is unresponsive to mitotane and chemotherapy, repeated surgical resection is the only alternative. However, given the infiltrative nature of the disease, complete resection is difficult. Image-guided tumor ablation with radiofrequency currently offers a valid alternative for these patients. Prognosis Complete tumor resection is the single most important prognostic indicator. Patients who have distant or local with gross or microscopic residual disease after surgery have a dismal prognosis. Long-term survival (5 years or more after the diagnosis) is about 75% for children after complete tumor resection. Among those who undergo complete tumor resection, tumor size has prognostic value. The estimated event-free survival is 40% for those with tumors weighing more than 200 g and 80% for those with smaller tumors. Children whose tumors produce excess glucocorticoid appear to have a worse prognosis than children who have pure virilizing manifestations. Classification schemes or disease staging systems (Table 2) are still evolving. Prognosis will likely be further refined by adding other predictive factors, including those from gene expression studies. Concluding Remarks Adrenocortical tumors remain difficult to treat, and little progress has been made in developing effective chemotherapeutic regimens. The rarity of ACT hinders the opportunity to conduct adequately powered clinical trials, including biological studies. Therefore, efforts must be coordinated and resources must be consolidated to advance our understanding and treatment of ACT. In this regard, a long-standing international ACT registry and tissue bank has been established [http://www.stjude. org/international-outreach/0,2564,455_2265,00.html]. Short-term goals are to establish tissue culture, xenograft transplants, and genetically engineered mouse models to explore novel therapies. Clinical investigators,

Childhood Cancer Childhood Adrenocortical Carcinoma. Table 2

Staging criteria for childhood adrenocortical tumor

Stage I II III IV

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Description Tumor totally excised, tumor size = Smad 5 BMP > ; Smad 8 ) Smad 2 TGFb, Activin Smad 3

Common DPC 4/Smad 4

Inhibitory Smad 6 Smad 7

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able to form a heterodimeric or trimeric complex with the “common-mediator” DPC4/Smad4 which then translocates into the nucleus. Here it can either up- or down-regulate the transcription levels of target genes by interacting with other nuclear factors and by recruiting transcriptional co-activators or co-repressors. This signaling cascade can be negatively regulated by the I-Smads (Smad6 and Smad7). Whereas Smad7 acts as a more general inhibitor of TGFβ family signaling Smad6 seems to preferentially block BMP signaling. I-Smads can compete with R-Smads for type I receptor binding and can therefore prevent the phosphorylationdependent activation of R-Smads. Furthermore, Smad7 can interact with the E3 ubiquitin ligases Smurf 1 and 2. Once the Smad7/Smurf complex is bound to the TGFβ receptor it induces TGFβ receptor degradation. Direct binding of I-Smads to R-Smads has also been shown, yielding R-Smads inactive. The expression of I-Smads appears to be regulated by TGFβ and BMP via an autoregulatory feed back loop. Furthermore, it has been shown that ▶interferon-γ (interferon-γ) (IFN-γ) via the Jak1/STAT1 pathway, and tumor necrosis factor alpha (TNFα) and interleukin 1 through NFκB (▶nuclear factor κB) /RelA can induce the expression of I-Smads to antagonize TGFβ signaling. Transcriptional Regulation through Smads Since Smad proteins have no intrinsic enzymatic activity, they exert their effector function as transcriptional

Deleted in Pancreatic Carcinoma Locus 4. Figure 2 Functional domains and sites of identified DPC4 mutations. In addition to the mad homology domains MH1 and MH2, DPC4 carries a ▶nuclear localization signal (NLS) domain and a ▶nuclear export signal (NES) domain responsible for constant shuttling of DPC4 between nucleus and cytoplasm, thus helping the cell to constantly sense the TGFβ receptor activation state. A number of candidate phosphorylation target sites (P) for kinase pathways such as the MAPK (▶MAP-Kinase) pathway have been described within the linker region which for example may modify nuclear accumulation rates of DPC4. The numbered squares forming the schematic DPC4 molecule also depict the 11 known exons within the DPC4 transcript.

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Deleted in Pancreatic Carcinoma Locus 4

regulators either directly by binding to specific promoter consensus sequences termed ▶Smad-binding elements (SBE) and/or indirectly by associating with transcription factors already bound to the promoter. Therefore, many but not all Smad responsive promoters have two adjacent DNA sequences. One provides the binding site for transcription factors that are cooperating with the ▶Smad complex, the other allows direct binding of the Smad complex to the DNA. While R-Smads provide the interface for the binding to ▶transcription factors, DPC4 makes the contact to SBE elements. DPC4 thereby stabilizes the formation of a higher order DNA binding complex which is able to recruit transcriptional co-activators or co-repressors. Many of the factors that cooperate with the Smad complex are regulated independently by other signaling cascades. The function of an active Smad complex can therefore be described as a co-modulator of transcription. It can modulate gene expression positively as well as negatively by integrating various incoming signals -including those mediated by the TGFβ ligand family. Therefore, it is not surprising that currently more than 1,000 genes are described to be either directly or indirectly regulated by DPC4. In addition, many of these DPC4 target genes can only be found in a certain cell type and growth state, again illustrating how much the cellular differentiation and signaling state determines the net gene expression regulation pattern of a DPC4 containing transcription complex. What makes DPC4/Smad4 Unique among the Other Smad Family Members? . DPC4 is the only human Co-Smad that is currently known. . It seems particular to DPC4 that it is, almost without exception, essential for the establishment of a functional active Smad complex, a fact that emphasizes its role as a “master switch” in the regulation of TGF-β-like signals. . Most somatic and all germ line mutations in human Smad genes identified to date target DPC4. Only very few somatic mutations were found in the human Smad2 gene, none were found in the other members of the human Smad gene family. Which Human Tumors Show Alterations of the DPC4 Gene? Changes, resulting in the inactivation of the DPC4 gene were found in approximately 50% of pancreatic carcinomas (▶pancreas cancer). Research carried out in a variety of other cancer types suggested that DPC4 may contribute primarily to the formation of pancreatic neoplasia, and to a lesser extend to ▶colon cancer ▶cervical cancer and biliary cancer (▶bile duct neoplasia) as well as the induction of non-producing ▶neuroendocrine tumors. However, such changes appear to play

only a minor role in the development of other tumor types such as head and neck, ▶lung cancer ▶ovarian cancer ▶breast cancer and ▶bladder cancer. The frequency of DPC4 mutations is markedly increased in metastatic colorectal carcinoma (35%) compared to non-metastatic colorectal carcinomas (7%). Furthermore, during pancreatic carcinoma development, a high incidence of biallelic DPC4-inactivation is generally not present before the carcinoma-in-situ stage, suggesting the loss of DPC4 function is critical for the tumor cell to develop characteristics such as the ability to invade into the surrounding tissue and to form metastasis. In addition, germline mutations of the DPC4 gene have been identified in patients with familial juvenile polyposis, an autosomal dominant disorder that is characterized by a predisposition to hamartomatous polyps as well as an increased risk for gastrointestinal carcinomas. How are Naturally Occurring DPC4 Mutations Interfering with the Smad Signaling Cascade Most DPC4 mutations identified to date are located within the C-terminal MH2 domain. Functional studies identified the MH2 domain as providing the binding properties to R-Smads, the latter being important for a functionally active Smad complex. It is therefore likely that compromising mutations of the MH2 domain structure restrict the formation of a functional Smad complex, thus preventing signal transduction to downstream components. In addition, a few mutations have been identified within the N-terminal MH1 domain which was shown to mediate the direct binding of DPC4 to DNA promoter sequences. Such mutations might interfere with Smad signaling by rendering the formation of the higher order Smad-DNA complex unstable. Furthermore, some DPC4 ▶missense mutations targeting the MH1 domain result in an instable protein due to a mutation-induced poly-ubiquitination of DPC4 and its subsequent proteasomal degradation. Does DPC4 Contribute to the Familial Risk for Pancreatic Cancer? Although the DPC4 gene is most frequently altered in ▶sporadic pancreatic carcinoma, to date no germline mutations were found in families with an increased risk of this type of carcinoma. DPC4 is therefore unlikely to play an important role as a heritable genetic risk factor in pancreatic carcinoma. How does DPC4 Contribute to Tumor Formation? Although Smad signaling (including DPC4/Smad4) is regarded as central to the TGFβ pathway, there are now numerous examples illustrating that DPC4 inactivation is not simply abolishing TGFβ responsiveness and thus providing the cell a growth advantage. This

Dendritic Cell-Based Tumor Vaccines

can partly be explained by the ability of TGFβ to modulate also Smad-independent pathways such as the Ras (▶RAS) -ERK, PI3K (▶PI3KSignaling) –AKT (▶AKTSignal Transduction Pathway in Oncogenesis) and Rac/Rho pathways. Thus, loss of DPC4 function is not able to completely abrogate TGFβ signaling rather than shifting the balance between DPC4/Smad-dependent and DPC4/Smad-independent TGFβ signaling pathways towards the DPC4/Smad-independent pathways. The output of the latter is dependent on the successful activation of the latent form of TGFβ ligands and intactness of the TGFβ receptors. The cellular context will further modulate the signaling state of the DPC4/Smad-independent pathways through regulating the activity status of their pathway target genes by integrating signals from other signaling pathways. Thus, loss of DPC4 function has been shown cell type dependent to be involved in altering a number of different cell behaviors relevant to tumor formation such as, cell growth rate by modulating the cell cycle and/or the rate of ▶apoptosis, altering the extracellular matrix components (▶extracellular matrix remodeling), the cell adhesion (▶adhesion) properties, and supporting ▶epithelial to mesenchymal transition, thereby facilitating tumor ▶invasion and ▶metastasis, Furthermore, other experiments provided evidence that loss of DPC4 function might promote tumor ▶angiogenesis by causing an increase in the concentration of angiogenic factors and/or a decrease its corresponding inhibitors. Additional insight of DPC4 function was provided by targeted mutagenesis in mice. Mice with two mutated alleles for DPC4 die at embryonic day 7.5, a result that underlines the importance of DPC4 in early embryonic development. DPC4 heterozygous mice develop gastric and duodenal polyps which resemble human juvenile polyps. Furthermore, knockout mice experiments have demonstrated a functional cooperation between the DPC4 and the APC (APC) (adenomatous polyposis coli) gene. In mice that were carrying defect copies of both genes, compared to mice carrying only the mutated APC gene, the induced colonic tumors displayed a much more aggressive phenotype. Lastly, data from primary human tumors and from mice experiments provided evidence that haploinsufficiency of the DPC4 locus may also contribute to progression of cancer. These data clearly support the importance of DPC4 in the suppression of tumorigenesis.

References 1. Hahn SA, Schutte M, Hoque AT et al. (1996) DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271:350–353 2. Howe JR, Roth S, Ringold JC et al. (1998) Mutations in the smad/dpc4 gene in juvenile polyposis. Science 280:1086–1088

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3. Miyaki M, Iijima T, Konishi M et al. (1999) Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis. Oncogene 18:3098–3103 4. Takaku K, Oshima M, Miyoshi H et al. (1998) Intestinal tumorigenesis in compound mutant mice of both Dpc4 (Smad4) and Apc genes. Cell 92:645–656 5. Alberici P, Jagmohan-Changur S, De Pater E et al. (2005) Smad4 haploinsufficiency in mouse models for intestinal cancer. Oncogene 25:1841–1851

Deletion Definition Is the loss of a chromosomal segment or gene. A chromosomal deletion can be terminal, i.e., involve the end of a chromosome, or it can be interstitial, in which case a segment from within the chromosome is lost. A decrease in specific DNA fragment copy numbers. ▶ArrayCGH

Dendrimer Definition

A type of ▶nanoparticle that is a highly branched polymeric molecule synthesized from monomers in a reproducible fashion that may have applications for drug delivery and imaging. ▶Nanotechnology

Dendritic Cell-Based Tumor Vaccines Definition The system of dendritic antigen-presenting cells derive from hematopoetic precursors and reside as sentinels of the immune system in all tissues, particularly in skin and mucous membranes. They are able specifically equipped with pathogen recognition receptors which enable them to recognize harmful microbial infections and take up foreign antigens. Activation of ▶dendritic cells triggers (i) their ▶migration to the regional lymph

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nodes, (ii) antigen processing and (iii) functional maturation for optimal antigen presentation and stimulation of T cell proliferation. ▶Dendritic cells are the key regulators of cellular immunity against both infected as well as malignant cells. They are therefore targets of many ▶cancer vaccine strategies both ex vivo (using cultured dendritic cells) as well as in vivo. ▶Melanoma Vaccines

Dendritic cells N ATHALIE C OOLS , V IGGO VAN T ENDELOO, Z WI B ERNEMAN Vaccine and Infections Disease Institute (VIDI) Laboratory of Experimental Hematology, University of Antwerp, Belgium

Definition

Dendritic cells are a special subset of ▶leukocytes that form a complex network of ▶antigen-presenting cells (APC) throughout the body. They play a principal role in the initiation of immune responses to invading micro-organisms (bacteria, fungi and viruses), malignant cells and allografts, by activating naïve lymphocytes, by interaction with innate cells and by the secretion of cytokines. At certain developmental stages they grow branched projections, the dendrites, hence the cell’s name.

Characteristics Origin and Function Dendritic cells (DC) were characterised for the first time by Steinman in 1973 based on their distinct morphology with different cytoplasmic extensions, such as dendrites, pseudopodia and ▶lamellipodia, which give the cell its star-shaped feature. Due to their pronounced morphology, DC have a large surface, ensuring close contact with neighbouring cells. Variations among the tissue distribution of DC and differences in their phenotype and function, indicate the existence of heterogenous populations of DC. DC originate from different hematopoietic lineages in the bone marrow (Table 1). A myeloid progenitor cell can differentiate in vivo to different DC populations: ▶Langerhans cells that migrate to the skin epidermis and interstitial DC that migrate to the skin dermis and various other tissues (airways, liver and intestine). Circulating or migrating DC are found in the blood and in the afferent lymphatics, respectively (the latter called veiled cells). Interdigitating DC are found in

the paracortex of lymph nodes in close proximity with T cells. In addition, monocytes represent an abundant source of DC precursors during physiological stress. Another subset of DC, plasmacytoid DC (pDC) originate from a lymphoid progenitor cell in lymphoid organs. By contrast, follicular DC (FDC) are probably not of hematopoietic origin, despite similar morphology and function to the above mentioned subsets of DC. FDC are APC of the B cell follicles in lymph nodes and central players in humoral immunity. DC express several different types of membrane molecules that determine their phenotypic and functional characteristics: 1. DC display a high surface density of antigenpresenting molecules, such as CD1a, ▶major histocompatibility complex (MHC) class I and class II molecules. The level of expression of these molecules is 10- to 100-fold higher compared to other APC (e.g. B cells). 2. In addition, mature DC have high expression levels of costimulatory and ▶adhesion molecules: CD40, ICAM-1/CD54, ICAM-3/CD50, LFA-3/ CD58, B7-1/CD80 and B7-2/CD86. Binding of these molecules with their respective receptors on T cells results in T cell activation and subsequently stimulates the expression of cytokines, cytokinereceptors and genes for cell survival. 3. Several members of the integrin family are expressed by DC. ▶Cadherins contribute to the generation of cell contacts and selectins are important for the motility of DC. 4. DC also express pathogen-recognition receptors, e.g. DEC-205, a macrophage-mannose receptor capable of binding bacterial carbohydrates and ▶toll-like receptors (TLR), recognising a variety of pathogen-associated molecular patterns (PAMP), such as carbohydrates, nucleic acids, peptidoglycans and lipoteichoic acids. 5. Cytokine and chemokine receptors are also important for DC function, since growth, differentiation and migration of DC as well as antigen processing and presentation is tightly regulated by cytokines and/or chemokines. The widespread distribution of DC and their expression of a variety of membrane molecules underline their sentinel function: they patrol the body to capture invading pathogens and certain malignant cells in order to induce efficient anti-microbial or antitumour ▶T-cell responses. In their in vivo steady state condition, immature DC are specialised in capturing antigens, i.e. they efficiently take up pathogens, apoptotic cells and antigens from the environment by phagocytosis, macropinocytosis or ▶endocytosis. However, immature DC remain tissue-resident, expressing only small amounts of (MHC) class II and of

Dendritic cells Dendritic cells. Table 1

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Different subsets of dendritic cells CD34+ hematopoietic stem cell Myeloid progenitor cell

Phenotype CD11c CD1a CD123 Birbeck granules Factor XIIIa Function Endocytosis IL-10* IL-12* IFN-α*

Lymphoid progenitor cell

Monocyte-derived DC

Langerhans cells

Interstitial DC

Plasmacytoid DC

+ ± – – ±

+ + – + –

+ – – – +

– – + – –

+ + + –

+ + – –

+ + + –

+ + + +

*After application of danger signals.

costimulatory molecules, which leads to T cell unresponsiveness. After encounter of a “danger” signal (e.g. TLR ligand) immature DC mature and migrate to the secondary lymphoid organs. Mature DC are considered to be immunogenic, mainly due to the marked upregulation of MHC class II and costimulatory molecules. This maturation step is believed to be a crucial event to regulate DC function and makes DC potent inducers of T cell immunity.

granulocyte-monocyte colony stimulating factor (GMCSF), tumour necrosis factor (TNF-α), stem cell factor (SCF), interleukin (IL)-3 and ▶Interleukin-6. Second, DC can be generated starting from monocytes using GMCSF and ▶Interleukin-4. Finally, DC can be directly harvested from the peripheral blood of a patient, where they reside at low percentages (0,1%). Next, cultivated DC can be loaded with the tumour antigen of importance in different ways:

Dendritic Cell-Based Immunotherapy Despite our immune system’s function to protect us from malignant cells, tumour cells grow undisturbed and, unless treated, are fatal to the host. The reasons for the failure to eliminate tumour burden in a majority of patients can be the consequence of different tumour escape mechanisms. For example, tumour-derived inhibitory factors (e.g. IL-10 and/or TGF-β) or tumour cell-induced T regulatory cells (▶Treg) might be involved in downregulating or altering immune function. The goal of cancer ▶immunotherapy is to resolve or circumvent these problems and generate tumourspecific immune responses. It is important to realise that immunotherapies will likely only be successful after reducing tumour mass via primary therapies: surgery, radio- and/or chemotherapy i.e. in a ▶minimal residual disease (MRD) setting. Because of their pivotal immune-stimulating capacity and their ability to activate naïve tumour-specific T cells, DC-based ▶cancer vaccines could have important applications in the future treatment of cancer. For this, it was necessary to cultivate DC with high yields. Several cultivation protocols were developed for in vitro generation of DC. First, DC can be differentiated from CD34+ hematopoietic progenitor cells using

1. DC can be grown in vitro in the presence of ▶tumorassociated antigens (TAA). This technique is called peptide pulsing and results in direct binding of the immunodominant epitope on an empty MHC class I molecule on the DC membrane. This circumvents the need for antigen uptake and processing and ensures the stimulation of tumour specific cellmediated cytotoxicity. However, the number of known TAA is still restricted and highly dependent on the human leukocyte antigen (HLA) haplotype of the patient. 2. DC can also be fused with the patient’s tumour cells in vitro or pulsed with tumour cell lysates. The former method combines sustained tumour antigen expression with the antigen-presenting and immunostimulatory capacities of DC. DC-tumour cell hybrids will also stimulate an active anti-tumoural immune response. 3. Tumour antigen can also be loaded on DC using plasmid DNA transfection or ▶viral vector mediated gene transfer. The former method results in only low transfection efficiencies. On the other hand, viral transduction, for example by using adenoviral or lentiviral, vectors is very effective with regard to transfection efficiency. However, the immunogenic character of the viral vector itself is a serious

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disadvantage. In both cases, DC will transcribe and process the tumour antigen. This will result in a cytotoxic immune response, necessary for immunological defence against cancer cells. 4. It is also possible to transfect DC using in vitro transcribed mRNA coding for tumour antigens or total tumour RNA. It has been shown that electroporation of RNA is the most effective nonviral transfection method for DC (▶non-viral vectors for cancer therapy). mRNA is brought directly into the cytoplasm and the cell’s metabolism will translate mRNA into proteins, which can be presented onto MHC class I molecules after processing. This will guarantee a specific cellmediated anti-tumoural immune response. In a clinical context, in vitro cultured and activated DC loaded with appropriate tumour antigens could be administered to cancer patients in a therapeutic setting (active specific immunotherapy). The aimed generation of anti-tumour immunity, mediated by DC, could be of importance for both treatment (as adjuvant to conventional therapies) as well as to prevent relapse in a MRD setting. On the other hand, tumour antigen-loaded DC can also be used for the ex vivo generation of tumourspecific cytotoxic T lymphocytes (CTL) in an autologous system. These tumour-specific CTL can, in their turn, be administered, to the patient to exert a direct cytotoxic effect on the patient’s cancer cells (passive or adoptive immunotherapy). The impact of a DC-based cancer vaccine is clear: an antigen-specific anti-tumour vaccine would influence both morbidity and mortality of various cancers. Currently, several phase I-II or III ▶clinical trials using TAA-loaded DC are ongoing worldwide in order to stimulate the patient’s immune system against tumour antigens. A number of these trials demonstrated some clinical and immunological responses (as evidenced by T cell proliferation, IFN-γ ▶ELISPOT and ▶delayed type hypersensitivity [DTH reaction] reaction) without any significant toxicity. However, despite the presence of expanded antigen-specific T cells in patients after vaccination, only a minor population of these patients showed a beneficial biologically relevant clinical response, i.e. tumour regression and increased disease-free survival. Until now, clinical trials using DC have only shown moderate, if any, success. Since DC possess the exceptional capacity to stimulate the patient’s own immune system against cancer, the reasons for the failure to eliminate tumour burden in a majority of patients needs to be carefully examined in ongoing and future trials. Dendritic Cells in Cancers DC can also infiltrate human tumours where they are involved in the induction of anti-tumour immune

responses. It is likely that the establishment of tumour-specific immune responses depends on the migratory capacity of DC from the tumour microenvironment to the draining lymph nodes, where tumour antigen presentation to T cells takes place. Moreover, by their expression of costimulatory molecules and several cytokines, such as IFN-α and IL-12, DC also mediate T cell survival by preventing T cell ▶apoptosis. In addition, mature DC have been reported to cause direct lysis, apoptosis as well as cell cycle arrest of cancer cells through the secretion of soluble factors. As a consequence, the presence of a high number of DC in the tumoural or peritumoural area, as well as in the draining lymph nodes of various human tumours has been shown to correlate with patients’ survival and a better prognosis. Decreased numbers or dysfunction (e.g. decreased expression of costimulatory molecules) of DC are reported in poor-prognosis tumours. Furthermore, tumour cells can secrete certain factors (e.g. IL-10 and TGF-β) that counteract DC maturation and migration and thus actively contribute to DC dysfunction. Occasionally, neoplasms of accessory immune cells (antigen-presenting cells, dendritic cells) can occur. These are primarily found in lymph nodes and extranodal lymphoid tissues (lymph node interdigitating cell sarcoma), but are also reported from other sites such as the skin (▶Langerhans cell histiocytosis). The incidence of dendritic cell tumours is very rare: until now, only a few dozens of cases have been reported in literature.

References 1. Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245–252 2. Lotze MT, Thomson AW (2001) Dendritic cells, 2nd edn. Academic Press, London 3. Van Tendeloo VF, Van Broeckhoven C, Berneman ZW (2001) Gene-based cancer vaccines: an ex vivo approach. Leukemia 15:545–558 4. Ponsaerts P, Van Tendeloo VF, Berneman ZN (2003) Cancer immunotherapy using RNA-loaded dendritic cells. Clin Exp Immunol 134:378–384 5. Gilboa E (2007) DC-based cancer vaccines. J Clin Invest 117:1195–1203

Densitometric Definition Pertaining to measurement of optical density in a material (e.g. amount of stain). ▶Malignancy-Associated Changes

Dental Pulp Neoplasms

Dental Pulp Definition Forms a functional and interdependent unit together with ist adjacent tissue, the dentin. Physiologic or pathologic reactions in one compartment will affect the other compartment as well. Dentin and the dental pulp are of mesectodermal origin. They are also called “pulpdentin complex”or “pulp-dentin organ”. ▶Dental Pulp Neoplasms

Dental Pulp Neoplasms K LAUS N EUHAUS MMA Department of Operative, Preventive and Paediatric Dentistry, School of Dental Medicine, University of Bern, Bern, Switzerland

Definition Are tumors that are located in the dental pulp.

Characteristics Dental pulp neoplasms (DPNs) are rare tumors of the dental pulp tissue which is not exposed to the oral cavity. Two types of DPNs can be distinguished: Type 1 originates from the dental pulp itself (primary DPN) and type 2 originates from tissue outside of the tooth (secondary DPN). Most DPNs are somewhat incidental findings in patients with a known tumor anamnesis. Therefore the number of histologic examples of DPNs is rather limited, and one also has to take into account articles from old literature in order to draw a complete clinical picture. History In the late nineteenth century, when systematic dental care and oral hygiene in general were considerably more deficient than today, dentists encountered numerous teeth with deep ▶caries and sometimes massive exposed pulp tissue. This phenomenon was called “pulpitis chronica sarcomatosa”, a chronically inflamed dental pulp supposedly caused by a sarcoma. Later it was found out that this pulpal alteration was in fact nothing to do with a sarcoma but rather was the result of colonization of the exposed dental pulp by free epithelium cells of the gums. This entity is a so-called “dental pulp polyp”. However, the first true description of a type 1 DPN was made in 1904 by V. A. Latham from

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Rogers Park, Illinois. He presented the case of a 56-yearold woman presenting with an upper right canine with a greenish-white tinge. The tooth was vital, symptomless and caries-free, i.e. the dental pulp was not exposed to the oral environment. After tooth extraction (for prosthodontic reasons) and histological processing, this canine proved to have an epithelioma of the pulp. The extraction socket was curetted and subsequently cleaned with iodine and carbolic acid. According to his report, Latham thus seems to have cured the patient from a tumor by simply extracting the tooth. Until today, descriptions of type 1 DPNs are very rare. First descriptions of type 2 DPNs also date back to the early 20th century where reports of involvement of dental pulps in patients with ▶breast cancer, ▶lymphoma or neuroma have been given. Three to thirty percent of tumors of the head and neck region (HNR) are associated with involvement of the dental pulp. Carcinomas are more likely to be associated with DPNs than sarcomas or any other type of tumors of the HNR. The maximum incidence of DPNs lies between the fifth and sixth decade of life. Inflammatory Pulp Reactions A DPN causes inflammatory reactions (▶inflammation) in the dental pulp. Chronic inflammation of the pulp may either lead to calcification of parts of the dental pulp tissue or to resorption of the surrounding hard tissue, i.e. ▶dentin. Calcifications – as regularly observed histological findings in dental pulps with a neoplasm – can be explained by the behavior of primary and secondary ▶odontoblasts. These cells are determined to secrete dental hard substance. If a bacterial impact is directed towards the pulp (as is the case with dental caries), the primary odontoblasts immediately start to produce tertiary dentin in the targeted area. Thus increasing the distance between the bacteria and the pulp, an early opening of the pulp chamber in the course of the carious process is evaded. Meanwhile, the chronic inflammation of the dental pulp in slowly progressing caries may lead to the calcification of parts of the pulpal tissue via secondary odontoblasts. These particular cells are differentiations of former pulpoblasts. Pulpoblast differentiation can be modified by ▶bone morphogenetic protein (BMP) 2, 4, and 11, ▶GDF, ▶TGF-β, or high calcium concentrations, all of which are present in dentin. It can also be modified by certain medicaments, which originally were only used in periodontal regenerative therapy but have now also been introduced in endodontic therapy as well as dental traumatology as a means of direct pulp capping. Radiotherapy As an additional point of discussion the possibility of therapeutically induced DPNs by radiotherapy has to

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be mentioned. It is a given fact that one of the risks of radiotherapy of the HNR consists in radiation-induced tumors (▶radiation-induced sarcomas after radiotherapy). Establishing a causal connection is often difficult due to a latency period of several years. However, teeth after radiotherapy sometimes show calcifications of the dental pulps, which can be detected in postirradiation radiographs. Since there has not been a study distinguishing between bacterial and abacterial calcifications as signs of chronic inflammations of the dental pulp in postirradiated cases, no predication can be given about a higher risk of DPN after radiotherapy. Animal Investigations As to DPN-cases, the pulp tissue reaction with respect to calcifications seems to be the same as in cases of dentin caries. At this point, observations made in experimental animal models become of interest: After several days calcification of the dental pulp tissue (with a simultaneous breakdown of the odontoblast layer) can be detected when inoculating the dental pulps of rodents with virulent sarcoma cells. Regular findings in these studies consist in massive development of intrapulpal dental hard substance like denticles, osteoids, or pulp stones. Also destruction of pulpal cells, particularly of the odontoblasts, by tumor tissue has been described in an animal study. In none of these investigations do the dental pulps survive longer than three weeks. Nevertheless, it is a matter of speculation whether this effect is really due to the sarcoma cells or rather to the increased extravasal pressure of the inflamed pulpal tissue. In these animal models, the sarcomata are able to infiltrate the dental pulps and to proliferate to adjacent tissue like ▶periodontium, mandibular bone and masseteric muscle. In later stages, ▶metastasis in the regional lymph nodes as well as in the sublingual, submandibulary and parotid glands can be found. The fact that rodent teeth are substantially different from human teeth must not be neglected. While rodent teeth are growing lifelong and have a largely open apex, human tooth formation literally comes to an endpoint in a constriction at the tip of the root(s). Clinical Relevance Since systematic autopsies of the jaws are no longer common, the entity of DPNs have somewhat moved out of the focus of scientific attention. Type 1 DPNs are certainly of small clinical relevance. In the dental pulp, fibroblasts, subodontoblastic progenitor cells, pericytes, stem cells, and, occasionally, Malassez epithelium remainders of the Hertwig root sheath are cells with mitotic competence and thus are able to undergo neoplastic alteration. A relatively high grade of differentiation of the pulpal tissue limits further differentiation of purported neoplasms.

Due to the restricted anatomical macroenvironment of a tooth and possibly further due to ▶microenvironmental interactions a type 1 DPN is more or less self-limited. Concerning the formation of a DPN, the capability of the dental pulp to regularly form calcifications under certain circumstances as well as the fact that one encounters a terminal blood supply in the pulp play a crucial role. Growth of a neoplasm will increase extravasal pressure within the dental pulp and thus stimulate secondary odontoblasts to secrete irritation dentin. A large amount of irritation dentin might influence the blood supply of the dental pulp and thus will probably lead to a hemorrhagic infarct. A growing tumor in the root canal system will contribute to this effect. While becoming necrotic in such a way, the dental pulp does not necessarily have to show clinical symptoms (such as tooth ache). Teeth with necrotic pulps will normally receive endodontic treatment (i.e. root canal therapy) or they will be “cured” by tooth extraction. It can be acclaimed that the specialty about a type 1 DPN lies in its possibility to be removed successfully and in a relatively easy way. The anatomic prerequisite of the root canal system presents the unique fact that while growing atumor is already limiting its further existence. The risk of metastasis of a DPN is not given in normal-sized teeth. The volume of the dental pulp chamber and the root canal system do not provide sufficient space for a tumor to gain a critical cell mass in order to disseminate clonal cells. Only teeth with incomplete root formation (as in children or adolescents) or ▶taurodonts, i.e. teeth with an abnormally large crown and roots, might provide enough space allowing a tumor to gain a critical cell mass. Large animal teeth, whose pulp chambers can surely provide enough space for a tumor (for instance in large mammalians), are not systematically screened for dental pulp diseases. Type 2 DPNs seem to be mere incidental findings in patients with tumors mainly of the HNR. This leads to the assumption that DPNs are normally symptomless and of relatively small clinical relevance. Nevertheless, type 2 DPNs may also lead to toothrelated symptoms (pain) as has been described in single case reports. Far more often (and clinically more important) is the opposite case when seemingly healthy teeth with no sign of caries, filling or a positive trauma history mimic toothache. The projected toothache is thus drawing off the attention of a true HNR tumor, which often leads to unnecessary root canal treatment or tooth extraction. Therefore, apart from regular or ofacial neuropathic or nociceptive pain conditions, differential diagnosis therefore should always consider a neoplasm in the HNR.

DES Mothers

In common classifications of dental pulp diseases, inflammation of the dental pulp due to neoplasms are neglected. However, animal tumor models (▶mouse models) should be reinvestigated for changes within the dental pulp.

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Deoxycytidine Kinase The rate-limiting enzyme in cytarabine metabolism, converting ara-C to ara-CMP. Also catalyzes the conversion of F-ara-A to F-ara-AMP.

References 1. Neuhaus KW (2007) Teeth: malignant neoplasms in the dental pulp? Lancet Oncol 8:75–8 2. Zajewloschin MN, Libin SI (1934) Histologische Untersuchungen der Zähne bei Neubildungen der Kiefer. Virchows Arch Pathol Anat Physiol Klin Med 293:365–380 3. Stewart EE, Stafne EC (1955) Involvement of the dental pulp by malignant tumors of the oral cavity. Oral Surg Oral Med Oral Pathol 8:842–55

2-Deoxy-D-Glucose Definition A glucose analog that inhibits glycolysis. ▶Jasmonates in Cancer Therapy

Dentin Definition

Dental hard substance located between ▶dental pulp and enamel/cementum. ▶Dental Pulp Neoplasms

Denys-Drash Syndrome Definition DDS; Is a rare disorder consisting of the triad of congenital nephropathy, ▶Wilms tumor, and disorders resulting from mutations in the Wilms tumor suppressor (WT1) gene. Nephropathy is a constant feature. ▶Nephroblastoma

Deoxyazacytidine

Dermoid Cyst Definition

▶Ovarian Teratoma.

DES ▶Diethylstilbestrol

DES Daughters Definition

Women exposed in utero to ▶diethylstilbestrol.

▶A5-aza-2′ Deoxycytidine

DES Mothers 2´-Deoxy-5-azacytidine ▶A5-aza-2′ Deoxycytidine

Definition Women administered pregnancy.

▶diethylstilbestrol

during

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DES Sons

DES Sons Definition

Men exposed in utero to ▶diethylstilbestrol.

Descriptive Epidemiology Definition The branch of cancer epidemiology that deals with the collection and analysis of data on the incidence, mortality, and survival of cancer in populations.

Desmocollin Definition

Dsc; a protein belonging to the desmosomal ▶cadherin family of cell ▶adhesion molecules. In humans three desmocollins (Dsc1–3) are known. Each is encoded by a distinct gene that is located in the desmosomal cadherin gene cluster on chromosome 18q21. Each desmocollin gene encodes two closely related proteins (the larger Dsc “a” protein and the smaller “b” protein) that differ only in the length of their C-terminal tails. The desmocollins are membrane spanning constituents of ▶desmosomes and are essential for desmosomal adhesion.

▶Cancer Epidemiology ▶Epidemiology of Cancer

Desmoglein Desert Definition Gene in hedgehog signaling. ▶Hedgehog Signaling

Definition

Dsg; a protein belonging to the desmosomal ▶cadherin family of cell ▶adhesion molecules. In humans four desmogleins (Dsg1–4) are known. Each is encoded by a distinct gene that is located in the desmosomal cadherin gene cluster on chromosome 18q21. The desmogleins are membrane spanning constituents of ▶desmosomes and are essential for desmosomal adhesion.

Des-Gamma-Carboxy Prothrombin Definition (DCP); Has been reported to be useful in the diagnosis of ▶Hepatocellular Carcinoma (HCC). This marker is also known as a protein induced by vitamin K absence or antagonist-II (PIVKA-II), or abnormal prothrombin. DCP was originally found in the blood of patients who were deficient in vitamin K or who were receiving a vitamin K antagonist. In 1984, Liebman et al. reported for the first time that serum DCP was elevated in patients with HCC.

Desmoglein-2 M ASAKAZU YASHIRO Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan

Synonyms Dsg2

Definition

Designer Foods ▶Nutraceuticals

Dsg2 is one of the calcium-binding transmembrane glycoprotein components of the cell-cell ▶adhesion molecules of the ▶desmosomes. Dsg2 is one of the ▶cadherin cell adhesion molecule superfamily in vertebrate epithelial cells.

Desmoglein-2

Characteristics Cell Junctions Epithelial cell-cell junctions consists of four junctions, ▶tight junctions, ▶adherens junctions, ▶desmosomes, and ▶gap junctions (Fig. 1). Two adhering-type junctions, the adherens junctions and the desmosomes, are responsible for strong cell-cell adhesion. Each of these junctions consists of a transmembrane cadherin and a complex cytoplasmic plaque that serve to link cadherin to actin microfilaments or the intermediate filament cytoskeleton. Desmosome Intercellular junctions known as desmosomes are multimolecular membrane domains that provide intercellular adhesion and membrane anchors for the intermediate filament cytoskeleton. Desmosomes are essential adhesion structures in most epithelia that link the intermediate filament network of one cell to its neighbor, thereby forming a strong bond. Desmosomes contain the desmosomal cadherins, desmoglein (Dsg) and ▶desmocollin (Dsc) that are linked to the intermediate filament cytoskeleton through interactions with ▶plakoglobin and ▶desmoplakin (Fig. 2). Desmoglein and Cancer Epithelial cell-cell adhesion is important in tumor development. Dsgs are transmembrane glycoproteins of the desmosome, a cell-cell adhesive structure prominent in epithelial tissues, which have been reported to be associated with tumor development. cDNA and protein

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studies have revealed that there are subfamilies of Dsg (types 1, 2 and 3) and Dsc (types 1, 2 and 3) [3]. Dsg2 and Dsc2 are widely expressed and are found together in desmosomes of the basal layer of stratified epithelia, simple epithelia, and nonepithelial cells such as in the myocardium of the heart and lymph node follicles, whereas Dsg3/Dsc3 and Dsg1/Dsc1 are more restricted to complex epithelial tissues. Although considerable overlap is exhibited in the distribution of these isoforms in stratified tissues, their expression is clearly differentiation-dependent. Dsg2, but not Dsg1 or Dsg3, is expressed in stomach epithelia. ▶Gastric cancers have been classified into two histological types: intestinal-type and diffuse-type. Diffuse-type gastric cancers show decreased cell-cell adhesion, which is associated with metastatic potential. These histological features indicate that a decrease in adhesive junctions may be involved in the emergence of diffuse-type gastric cancers. A decrease in E-cadherin has been reported to be one cause of the decrease in adhesive junctions, but not all diffuse-type gastric cancers show such a decrease. Decreased expression of Dsg2 is associated with diffuse-type gastric cancers and poor ▶prognosis in gastric carcinoma. Adherens Junctions The adherens junction is composed of a classic cadherin (e.g. E-, P- or N-cadherin) linked to ▶β-catenin or plakoglobin [5]. Thus, plakoglobin is found in both adherens junctions and desmosomes, while β-catenin is restricted to the adherens junction. Alpha-catenin links the cadherin/catenin complex to the actin cytoskeleton

Desmoglein-2. Figure 1 Cell junctions. Epithelial cell-cell junctions consist of four junctions, tight junctions, adherens junctions, desmosomes, and gap junctions.

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Desmoid Tumor

Definition Desmoid (meaning tendon-like) tumors are a heterogeneous group of rare connective tissue neoplasms, which can occur at almost any anatomical location. Desmoids have been classified as fibromatoses, along with pathologies such as palmar fasciitis, which are due to proliferation of well differentiated fibroblasts and are locally infiltrative and tend to recur after excision, but do not metastasize.

Characteristics Desmoglein-2. Figure 2 Desmosomes. Desmosomes contain the desmosomal cadherins, desmoglein and desmocollin that are linked to the intermediate filament cytoskeleton through interactions with plakoglobin and desmoplakin.

through interactions with ▶α-actinin, vinculin, ZO-1 and actin filaments. Lost or reduced plakoglobin expression has been observed in tumor tissues and metastatic lesions, and has been linked to poor prognosis in a variety of tumors.

References 1. Wahl JKr, Nieset JE, Sacco-Bubulya PA et al. (2000) The amino- and carboxyl-terminal tails of (beta)-catenin reduce its affinity for desmoglein 2. J Cell Sci 113 (Pt 10):1737–1745 2. Tselepis C, Chidgey M, North A et al. (1998) Desmosomal adhesion inhibits invasive behavior. Proc Natl Acad Sci USA 95:8064–8069 3. Buxton RS, Cowin P, Franke WW et al. (1993) Nomenclature of the desmosomal cadherins. J Cell Biol 121:481–483 4. Yashiro M, Nishioka N, Hirakawa K (2006) Decreased expression of the adhesion molecule desmoglein-2 is associated with diffuse-type gastric carcinoma. Eur J Cancer 42:2397–2403 5. Jou TS, Stewart DB, Stappert J et al. (1995) Genetic and biochemical dissection of protein linkages in the cadherincatenin complex. Proc Natl Acad Sci USA 92:5067–5071

Desmoid Tumor S UE C LARK The Polyposis Registry, St Mark’s Hospital Harrow, UK

Synonyms Aggressive fibromatosis; Mesenteric fibromatosis; Gardner syndrome

Desmoids are rare, accounting for less than 0.1% of all tumors, and have an annual incidence of 2–4 per million. While most occur sporadically, 2% are associated with ▶familial adenomatous polyposis (FAP), an autosomal dominantly inherited cancer predisposition syndrome due to mutation of the tumor suppressor gene APC (▶APC gene in familial adenomatous polyposis). Desmoids are over 1,000 times more common individuals with FAP than in the population in general, occurring in about 10–20% of them, and are an important cause of death in this group. It is useful to classify desmoid tumors as being either sporadic or FAP-associated, and by their location into intra-abdominal, abdominal wall or extra-abdominal. Pathology Both sporadic and FAP-associated desmoids have been shown to be clonal proliferations of myofibroblasts. Those associated with FAP result from acquired mutations in the ▶wild-type copy of APC. Somatic loss of the β-catenin gene has been described in sporadic desmoids, and APC mutation has also been identified in some cases ▶APC/β-catenin pathway. Thus abnormal activation of the Wnt pathway seems to have an important role in desmoid tumorigenesis ▶Wnt Signaling. A variety of complex chromosomal abnormalities, including trisomy 8 and gain of 1q21, has also been found in some tumors. There is no true capsule, and the desmoid compresses and infiltrates surrounding tissues as it grows. Desmoids range in size from a few centimetres to large masses weighing several kilograms. A photograph of a mesenteric desmoid tumor taken at surgery can be found in ▶APC gene in familial adenomatous polyposis. Growth rates are very variable. There have been reports of spontaneous resolution, and some desmoids grow relentlessly. The majority, however, either display cycles of growth and resolution or stabilize. The cut surface is usually pale and whorled. There may be central hemorrhage, necrosis or cystic degeneration. Histologically desmoids consist of mature, highly differentiated spindle shaped fibroblasts in an abundant collagen matrix. The histological appearances are not

Desmoid Tumor

necessarily diagnostic, and need to be interpreted in the light of the macroscopic findings. Aetiology Trauma, sex hormones and genetics have all been implicated in their aetiology. Many sporadic abdominal wall desmoids seem to arise in women in pregnancy, perhaps as a result of low-grade trauma of stretching, coupled with high levels of ▶female sex hormones. There have been numerous reports of desmoids arising at sites of surgical wounds, although many, particularly at extra-abdominal sites, seem to occur in the absence of any previous trauma. The higher incidence of desmoids in females, association with pregnancy, presence of estrogen receptors, and results of some experimental studies on desmoid cell lines all suggest that estrogens may have a role in stimulating desmoid development. Desmoids are very much more common in individuals with FAP. Within this group some familial clustering has been observed, in part explained by a ▶genotype-phenotype correlation in which families with an APC mutation 3′ of codon 1444 have an attenuated colorectal phenotype but a high risk of desmoid development. There is also evidence of the influence of as yet unidentified modifier genes. Clinical Features Desmoids most commonly occur in young adults (mean age of onset around 30 years), but have been described in children and even babies. Sporadic desmoids are more frequent in women than men (reported gender ratio 2–5:1), but in FAP there is a less marked gender difference. Sporadic desmoids are found predominantly in the abdominal wall (50%) and at extra-abdominal sites (40%), whereas about 80% of desmoids associated with FAP are within the abdomen, most in the ▶small bowel mesentery. It is not uncommon for an individual to develop desmoids at multiple sites. Intra-abdominal desmoids characteristically arise in the small bowel mesentery. Potential “desmoid precursor lesions,” consisting of small plaques of peritoneal thickening, have been observed in patients with FAP. It is thought that these enlarge, causing a diffuse thickening and puckering of the mesentery which can be seen on ▶CT scans. In some cases a frank desmoid mass develops. Most extra-abdominal desmoids cause symptoms because of their bulk and resulting mechanical effects. At some sites, for example in the neck, they can compress nerves and blood vessels. The overlying skin may ulcerate and abdominal wall desmoids occasionally adhere to and erode abdominal organs. Intraabdominal desmoids can cause major morbidity and even death, usually due to ureteric obstruction, bowel

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obstruction or perforation, either due to direct erosion or to compromise of the vascular supply. ▶CT and ▶MRI are the most useful imaging modalities, showing both tumor size and relationship to neighboring structures. Signal intensity on T2 weighted MRI may reflect cellularity and is correlated with tumor growth. Treatment The treatment of desmoids is difficult and controversial. There are numerous case reports and small uncontrolled series in the literature, but these are difficult to interpret, particularly as the natural history of these tumors is so variable. The drugs most widely used are ▶non-steroidal antiinflammatory drugs (NSAIDs) (particularly sulindac), and anti-estrogens (▶tamoxifen or toremifene). Overall the response rates to a variety of drugs in these classes is claimed to be in the region of 50%, but in reality is likely to beconsiderably less thanthis. There have beena handful of reports of acute desmoid necrosis, with abscess formation or bowel perforation in some, occurring in the weeks after initiation of drug treatment. As NSAIDs have little in the way of adverse effects they are often used as first-line treatment. The mechanism of action in this setting is not clear, but there is some evidence that Cox-2 inhibition my inhibit desmoid growth ▶celecoxib, ▶cyclooxygenase-2 in colorectal cancer. There have been no trials of Cox-2 inhibitors used therapeutically. Anti-estrogens can be used alone or in combination with NSAIDs. Surgery is widely accepted as the first line treatment for extra-abdominal and abdominal wall tumors. Recurrence rates are high (20–80%), but unaffected by use of prosthetic mesh in reconstruction. Serious morbidity and mortality rates are generally very low, although some sites, such as the neck, pose particular challenges. There are some reports suggesting that radiotherapy given postoperatively might reduce recurrence rates. Excision of intra-abdominal desmoids is also associated with frequent recurrence, but carries a substantial risk of perioperative mortality or major morbidity. The commonest reason for this is that the tumors lie close to or encase the superior mesenteric blood vessels, so that the blood supply of a large part of the intestine may be damaged or deliberately sacrificed during surgery. This may result in the need for lifelong parenteral nutrition, and in a handful of cases small bowel transplantation has been performed in these circumstances. Careful case selection, using CT angiography and multiplanar reconstruction, together with accumulation of expertise in a specialized institution has been shown to produce better surgical results in the last 10 years. Generally, however, major resection of intra-abdominal desmoids should be avoided. Ureteric obstruction can be successfully overcome by ▶stenting, and intestinal obstruction

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or fistulation may be managed in many cases, at least acutely, by defunctioning. Cytotoxic chemotherapy has been used to treat life-threatening desmoids. Response rates of 50% have been obtained using doxorubicin and dacarbazine in combination, and also with a less toxic regimen of methotrexate and vinblastine. In view of the potential toxicity of this type of treatment, it should probably be reserved for progressive, inoperable desmoid tumors in which other treatments have failed. ▶Aggressive Fibromatosis in Children

References 1. Clark SK, Phillips RKS (1996) Desmoids in familial adenomatous polyposis. Br J Surg 83:1494–1504 2. Hosalkar HS, Fox EJ, Delaney T et al. (2006) Desmoid tumours and current status of management. Orthop Clin North Am 37:53–63 3. Okuno S (2006) The enigma of desmoid tumours. Curr Treat Options Oncol 7:438–443 4. Reitamo JJ, Scheinin TM, Hayry P (1986) The desmoid syndrome. New aspects in the cause, pathogenesis and treatment of the desmoid tumour. Am J Surg 151:230–237 5. Sturt NJH, Clark SK (2006) Current ideas in desmoid tumours. Fam Cancer 5:275–285

Desmoplakin Definition DP; a protein belonging to the plakin family of cytolinkers. Desmoplakin is found in ▶desmosomes and is essential for desmosomal adhesion. The desmoplakin gene encodes two closely related proteins (the larger DPI and the smaller DPII) that differ only in the length of their central rod domain.

Definition Desmoplasia is the formation of fibrous connective tissue by proliferation of ▶fibroblasts. Desmoplasia is a key component of solid tumor stroma (Fig. 1).

Characteristics Tumors have many parallels to wounds, including similar inflammatory and desmoplastic responses, and fibroblasts are the key cellular component in development of desmoplasia. Fibroblasts are recruited into the wound or tumor, secrete and remodel ▶extracellular matrix (ECM) (▶Extracellular Matrix Remodeling), and serve as scaffolding for other cell types in connective tissue. As fibroblasts incorporate into a tumor environment, they undergo a phenotypic change and acquire an “activated fibroblast” appearance, which are also known as a ▶myofibroblasts or tumor-associated fibroblasts. Myofibroblasts have similar markers to fibroblasts, but myofibroblasts upregulate proteins such as α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP-1), and ▶fibronectin fibrils. During wound repair, the number of myofibroblasts return to a normal level upon wound resolution. In contrast to wound repair, ▶tumor microenvironments simulate a chronic wound in many ways. Thus, local fibroblasts and those that were recruited into the expanding stroma are continuously exposed to activation signals. Activated fibroblasts expand and contribute to an increased stromal response known as desmoplasia. Desmoplasia can be associated with increased tumor stage and poor prognosis in ▶breast cancer patients, but it is unclear whether fibroblasts are active inducers or passive participants in cancer progression. It is clear, however, that activated fibroblasts play a large role in the expanding tumor stroma (▶Stromagenesis). Fibroblastic stromal cells and desmoplasia have been linked to several activities that promote cancer growth

▶Desmosomes ▶Maculae Adherents

Desmoplasia S ASSER A. K ATE , B RETT M. H ALL Department of Pediatrics, Columbus Children’s Research Institute, The Ohio State University, Columbus, OH, USA

Synonyms Stroma; Stromal cell response; Schirrous (archaic)

Desmoplasia. Figure 1 Hematoxolin and Eosin (H&E) stain. Tumor fibroblasts (i.e., desmoplasia) appear pink.

Desmoplasia

and ▶metastasis (▶Seed and Soil) including ▶angiogenesis, ▶epithelial to mesenchymal transition (EMT), and progressive ▶genetic instability. Additionally, fibroblastic stromal cells can dysregulate anti-tumor immune responses, as exemplified by experiments demonstrating that ▶allogeneic murine tumor cells, when co-injected with fibroblastic stromal cells, can engraft across immunologic barriers. Together, these studies suggest that tissue-specific fibroblasts are influential players in progression of metastatic cancer. However, with the exception of promoting epithelial to mesenchymal transition, the direct biological impact on cancer cells themselves has been difficult to distinguish from indirect mechanisms such as enhanced support for angiogenesis or recruitment of inflammatory cells. The origins of desmoplastic fibroblasts are not fully understood. Some studies have suggested that stromal cell fibroblasts are recruited to the expanding tumor mass from local tissue fibroblasts. However, other experimental evidence supports that additional tumorassociated fibroblasts can be recruited from peripheral fibroblast pools, such as ▶bone marrow-derived mesenchymal stem cells (MSC) or fibrocytes. It has been shown that once fibroblasts are recruited into the expanding stroma they change their phenotype and may also undergo selective genetic alterations, which may drive additional tumor growth. Desmoplastic tumor fibroblasts have also been shown to carry unique genetic lesions when compared to those found

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in expanding tumor cells. These observations offer an additional insight into potential mechanisms for how genetic lesions can induce tumor cell expansion. The mechanism for recruitment of desmoplastic fibroblasts into a developing tumor remains poorly defined. Yet, several groups have shown that ▶plateletderived growth factor (PDGF) can contribute to the formation of desmoplasia. In a ▶xenograft model using the human breast carcinoma cell line MCF-7 expressing the cellular oncogene, c-ras, investigators demonstrated that blocking tumor PDGF inhibited the formation of desmoplasia. Others have shown that blocking TGF-α, TGF-β, IGF-I, and IGF-II had no effect on the desmoplastic response. Since these models used murine xenografts it remains unclear whether PDGF is as critical for development of desmoplasia in human carcinomas (▶Epithelial Tumorigenesis). One important way that desmoplastic fibroblasts can contribute to tumor growth and metastasis is through the production of multiple growth factors (▶Fibroblast Growth Factors). ▶Paracrine growth factors such as the stroma derived factor 1 (SDF-1/CXCL12) (▶angiogenesis), vascular endothelial growth factor (▶VEGF) (angiogenesis), ▶fibroblast growth factor (FGF) family, ▶hepatocyte growth factor (HGF), ▶transforming growth factor beta (TGF-β) family, ▶interleukin-6 (IL-6), and epidermal growth factor (EGF) have all been linked to increased tumor growth. Desmoplastic fibroblasts also contribute to tumor stroma through the

Desmoplasia. Figure 2 The tumor microenvironment is composed of many cell types that support tumor cell growth and survival.

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production of fibrous connective tissues and extracellular matrix proteins (▶Fibronectin) (▶Focal Adhesion Kinase (FAK)). ▶Collagen production is a hallmark feature of desmoplasia. As fibroblasts convert to myofibroblasts or tumor-associated fibroblasts, parallel increases in production of collagen are observed. A pathologist can readily visualize increased levels of tumor collagen using standard histology procedures (▶Pathology), and collagen types I and IV are the most prevalent forms of collagen found within most desmoplastic reactions. Collagen bundles interact with extracellular matrix and cell surface proteins such as ▶integrins (▶Cell Adhesion Molecules) (Focal Adhesion Kinase (FAK)) to influence the stiffness of a given tumor ▶microenvironment. Desmoplasia varies extensively between tumors and even within the same tumor. Some studies have suggested that desmoplasia is a defensive mechanism used to wall off the expanding tumor, but other data demonstrate that desmoplasia is associated with increased tumor growth, invasion, and metastasis. It is unclear, however, which underlying mechanisms determine the extent to which desmoplasia may promote tumor progression. As investigators continue to recognize the importance of the tumor microenvironment (Fig. 2), more detailed studies will allow clarification of the biological impact of desmoplasia in tumor development, survival and metastasis.

Desmoplastic Medulloblastoma Definition

Histological subtype of ▶medulloblastoma characterized by a network of reticulin fibers leaving pale islands of typical medulloblastoma cells. Predominant histological medulloblastoma variant in ▶BCNS or ▶Gorlin syndrome.

Desmoplastic Melanoma ▶Cutaneous Desmoplastic Melanoma

Desmoplastic Small Round Cell Tumor S EAN B ONG L EE

▶Cutaneous Desmoplastic Melanoma ▶Stromagenesis ▶Stem Cell Plasticity

Genetics of Development and Disease Branch, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

References

Synonyms

1. Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer initiation and progression. Nature 432:332–337 2. Mahadevan D, Von Hoff DD (2007) Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Mol Cancer Ther 6(4):1186–1197 3. Walker RA (2001) The complexities of breast cancer desmoplasia. Breast Cancer Res 3:143–145 4. Zipori D (2006) The mesenchyme in cancer therapy as a target tumor component, effector cell modality and cytokine expression vehicle. Cancer Metastasis Rev 25:459–467 5. Kunz-Schughart LA, Knuechel R (2002) Tumor-associated fibroblasts (part I): active stromal participants in tumor development and progression? Histol Histopathol 17(2):599–621

Desmoplastic Definition A growth of fibrous or connective tissue around the tumor. ▶Desmoplastic Small Round Cell Tumor

Small round-cell tumor; Malignancy of small round blue cell type

Definition DSRCT; Is a rare and highly aggressive tumor occurring mostly in the abdominal peritoneal cavity of adolescents and young adults. In rare cases, the tumors can also be found in other sites such as pleural cavity, pelvis, bone, and head and neck region. DSRCT belongs to a group of undifferentiated small round cell tumors, which include ▶Ewing sarcoma/primitive peripheral neuroectodermal tumor (PNET)/Askin’s tumor and ▶rhabdomyosarcoma. DSRCT is invariably defined by a ▶chromosomal translocation involving chromosomes 11 and 22, t(11;22)(p13;q12), leading to a fusion of two unrelated genes, ▶EWS and ▶WT1, into a single ▶chimeric gene.

Characteristics Clinical and Pathological Features DSRCT was first described in 1989 and is a poorly understood cancer that primarily affects young adults in their second and third decades of life. DSRCT occurs

Desmoplastic Small Round Cell Tumor

predominantly in males than females but the reason for this is unknown. Symptoms of DSRCT are usually associated with abdominal pain or pain in the primary site of tumor involvement, distention, and palpable mass. Local invasion or metastasis to liver, lungs, and bone is commonly found at diagnosis. DSRCT displays distinct histological and immunological features. Most of DSRCT cases are presented as tumors in the serosal surface of abdominal cavity, displaying nests of tumor cells surrounded by dense stromal components (hence the term ▶desmoplastic) containing spindle-shaped fibroblasts and hyperplastic blood vessels. Though rare, the primary tumors in sites other than abdominal region have been documented. The tumors are positive for various cell lineage markers, such as epithelial membrane antigen, keratin (epithelial), desmin (muscle), and neuron-specific enolase (neural). Thus, the tumor cell origin of DSRCT is not known. DSRCT is a clinically aggressive tumor with a high risk of recurrence and an overall poor prognosis. The most recent report on the comparison of different treatments of DSRCT patients suggests that compared to patients who received conventional treatments, a multimodal therapy, which include high-dose multiagent chemotherapy, aggressive debulking surgery, and radiotherapy, can prolong overall survival at 3 years (55%) and may provide a possibility of achieving a long-term survival, albeit at a low rate. The two key elements of the multimodal approach are the use of high-dose polychemotherapy, so-called ▶P6 protocol, and greater than 90% removal of tumor by surgery. P6 protocol consists of seven courses of high-dose alkylating agents ▶cyclophosphamide, ▶doxorubicin, ▶vincristine, ifosfamide, and ▶etoposide. This is followed by aggressive ▶debulking surgery, which was shown to be the major determinant in patient survival. Postoperative radiotherapy also contributed to improved survival. Although the multimodal therapy can improve survival at 3 and 5 years, the prognosis of DSRCT still remains extremely low (median survival of 2.5 years).

Molecular Diagnosis Although clinical, histologic, and immunologic features of DSRCT are distinct, a definitive diagnosis of DSRCT can be provided by genetic techniques. FISH technique, using fluorescently labeled genomic DNA probes derived from EWS and WT1, can be used to identify the specific t(11;22)(p13;q12) translocation of DSRCT. Alternatively, a definitive DSRCT diagnosis can be made with the use of reverse transcriptase-polymerase chain reaction (▶RT-PCR) technique to amplify and detect the DSRCT-specific EWS/WT1 hybrid mRNA transcripts using DNA primers specific for EWS and WT1 genes. This is an extremely sensitive detection

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method that can provide accurate diagnosis with limiting tumor materials. Molecular Genetics Molecular genetic studies revealed that all cases of DSRCT harbor a balanced reciprocal chromosomal translocation, t(11;22)(p13;q12) (▶reciprocal translocation) (Fig.1). The breakpoint in chromosome 22 has been mapped to the intron 7 of Ewing sarcoma gene, EWS, (breakpoints in other sites of EWS, such as in introns 8 and 10, have also been observed in rare cases), while the other breakpoint in chromosome 11 has been invariably mapped to the intron 7 of ▶Wilms tumor gene, WT1. This DSRCT-specific chromosomal translocation between EWS and WT1 results in a fusion of the N-terminal domain (NTD) of EWS to the C-terminal DNA-binding domain of WT1. EWS gene was first isolated from the Ewing sarcoma chromosomal breakpoint, where the translocation generates a fusion between EWS and an ETS-family transcription factor gene, FLI-1. EWS encodes a putative RNA-binding protein with presumptive roles in transcription and splicing. The NTD of EWS mediates potent transcriptional activation when fused to a heterologous DNA-binding domain, while its C-terminal domain, which is lost in the translocation gene product, is involved in RNA recognition. WT1 encodes a transcription factor which is mutated in a subset of Wilms’ tumor, a childhood kidney cancer. WT1 encodes four Cys2-His2 zinc-fingers in the C terminus that mediate sequence-specific DNA binding and the NTD containing both transcriptional activation and repression domains. WT1 is subjected to two ▶alternative RNA splicing events, one of which involves the usage of two alternative splice donor sites at the end of exon 9, leading to inclusion or exclusion of three amino acids, lysine, threonine, and serine (termed KTS), between the zinc-fingers 3 and 4 (Fig.1). The KTS insertion leads to a markedly decreased DNA-binding affinity of WT1. In all EWS/WT1 translocations examined, only the last 3 exons of WT1 (exons 8–10) encoding the last three zinc-fingers are fused to the NTD of EWS (Fig. 1), while the first zinc-finger of WT1 is invariably lost. The alternative KTS splicing of WT1, however, is preserved. As a result, EWS/WT1 produces two isoforms: EWS/WT1(−KTS) and (+KTS) that differs in the DNA binding affinity and specificity (Fig. 1). In vitro study has shown that only the EWS/WT1(−KTS) isoform, but not the EWS/WT1(+KTS), possesses the oncogenic activity in ▶NIH3T3 ▶transformation assay. DSRCT is a rare disease and has been recognized only recently as a distinct cancer type. Therefore, not much is known about the mechanisms of DSRCT but molecular details are starting to emerge. The novel fusion protein EWS/WT1(±KTS) acts as an aberrant transcription factor to presumably initiate the oncogenic

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Desmoplastic Small Round Cell Tumor

Desmoplastic Small Round Cell Tumor. Figure 1 Schematic representation of DSRCT-specific chromosomal translocation. A reciprocal balanced chromosomal translocation that results in the fusion of EWS gene to WT1 gene is shown. The arrow indicates the promoter of EWS which drives the transcription of the fusion gene and the boxes mark the exons. Alternative KTS splicing (grey box, KTS) within the exon 9 of WT1 is shown. Two isoforms of the fusion product are shown separately. See text for details.

process. To date, a number of direct transcriptional targets of EWS/WT1(−KTS) have been identified, which include PDGF-A (platelet-derived growth factor A), IGFR1 (insulin-like growth-factor receptor 1), IL2RB (interleukin 2 receptor beta), BAIAP3 (BAI1-associated protein 3), a potential regulator of growth-factor release, and TALLA-1 (T-cell acute lymphoblastic leukemiaassociated antigen 1), a gene encoding a tetraspaninfamily protein. There is only one target gene identified for EWS/WT1(+KTS), which is LRRC15 (leucine-rich repeat containing 15), a gene implicated in cell invasion. All of these target genes are not transcribed by the native WT1 and thus represent EWS/WT1-specific transcripts. Identification of the EWS/WT1 target genes may provide clues to the molecular and cellular pathways that are central to DSRCT. For example, expression of IGFR1 and IL2RB can promote proliferation and survival of the tumor cells, while expression of PDGF-A and BAIAP3 by the tumor cells can enhance recruitment and proliferation of surrounding fibroblasts and stromal tissues, which may further enhance the growth of the tumor cells

and may explain the dense stroma (desmoplastic feature) associated with DSRCT. Some of these target genes may also have diagnostic and therapeutic values, but it will require further evaluation.

References 1. Gerald WL, Rosai J (1989) Desmoplastic small round cell tumor with divergent differentiation. Pediatr Pathol 9:177–183 2. Ladanyi M, Gerald WL (1994) Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res 54:2013–2840 3. Gerald WL, Ladanyi M, de Alava E et al. (1998) Clinical, pathologic, and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic small round cell tumor and its variants. J Clin Oncol 16:3028–3036 4. Lal DR, Su WT, Wolden SL et al. (2005) Results of multimodal treatment for desmoplastic small round cell tumors. J Pediatr Surg 40:251–255 5. Gerald WL, Haber DA (2005) The EWS-WT1 gene fusion in desmoplastic small round cell tumor. Semin Cancer Biol 15:197–205

Desmosomes

Desmosomal Cadherins Definition A sub-family of the cadherin super-family of cell ▶adhesion molecules. In humans, the desmosomal cadherin family comprises four ▶desmogleins and three ▶desmocollins. The desmosomal cadherins are constituents of desmosomes and are essential for desmosomal adhesion. ▶Desmosomes

Desmosomes M ARTYN A. C HIDGEY Division of Medical Sciences, University of Birmingham, Clinical Research Block, Queen Elizabeth Hospital, Birmingham, UK

Synonyms Maculae adherents

Definition Desmosomes are intercellular junctions that mediate cellular ▶adhesion and maintain tissue integrity. They are found in ▶epithelial cells, myocardial and Purkinje fiber cells of the heart, arachnoid cells of brain meninges and follicular dendritic cells of lymph nodes.

Characteristics Desmosomes are localized at sites of close cell-cell contact (Fig. 1a). They are less than 1 μm in diameter, have a highly organized structure at the ultrastructural level and act as anchoring points for intermediate filaments of the cell cytoskeleton (Fig. 1b). By linking intermediate filaments of adjacent cells desmosomes confer structural continuity and mechanical strength on tissues. Desmosomes are particularly prevalent in tissues, such as the epidermis and heart, that experience mechanical stress. The proteins that form desmosomes belong to three genes families, the ▶desmosomal cadherins, the ▶armadillo family and the ▶plakin family of cytolinkers. Desmosomal Cadherins The desmosomal cadherins are the membrane spanning ▶cell adhesion molecules of desmosomes. In humans there are seven, four ▶desmogleins (Dsg1–4) and three ▶desmocollins (Dsc1–3). Each desmoglein and

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desmocollin is encoded by a distinct gene that is located in the desmosomal cadherin gene cluster on chromosome 18q21. All three desmocollin genes encode a pair of proteins, a larger “a” protein and a smaller “b” protein, that are generated by alternative splicing; the role of the smaller protein in desmosomal adhesion is not yet clear. All desmosomes contain at least one desmoglein and one desmocollin and both are required for adhesion. The desmosomal cadherins show tissue-specific patterns of expression with Dsg2 (▶desmoglein-2 adhesion molecule) and Dsc2 ubiquitously expressed in tissues that produce desmosomes and the others largely restricted to stratified epithelial tissues. The extracellular domains of desmosomal cadherins produced by adjacent cells interact in the intercellular space. Within the cell desmosomal cadherin cytoplasmic domains associate with armadillo proteins (Fig. 1c). Armadillo Family Armadillo proteins that are found in desmosomes include ▶plakoglobin (γ-Catenin) and ▶plakophilins. Plakoglobin is indispensable for desmosome function and interacts with desmosomal cadherins, plakophilins and ▶desmoplakin. Plakoglobin is also found in ▶adherens junctions where it is interchangeable with a closely related armadillo protein, β-catenin. In addition to its structural role in adherens junctions, β-catenin acts as a signaling molecule in the ▶APC/β-catenin pathway. There is a strong possibility that plakoglobin also has a signaling function in this pathway although its role has yet to be fully defined. There are three plakophilins (PKP1–3) that exhibit complex tissuespecific patterns of expression. All three plakophilins show dual localization in desmosomes and in the nucleus. The plakophilins have an important structural role in desmosomes and, because of their nuclear localization and similarity to other armadillo proteins, it is possible that they act as signaling molecules. Plakin Family ▶Plakin family proteins bind intermediate filaments and several, including desmoplakin, plectin, envoplakin and periplakin, localize to desmosomes. Of these only the presence of desmoplakin is obligatory for normal desmosomal adhesion. It is a dumbbell shaped molecule with two globular domains separated by a coiled-coil rod domain and is thought to exist as a homodimer. The desmoplakin gene encodes two proteins (DPI and DPII) that are generated by alternative splicing and differ only in the length of their central rod domain; the role of DPII, the smaller of these proteins, is unclear. The N-terminal end of desmoplakin binds to plakoglobin and plakophilins whereas its C-terminal end binds to intermediate filaments. In epithelial tissues desmoplakin anchors keratin intermediate filaments to the membrane, but in myocardial and

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Destruction Box

Purkinje fiber cells it interacts with desmin intermediate filaments and in arachnoid and follicular dendritic cells it associates with vimentin intermediate filaments. Null Mutations in Mice ▶Genetic ablation studies in mice have shown the importance of desmosomes for embryonic development and normal tissue biology. ▶Knock-out mice of either Dsg2, Dsc3 or desmoplakin display early embryonic lethality at around implantation or before. Mice without either PKP2 or plakoglobin survive longer but die during mid-gestation as a result of heart defects. Embryonic survival is not affected by absence of either Dsg3, Dsg4 or Dsc1 but loss of these molecules does result in defects in keratinocyte adhesion and skin and hair abnormalities. Clinical Relevance Loss of desmosomal adhesion can result in skin blistering diseases. Pemphigus is an autoimmune blistering disease that is caused by pathogenic ▶autoantibodies against desmogleins. Staphylococcal scalded-skin syndrome is caused by toxins with serine protease activity that are released by the bacterium Staphylococcus aureus and specifically cleave Dsg1. Mutations in DNA encoding desmosomal constituents result in a variety of diseases that can affect the skin, hair and heart, and sometimes all three. No mutations in desmosomal cadherins, plakophilins or desmoplakin have been found so far in cancer. However, many reports have documented altered expression of desmosomal constituents in tumorigenesis. For example, loss of Dsg2, Dsc2 and Dsc3 occurs in ▶gastric ▶colorectal and ▶breast cancer respectively. By contrast Dsg2 is overexpressed in ▶skin cancer and Dsg3 is overexpressed in head and neck cancer. PKP3 levels are elevated in ▶lung cancer and loss of desmoplakin has been correlated with progression in a variety of epithelial tumors. A causal relationship between these changes and cancer has yet to be established. Mutations in plakoglobin, concomitant with strong nuclear accumulation, have been linked to the pathogenesis of prostate cancer. Nuclear accumulation and improper activation of transcriptional targets as a result of a failure to degrade cytoplasmic β-catenin has been implicated in FAP (▶APC gene in Familial Adenomatous Polyposis), a familial syndrome that predisposes to ▶colon cancer, and sporadic colon cancer. It remains to be seen whether plakoglobin has, in common with β-catenin, pro-proliferative effects in cancer. In many cancers loss of expression of plakoglobin has been observed and it may be that plakoglobin is anti-proliferative in some cell types. There is little doubt that plakoglobin plays a role in cancer but whether this is related to its participation in desmosomes remains unclear.

Overall it appears that the importance of desmosomes in cancer is twofold. Firstly, as mediators of cell-cell adhesion reduced expression of desmosomal constituents could lead to loss of cell-cell adhesion, ▶epithelialmesenchymal transition, increased invasiveness and metastasis. Secondly, desmosomes may act as signaling centers and variations in expression levels of desmosomal proteins could trigger intracellular signaling cascades that contribute to cancer pathogenesis.

References 1. Chidgey M (2002) Desmosomes and disease: an update. Histol Histopathol 17:1179–1192 2. Garrod DR, Merritt AJ, Nie Z (2002) Desmosomal cadherins. Curr Opin Cell Biol 14:537–545 3. Getsios S, Huen AC, Green KJ (2004) Working out the strength and flexibility of desmosomes. Nat Rev Mol Cell Biol 5:271–281 4. Green KJ, Gaudry CA (2000) Are desmosomes more than tethers for intermediate filaments? Nat Rev Mol Cell Biol 1:208–216 5. Kottke MD, Delva E, Kowalczyk AP (2006) The desmosome: cell science lessons from human diseases. J Cell Sci 119:797–806

Destruction Box Definition DB; Amino acid sequence that when present in a protein in appropriate location confers the ability to be recognized by ▶E3-ubiquitin ligases and the subsequent degradation by the ▶proteasome. Usually, DB sequences can act in heterologous contexts. ▶Snail Transcription Factors ▶Ubiquitination

Detachment-induced Cell Death ▶Anoikis

Determination of Tumor Extent and Spread ▶Staging of Tumors

Detoxification

Detoxification J OHN D. H AYES Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, UK

Synonyms Detoxication; Drug metabolism; Xenobiotic metabolism; Carcinogen metabolism; Xenobiotic biotransformation

Definition Metabolic and transport processes used to chemically inactivate noxious compounds and eliminate them from cells for subsequent excretion from the body.

Characteristics Humans are continuously exposed to foreign chemicals (xenobiotics (▶Xenobiotic)) through administration of medicines, the consumption of food and drink, and air breathed. Protection against the detrimental effects of xenobiotics is achieved by the concerted actions of a battery of proteins that metabolize, transport and ultimately pump out of cells modified forms of the compounds originally encountered. This process is called detoxification, or detoxication (in instances where no toxicity occurs). Although detoxication occurs primarily in the liver, all cells possess some capacity to metabolize and eliminate unwanted chemicals. The xenobiotics subject to this process are numerous and include mycotoxins, phytoallexins, pesticides, herbicides, environmental pollutants, cytotoxic anti-cancer agents and many pharmacologically-active drugs. Detoxication processes also confer protection against harmful compounds of endogenous origin, many of which arise as a consequence of interaction with reactive oxygen species, such as the superoxide anion, produced normally in the body. Detoxication is achieved in two distinct stages, the first involving metabolism of the xenobiotic, and the second involving energy-dependent efflux of the xenobiotic from the cell. Historically, description of xenobiotic biotransformation has been divided into phase 1 and phase 2 metabolism, and consequently efflux of xenobiotics is referred to as phase 3 of detoxication. . Phase 1 drug metabolism involves an initial chemical modification of the xenobiotic that results in the introduction, or exposure, of a functional chemical group (e.g. –OH, –NH2, –SH, –COOH) into the compound. This usually entails enzyme-catalyzed oxidation reactions by ▶cytochrome P450 (CYP) or flavin monooxygenase. . Phase 2 drug metabolism often involves a second chemical alteration of the xenobiotic, usually at the

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same region of the molecule where the functional group was introduced. This is performed by enzymes catalyzing conjugation reactions (such as ▶glutathione S-transferase (GST), ▶N-acetyltransferase (NAT), ▶sulfotransferase (SULT) and ▶UDP-glucuronosyl transferase (UGT). It should be noted that use of the terms phase 1 and phase 2 to define the detoxication enzymes is somewhat arbitrary and does not necessarily reflect the pathway of biotransformation of all chemicals. Thus, a number of xenobiotics are subject to several modifications by the phase 1 CYP isoenzymes before serving as substrates for the phase 2 enzymes. Alternatively, some xenobiotics do not require modification by phase 1 enzymes before metabolism by phase 2 enzymes, and others are subject to modification by more than one phase 2 drugmetabolizing enzyme. As a result of differences in drug metabolism, the group of enzymes catalyzing reduction of hydrolysis reactions (e.g. ▶aldehyde dehydrogenase (ADH), ▶aldo-keto reductase (AKR), ▶epoxide hydrolase (EPHX) and ▶NAD(P)H-quinone oxidoreductase (NQO)) are variously referred to as phase 1 or phase 2 detoxication, depending on the individual xenobiotic being considered and the preferences of research workers. Clearly, these enzymes provide a highly flexible metabolic defense that has evolved to protect against a diverse spectrum of chemicals. . Finally, phase 3 of detoxication involves ATPdependent elimination of the parent compound or modified xenobiotic by proteins that are drug efflux pumps (e.g. ▶multidrug resistance protein (MDR) and ▶multidrug resistance-associated protein (▶Multidrug resistance protein) (▶MRP)). As a consequence of the combined actions of phase 1 and phase 2 enzymes, a diverse spectrum of xenobiotics acquires a limited number of molecular “tags” (i.e. acetate, glutathione, glucuronide or sulfate moieties) that are recognized by the MRP trans-membrane pumps. Furthermore, the xenobiotic metabolites produced by phase 1 and phase 2 are usually more soluble, and easily excreted, than the parent compound. Whilst the ability of CYP to oxidize xenobiotics is generally desirable, as it facilitates further metabolism and elimination of harmful chemicals, it can sometimes result in the generation of highly reactive products that may not be readily detoxified. In such instances, modification of intracellular macromolecules will occur resulting in necrosis, ▶apoptosis or malignant ▶transformation. As an example of the interplay between toxification and detoxification reactions, a scheme depicting metabolism of ▶aflatoxin B1 (AFB1), modification of macromolecules by AFB1 metabolites, and efflux of the AFB1-glutathione conjugate from a cell is shown in the illustration (Fig. 1).

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Detoxification. Figure 1 Detoxification pathways for aflatoxin B1. The mycotoxin is converted to the ultimate carcinogen AFB1-8,9-epoxide, by the actions of the hepatic phase 1 CYP enzyme system. The epoxidated AFB1 is highly reactive, and if it is not detoxified it will form DNA adducts that may cause hepatocarcinogenesis. The phase 2 GST enzymes can achieve detoxification of this unstable intermediate, and the resulting AFB1-glutathione conjugate is eliminated from the liver cell by MRP. In addition, AFB1-8,9-epoxide can rearrange to form a dialdehyde-containing metabolite which will covalently modify proteins by forming Schiff’s bases. The dialdehyde can be reduced by phase 2 AKR to yield a dialcohol that may be a substrate for SULTor UGT before being transported out of the cell, presumably by MRP.

Genetic Variation Numerous proteins have evolved that detoxify drugs, and certain of the families listed above comprise over twenty genes. In total, the human probably possesses between 100 and 150 genes encoding detoxication proteins. Substantial variation can occur in the levels of these proteins in tissues from different individuals, and

this can result in increased sensitivity of cells to chemical insult. In part, this inter-individual variation is due to ▶genetic polymorphisms. By definition, such differences must be present in at least 1% of the population in order to be considered a ▶genetic polymorphism. In some instances the variation involves deletion of detoxication genes with complete loss of

De-ubiquitinase

specific functions, whereas in other instances point mutations result in alteration of protein structure causing only a modest attenuation of activity. In other cases mutations alter the regulatory regions of genes causing altered expression of normal protein. Detoxication genes that are polymorphic in the human include those for the enzymes CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP2E1, AKR1C4, GSTM1, GSTP1, GSTT1, NAT2, SULT1A1, SULT1E1, SULT2A1, UGT1A1, UGT1A4, UGT1A6 and UGT2B7, EPHX and NQO1, as well as the MRP2 efflux pump. It is clear additional polymorphisms remain to be identified. Cellular Regulation In addition to genetic polymorphisms, induction of detoxication proteins by xenobiotics and environmental agents is a further mechanism that can cause interindividual differences in detoxification capacity. Induction of detoxication proteins represents an ▶adaptive response to chemical and ▶oxidative stress, which can be brought about by synthetic drugs or by naturally occurring compounds such as coumarins, indoles and isothiocyanates that are found in edible plants. Increased expression provides short-term resistance to toxic xenobiotics. Enzyme induction also results in increased metabolism of therapeutic drugs. Many of the enzymes and pumps such as CYP, GST, ADH, AKR, NQO and MRP are inducible, often by transcriptional activation of genes encoding the proteins. The promoters of these genes contain enhancers that enable a transcriptional response to a diverse spectrum of chemical agents. The enhancers that are involved in induction of detoxication proteins include ▶AP-1 binding sites, the antioxidant responsive element, the xenobiotic responsive element, the phenobarbital responsive enhancer module, progesterone X receptor and peroxisome proliferator-activated receptor enhancer. Clinical Relevance It is apparent from studies into the mechanisms of selective toxicity between species that variation in the activity of detoxication proteins influences sensitivity to chemical insult. Increasing evidence suggests that genetic polymorphisms in detoxication enzymes can confer an inherited predisposition to a number of malignant diseases that are influenced by environmental factors (e.g. lung and colorectal cancer). They may also confer a predisposition to adverse drug reactions. Induction of some phase 2 detoxication systems is believed to represent a major mechanism of cancer ▶chemoprevention, and is thought to explain in part the epidemiological data suggesting that consumption of diets rich in fruit and vegetables protect against certain malignant diseases.

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Acquired ▶drug resistance to chemotherapy is a major problem in the treatment of many cancers. There is overwhelming evidence that the overexpression of several detoxication proteins, particularly GST, MDR and MRP, contributes to the drug-resistant phenotype.

References 1. Guengerich FP, Shimada T (1991) Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chemical Res Toxicol 4:391–407 2. Hayes JD, Pulford DJ (1995) The glutathione S-transferase supergene family: regulation of GST and contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 30:445–600 3. Klaassen CD (ed), Amdur MO, Doull J (eds emeriti) (1996) Casarett and Doull’s toxicology: the basic science of poisons. McGraw-Hill, New York 4. Dinkova-Kostova AT, Massiah MA, Bozak RE et al. (2001) Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups. Proc Natl Acad Sci USA 98:3404–3409 5. Hayes JD, McLellan LI (1999) Glutathione and glutathionedependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 31:273–300 6. Borst P, Evers R, Kool M et al. (2000) A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 92:1295–1302

Detoxication Definition Synonym Detoxification; Process, or processes, of chemical modification which make a toxic molecule less toxic. ▶Toxicological Carcinogenesis

De-ubiquitinase Definition DUB; An enzyme that can specifically remove ▶ubiquitin proteins from substrates through an enzymatic cascade that cleaves an isopeptide bond. ▶Herpesvirus-Associated Ubiquitin-Specific Protease (HAUSP) De-Ubiquitinase ▶Ubiquitination

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De-ubiquitinating Enzymes

De-ubiquitinating Enzymes Definition Are a large family of enzymes that cleave chemical bonds formed at the C-terminus of ▶ubiquitin. By virtue of their action, the ▶ubiquitination of a given protein is reversible. Ubiquitin-protein conjugates are not always degraded by the proteasome, an alternative fate is the protein is spared from degradation through the activity of any of a large family of de-ubiquitinating enzymes. De-ubiquitinating enzymes can remove ubiquitin from ubiquitin-protein conjugates. These enzymes break down abundant multiubiquitin chains that are nor attached to any substrate and produce mature ubiquitin from the precursor forms in which it is synthesized. A second alternative fate for ubiquitinprotein conjugates is that ubiquitinated cell surface proteins may be targeted for endocytosis and eventual degradation via the lysosome rather than the proteasome.

Dezocitidine ▶A5-aza-2′; Deoxycytidine

dFdC ▶Gemcitabine

DHT Definition Dihydrotestosterone.

Development of New Lymphatic Vessels

▶Cyclin G-Associated Kinase ▶Dihydrotestosterone Receptor

▶Lymphangiogenesis

Dexrazoxane

DIA ▶Leukemia Inhibitory Factor

Definition Is an iron chelator, is a bisdioxopiperazine with cardioprotective and antineoplastic activities. ▶Adriamycin

Dexrazoxane Definition

A cyclic derivative of ▶EDTA used to protect the heart against the cardiotoxic side effects of anthracycline chemotherapy. ▶Chemoprotectants

Diabetes Definition Diabetes mellitus; People who are Type 1 Diabetes mellitus must use manufactured ▶insulin, usually in an injectable form, to replace the natural insulin that is no longer produced by their body (for instance as the result of beta-cell degeneration). People with Type 2 Diabetes sometimes need to use insulin when their cells become too resistant to the insulin that they produce naturally and when oral medications are no longer working.

Diabody

Diabetes Type 2 Definition Type 2 Diabetes, is a metabolic disorder characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. A disorder of glucose and insulin metabolism that is characterized by inappropriately increased blood glucose levels and resistance of tissues to the action of insulin. Insulin levels are elevated during early stages of the disease. Previously referred to as adult-onset diabetes or non-insulin-dependent diabetes. ▶Obesity and Cancer Risk ▶Adiponectin

Diabody S HUJI O ZAKI Department of Medicine and Bioregulatory Sciences, The University of Tokushima Graduate School of Health Biosciences, Tokushima, Japan

Synonyms Engineered antibody; Single-chain Fv dimer; Multimeric antibody fragments

Definition Diabody is a noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker. Another form of diabody is single-chain (Fv)2 in which two scFv fragments are covalently linked to each other.

Characteristics Advances in antibody technology are enabling the design of antibody-based reagents for specific purposes in cancer diagnosis and ▶monoclonal antibody therapy. First, to minimize the immunogenicity and enhance the efficacy in human use, mouse monoclonal antibodies are engineered to ▶chimeric antibodies or ▶humanized antibodies by grafting to the human constant region or framework. Moreover, fully human antibodies are developed by the use of ▶transgenic mice or phage display technology. Second, monoclonal antibodies are designed as immunoconjugates to deliver the cytotoxic agents such as chemotherapeutic drugs, toxins, enzymes,

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and radioisotopes. These therapeutic antibodies have emerged as potent agents and are used worldwide for cancer therapy. More recently, engineered antibody fragments have been investigated as alternative reagents because of their unique properties resulting from the structure. Structure A variety of antibody fragments are developed including single VH domain, Fab, scFv, and multimeric formats such as multivalent scFvs (diabody, triabody, and tetrabody), bispecific scFv, and minibody (scFv– CH3 dimer) (Fig. 1). In scFv fragments, the VH domain binds to its attached VL domain when the linker is flexible and long enough (a length of at least 12 amino acids). For example, the linker sequence of (Gly4Ser)3 provides sufficient flexibility for the VH and VL domain to form Fv comparable to the parent antibody. In contrast, when the linker is shortened to less than 12 residues (e.g., five amino acids of (Gly4Ser)), the VH and VL domains are unable to bind each other and instead the scFv fragment form a noncovalent dimer by another scFv molecule [(scFv)2 diabody]. Shortening of the linker length between the VH and VL domains (less than three residues) promotes the assembly of trimeric or tetrameric structures (triabody or tetrabody). However, this multimer formation also depends on the V-domain orientation either VH–VL or reverse VL–VH orientation in the scFv constructs. The bivalent Fv fragment can also be designed by linking two scFv domains covalently as a single chain version [sc(Fv)2 diabody]. The sc(Fv)2 version is more stable than (scFv)2, and this structure may form a noncovalent dimer [sc(Fv)2]2. The capacity of multivalent binding of these fragments offers a significant opportunity to design multifunctional antibody reagents. The diabody structure is used to form ▶bispecific antibodies by linking different VH and VL domains of two antibodies (e.g., VHA–VLB and VHB–VLA). However, when two different polypeptides are produced within a single cell, purification steps are necessary to obtain the active heterodimeric antibody among the inactive homodimers. Therefore, bispecific sc(Fv)2 version is developed by connecting two different scFv domains with the middle-length linker (e.g., VHA–VLB–VHB–VLA or VHA–VLA–VHB–VLB). Pharmacokinetics and Distribution The ▶pharmacokinetics of these antibody fragments is markedly different from intact IgG antibodies that exhibit prolonged circulation (t1/2 of up to 3 weeks). The lower molecular weight constructs (below than 60 kDa) are subject to be excreted by renal clearance, resulting in a shorter serum half-life than intact IgG. In most cases, the t1/2 values of scFv and diabody are extremely short such as 2 and 6 h, respectively.

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Diabody

Diabody. Figure 1 Schematic structure of intact IgG antibody and engineered antibody fragments. The variable regions of heavy (VH) and light chains (VL) contribute to the antigen binding. The VH and VL domains can be connected by a peptide linker to form single-chain Fv (scFv). The scFv fragments can form multimers such as (scFv)2 diabody, triabody, and tetrabody depending on the linker length and the V-domain orientation. The bivalent sc(Fv)2 can be generated by connecting two scFvs covalently. Bispecific diabodies can also be engineered by using two different Fv domains.

This rapid pharmacokinetics is the most favorable for imaging applications and ▶radioimmunotherapy because of the lower background levels in normal tissues. ▶Drug biodistribution studies of radiolabeled scFv and sc(Fv)2 have shown high tumor-to-blood ratios in xenograft models compared with intact IgG antibodies. The fast blood clearance of antibody fragments contributes to avoid undesired toxicity, and these fragments have the remarkable advantage for ▶targeted drug delivery of toxins or radioisotopes. In addition, antibody fragments show better penetration into the tumor mass, but these smaller constructs have shorter retention to tumor cells at the same time. Thus, the valance of penetration and retention of antibodies is an important factor for therapeutic use, especially in solid tumors. The Fab and scFv fragments are monovalent and exhibit poor retention on target cells, but multivalent forms of these fragments such as diabody, triabody, and tetrabody exhibit dramatically increased affinity and high tumor retention compared with the parent scFv. The ideal tumor-targeting reagents are intermediate-sized multivalent antibodies such as bivalent diabodies that show a longer half-life as well. In another approach, the Fc portion is fused to antibody fragments to control the serum levels of the antibody. The scFv–Fc or scFv–CH3 fusion antibodies (minibodies) are expected to have a more prolonged half-life and increased tumor accumulation in vivo. The serum half-life of antibody fragments can also be extended by modification such as linkage to polyethylene glycol (▶PEG).

Agonistic Activity In terms of mechanism of action, intact IgG antibodies kill tumor cells mainly by Fc-mediated effector functions such as ▶antibody-dependent cell-mediated cytotoxicity (ADCC) and ▶complement-dependent cytotoxicity. In contrast, antibody fragments have the compact structures without the Fc portion, and have unique characteristics for using cancer treatment. The two binding sites of diabodies are located at a distance of about 70Å less than half for those of intact IgG antibodies. Therefore, diabodies can place the antigens more closely to each other than by the parent IgG antibodies, which efficiently induces the ligation of target molecules on the cell surface. When the targets are functional receptors, the diabody can mediate a direct effect or signal transduction in tumor cells, including the stimulation of ▶apoptosis or cell death. For example, we and our collaborators have generated (scFv)2 and sc(Fv)2 diabodies that recognize CD47 or ▶HLA class I molecules. These diabodies can crosslink the target antigens and show the enhanced cytotoxic activities against hematological malignancies such as leukemia, lymphoma, and myeloma cells when compared with the original IgG antibodies. Thus, enhancement of the cross-linking potential is one of the important bioactivity of antibody fragments. Application of Diabodies A variety of target antigens have been evaluated for therapeutic purposes including CD19, CD20, CD22,

Dicer

epithelial cell adhesion molecule (Ep-CAM), epidermal growth factor receptor (EGFR), HER2, MUC1, and carcinoembryonic antigen (▶CEA). Several types of diabodies and minibodies are engineered for targeting these candidate antigens on tumor cells. Immunotoxins are also constructed to deliver the cytotoxic agents, radioisotopes, enzymes, cytokines, and liposomes by using antibody fragments. Previous studies have shown the effectiveness of these reagents in preclinical and clinical trials. ▶Bispecific antibodies that comprise two different binding specificities have been studied extensively in cancer diagnosis and therapy. Most of the bispecific reagents are designed for the retargeting of effector cells such as cytotoxic T lymphocytes and NK cells. Recombinant bispecific diabodies such as anti-CD19 x anti-CD3, anti-Ep-CAM x anti-CD3, and anti-HER2 x anti-CD3 have been used in the immunotherapy of ▶B-cell lymphoma ▶breast ▶ovarian and ▶colorectal cancer. Another strategy of bispecific antibodies is the recruitment of effector molecules including toxins, drugs, ▶prodrugs, ▶cytokines, and radionuclides in vivo. First, the tumor cells are targeted by the tumorspecific binding site of the diabody. After the unbound diabody is cleared from the serum, cytotoxic drugs or radiolabeled hapten are administered to be captured by another binding site of the bound diabody.

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Diacylglycerol Definition DAG; Is a lipid with two acyl chains esterified to sn-1 and sn-2 position of glycerol backbone; it is produced by phospholipase C and is involved in selective activation of isoforms of ▶protein kinase C (PKC). ▶Lipid Mediators ▶Protein Kinase C Family

Diagnostic Biomarkers Definition Markers to assess the presence or absence of cancer.

Diagnostic Pathology Future Directions In principle, selection of target molecules and modification of antibody constructs are key issues of antibodybased strategies in clinical utility. Based on the properties of pharmacokinetics, biodistribution, and manufacturing production, engineered antibody fragments have been investigated as alternative reagents to target cancer cells. Although the efficacy of these antibody fragments needs to be evaluated in clinical settings, the drastic potential of agonistic activity or multivalent activity of these reagents will provide new promises for development of the next generation of antibody drugs in cancer diagnosis and treatment.

References 1. Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136 2. Jain M, Kamal N, Batra SK (2007) Engineering antibodies for clinical applications. Trends Biotechnol 25:307–316 3. Beckman RA, Weiner LM, Davis HM (2007) Antibody constructs in cancer therapy. Cancer 109:170–179 4. Batra SK, Jain M, Wittel UA et al. (2002) Pharmacokinetics and biodistribution of genetically engineered antibodies. Curr Opin Biotechnol 13:603–608

▶Pathology

Dibasic Processing Enzyme ▶Furin

Dicer Definition An RNaseIII type enzyme that cleaves perfect or partially double-stranded RNA molecules. The hallmark of Dicer cleavage is the production of double-stranded short RNA molecules consisting of two 21 nucleotide RNA strands annealing to each other through 19 base pairs and have a two nucleotide overhang at the 3′-ends. Dicer produces ▶siRNAs from long double-stranded RNAs in lower

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animals and plants and generates ▶microRNA duplexes from stem-loop structure pre-miRNAs.

Dietary Micronutrient Definition

DIE ▶Endometriosis

Diet Definition Dietary factors may contribute to enhancing risks for the various cancers. These factors are heterogeneous. In many cases individual compounds have been suggested to be involved but little definitive evidence is available. The individual risk factors are specific for different tissues, and the state of current knowledge has recently been published. In general diets high in total fat or animal fat are considered causative for several common tumors including those arising from tissues of the gastrointestinal tract. In contrast, vegetables and fruits are considered to be protective for many tissues and for all cancers discussed in the review listed below. Phytoprotectants are implicated as contributing to risk reduction by different mechanisms, but it has not been possible to pinpoint individual compounds as the responsible factors. Tissues that seem to be most protected are esophagus, stomach, colon, lung, pancreas and bladder. The least clear cut protection is achievable in the hormone dependent tissues, prostate and breast, although a dietary component can not be excluded for these tumors. Altogether the estimates indicate that at least 35% of all human tumors are dietary related, which means a large proportion of tumors could be prevented by adequate dietary regimens; biomarkers See also: World Cancer Research Fund and American Institute for Cancer Research; Food, Nutrition and the Prevention of Cancer: a global perspective. Washington DC: American Institute for Cancer Research, 1997 ▶Biomarkers

Dietary Essential Minerals ▶Mineral Nutrients

A trace element, present in the diet that is required for maintenance of normal health. ▶Ultra trace Minerals

Dietary Supplement Definition An agent intended to supply nutrients, vitamins, minerals, and essential elements. ▶Chemoprotectants

Diethylstilbestrol R OSEMARIE A. U NGARELLI , C AROL L. R OSENBERG Boston Medical Center and Boston University School of Medicine, Boston, MA, USA

Synonyms DES

Definition Diethylstilbestrol is a synthetic non-steroidal estrogen with biological properties similar to endogenous estrogens such as estradiol-17-beta and estrone (▶Estradiol).

Characteristics Pharmacology Diethylstilbestrol is administered orally, is lipid-soluble, and readily absorbed from the proximal gastrointestinal tract. It is metabolized via the hepatic microsomal system to dienestrol, and quinone and epoxide intermediates. It crosses the placenta and is thought to be metabolized by the fetus. Initial Use and Early Epidemiologic Studies Diethylstilbestrol (DES), first manufactured by Dodds and associates in London in 1938, was used to treat several gynecologic conditions. In particular, it was

Diethylstilbestrol

prescribed for the treatment of frequent or threatened miscarriages. As early as 1953, Dieckmann and colleagues demonstrated that DES did not improve pregnancy outcomes. (In fact, in a later re-analysis of these data in 1978, Brackbill and Berendes showed that women exposed to DES had higher risks of premature births, perinatal death, and miscarriages than women who were given placebo.) Other studies also found DES to be ineffective in preventing adverse pregnancy outcomes, but physicians continued to prescribe the drug to try to maintain high-risk pregnancies because its use seemed logical and it was well-established. It was administered to approximately 5–10 million pregnant women in the United States between 1940 and 1971. It remained in use in Europe until the early 1980s. In 1971, Herbst and colleagues established a strong connection between DES exposure in utero and subsequent development of clear-cell adenocarcinoma of the vagina and cervix in young women, aged 14–21 years. The incidence of this cancer in women whose mothers had been administered DES during pregnancy (▶DES daughters) is estimated to range from 1.4 cases per 1,000 exposed to one case per 10,000 exposed persons (▶Cervical cancer). Previously, clear-cell adenocarcinoma of the vagina and cervix had been observed only rarely, primarily in post-menopausal women (over age 50) not exposed to DES. Consequently, the U.S. Food and Drug Administration issued a drug bulletin recognizing DES as a ▶transplacental carcinogen, and banned its use during pregnancy (▶Carcinogen). Since then, DES exposure has been observed to cause a range of teratogenic and neoplastic changes in humans and animals. It is used now mainly for treatment of a small subset of hormonally responsive refractory cancers. However, DES exposure can serve as a model for evaluating the potential effects of xenoestrogens (▶Hormonal carcinogenesis; ▶estrogenic hormones). Therefore, its investigation remains important and

Diethylstilbestrol. Table 1

DES Mothers DES Daughters

DES Sons

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should not be limited to the study of the consequences of a specific, unintentionally deleterious administration. Animal Studies DES has been studied extensively in animal models. In the Syrian hamster model, exposure to DES induces neoplasms of the liver and kidney, as well as aneuploidy (particularly chromosome gains) in the renal neoplasms (▶Aneuploidy, ▶chromosome instability). DES exposure in rats elicits tumors of the reproductive tract, pituitary and mammary glands. In addition, Green and colleagues have observed that DES metabolites produce DNA adducts (▶Adducts to DNA) and, ultimately, cancer in the breast of female ACI rats. Tumors of the reproductive tract and mammary glands are seen in the murine model as well, along with alterations in the genetic pathways governing uterine differentiation. Data from Newbold and colleagues suggest that an increased susceptibility to tumor formation is transmitted along the maternal lineage to subsequent generations, both male and female. Thus, not only is the developing organism sensitive to the endocrine-disrupting chemical, but transgenerational effects are plausible as well. Neoplastic Effects in Humans The only increased risk of hormone-dependent cancers observed in women who took DES during pregnancy (▶DES mothers) is ▶breast cancer (Table 1). Hatch and colleagues found that DES mothers had a 30% increased rate of breast cancer compared to the general population. In contrast, DES daughters have an increased risk of two types of hormone-dependent cancers, clear cell adenocarcinoma of the vagina and cervix (mentioned above) and breast cancer (▶Breast cancer). Clear cell adenocarcinoma of the vagina and cervix generally presents in these women when they are in their teens and twenties; however, because some women have been diagnosed in their thirties and forties, concerns have arisen about whether

Effects of diethylstilbestrol exposure Non-neoplastic effects

Neoplastic effects

Increased risk of • Adverse pregnancy outcomes Increased risk of • Adverse pregnancy outcomes • Infertility • Reproductive tract structural abnormalities • Vaginal adenosis Increased risk of • Epididymal cysts • Cryptorchidism • Testicular hypoplasia • Semen and sperm abnormalities

Increased risk of • Breast cancer Increased risk of • Clear-cell adenocarcinoma of vagina and cervix • Breast cancer over age 40

No increased risk of hormone-related cancers

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another increase in risk will occur as DES daughters approach the age at which this type of cancer is seen in the general population (the postmenopausal period). Until recently, there was a paucity of information on breast effects in DES daughters. However, the National Cancer Institute’s Continuation of Follow-Up of DESExposed Cohorts has proven a rich source of information to study the long-term effects of in utero estrogen exposure in humans. These cohorts include over 4,000 women who had documented in utero exposure to DES and more than 2,000 unexposed women from the same record sources; all have been followed from 1994 or earlier. As women exposed in utero to DES have begun to reach the ages at which breast cancer is more common, it appears that these women have an increased risk. In the most recent data from the National Cancer Institute collaborative follow-up study of DES health effects (Continuation of Follow-Up of DES-Exposed Cohorts), Palmer and colleagues found that at ages 40 and older, DES daughters had double the risk of breast cancer of unexposed women; no association was seen prior to age 40. To date, males exposed in utero to DES (▶DES sons) do not exhibit an increase risk of developing hormonerelated cancers. However, DES sons do display nonneoplastic abnormalities (see below) that place then at increased risk of developing testicular cancer regardless of DES exposure (▶Testicular cancer). Non-Neoplastic Effects in Humans In utero exposure to DES has been shown to elicit a variety of non-neoplastic reproductive tract abnormalities, including structural cervical, vaginal, or uterine abnormalities, in addition to changes in the vaginal epithelium such as adenosis (see Table 1). DES dosage and the stage of pregnancy during which the drug was administered appears to be directly linked to the severity of the adenosis, with the most severe manifestations seen in DES daughters whose mothers took the drug during their first trimesters. It is unclear whether these areas of adenosis progress to vaginal clear cell adenocarcinoma. DES daughters have an increased risk of poor pregnancy outcomes, such as ectopic pregnancy or miscarriage, and also have a higher incidence of infertility than the general population. DES sons are more likely than unexposed men to exhibit genital abnormalities such as epididymal cysts, cryptorchidism, and testicular hypoplasia. Although DES sons show an increased incidence of semen and sperm abnormalities, they have not demonstrated an increased risk of infertility, but this is still under investigation. Mechanism of Toxicity The mechanism by which estrogens in general, and DES in particular, exert their toxic and carcinogenic

effects is not fully understood. Both proliferative and genotoxic mechanisms have been postulated. Classically, estrogen exerts its effects through interaction with the estrogen receptor α (ERα), which stimulates cell proliferation and inhibits apoptosis (▶Estrogen receptor). ERα may also interact with other receptors (such as ERβ, insulin-like growth factor 1 receptor and epidermal growth factor receptor) to influence proliferation, or may act through non-genomic pathways, since it is found in non-nuclear subcellular fractions such as the plasma membrane and the mitochondria. An alternative or additional mechanism that may mediate estrogen’s and perhaps DES’ toxic effects is through the metabolites’ genotoxic capacity. ▶Estrogens, and specifically DES, can be oxidatively metabolized into potentially genotoxic intermediates. Estrogen and its metabolites have been reported to induce DNA damage (▶DNA damage), manifesting as ▶allele imbalance and ▶DNA amplification in human breast epithelial cells in vitro (▶Amplification). DES metabolites have been reported to produce DNA adducts and cancer in mammary glands of female rats. DES is a strong mitotic inhibitor in cell lines, blocking equatorial plate formation, tubulin polymerization, and spindle assembly. This may, in turn, induce ▶aneuploidy. Tumors associated with DES exposure exhibit ▶genetic instability, such as whole and partial chromosome gains in vitro and in vivo, in the Syrian hamster model. ▶Microsatellite instability has been reported in human vaginal clear cell adenocarcinomas associated with in utero DES exposure, as well as in murine endometrial carcinomas after DES treatment. In contrast to the substantial microsatellite instability seen in human vaginal clear cell adenocarcinomas associated with in utero DES exposure, breast neoplasms in DES daughters do not exhibit an increased amount of microsatellite instability. Breast tumors of DES mothers have not been investigated. In fact, little microsatellite instability was observed in breast tumors in both exposed and unexposed women, which is consistent with previous results from unselected human breast cancers, confirming that microsatellite instability is unusual in human breast cancers and suggesting that prenatal DES exposure does not affect ▶DNA mismatch repair mechanisms in the breast. Similarly, equivalent amounts of allele imbalance have been observed in breast tissue regardless of exposure, which differs from findings in animal models and in vitro systems. Therefore, the effect of in utero DES exposure may be tissue, timing and/or species specific, as is the case with other hormonal agents such as ▶tamoxifen, which has variable effects on human endometrium and mammary tissue. It remains under investigation as to whether the potential effects of in utero DES exposure on human breast carcinogenesis

Diffuse Large B-Cell Lymphoma

are mediated by enhanced proliferation, by alternative genotoxic effects, or by other pathways entirely.

Summary The unfortunate consequences of DES administration to pregnant women have yielded clinical and scientific insights into estrogen’s effects on developing and mature tissues. Its continued investigation should provide additional clinical and mechanistic information about these effects, and have relevance to understanding the effects of exposure to xenoestrogens.

References 1. Giusti RM, Iwamoto K, Hatch EE (1995) Diethylstilbestrol revisited: a review of the long-term health effects. Ann Int Med 122:778–788 2. Larson PS, Ungarelli RA, De Las Morenas A et al. (2006) In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium. Cancer 107(9):2122–2126 3. Newbold RR, Padilla-Banks E, Jefferson WN (2006) Adverse effects of the model environmental estrogen diethylstilbestrol are transmitted to subsequent generations. Endocrinology 147(6 Supp1):S11–S17 4. Schrager S, Potter BE (2004) Diethylstilbestrol Exposure. Am Fam Physician 69:2395–2402 5. Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast cancer. New Eng J Med 354:270–282

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dramatically during differentiation, but the genetic material remains the same, with few exceptions.

Diffuse, Small Cleaved Cell Lymphoma ▶Mantle Cell Lymphoma

Diffuse Large B-Cell Lymphoma M ICHAEL B. M ØLLER Department of Pathology, Division of Hematopathology, Odense University Hospital, Odense, Denmark

Synonyms KIEL classification: Centroblastic, B-immunoblastic, B-large cell anaplastic; Working Formulation: Diffuse large cell, Large cell immunoblastic, Diffuse mixed small and large

Definition

Diferuloylmethane ▶Curcumin

Diffuse large B-cell lymphoma is a ▶non-Hodgkin lymphoma entity composed of malignant large lymphoid cells with blastic morphologic features, expression of B-cell markers, and with a diffuse growth pattern. The postulated cells of origin are germinal or post germinal centre B-cells. This lymphoma entity is morphologically, clinically and genetically heterogeneous.

Characteristics

Differentiation Definition A process whereby a cell undergoes morphological transition from a cell which is capable of undergoing cellular division to a state where the cell becomes postreplicative. Often accompanied by changes in cellular function. The process during which young, immature (unspecialized) cells take on individual characteristics and reach their mature (specialized) form and function. Describes the process by which cells acquire a “type or assignment.” The morphology of a cell may change

Diffuse large B-cell lymphoma is the most common type of lymphoma comprising 30–40% of adult nonHodgkin lymphomas and approximately 20% of non-Hodgkin lymphomas in childhood and adolescence. Diffuse large B-cell lymphoma can be seen in all age groups, but the incidence increases with age. The median age at diagnosis is approximately 65 years. There is a slight male preponderance. In most patients the tumor resides in lymph nodes, but 40% of patients have predominant extranodal disease. Virtually any extranodal site may be involved, but the most frequently involved organs include the gastrointestinal tract, soft tissue, thyroid, skin, central nervous system, liver, bone, gonads, breast, kidney, lung and salivary glands. So-called transformed diffuse large B-cell

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lymphomas arise from indolent lymphomas such as small lymphocytic lymphoma/▶chronic lymphocytic leukaemia, ▶marginal zone B-cell lymphoma and ▶follicular lymphoma. The aetiology of most diffuse large B-cell lymphoma cases is unclear. However, patients with immunodeficiency such as human immunodeficiency virus-infected patients or patients receiving immunosuppressive therapy are at increased risk of developing lymphoma. Diffuse large B-cell lymphoma is the most frequent lymphoma arising in this setting, and these lymphomas are often ▶EBV associated. Diagnosis The typical clinical presentation of diffuse large B-cell lymphoma patients with nodal disease is rapidly enlarging ▶lymphadenopathy. Patients with extranodal presentation of the disease often have symptoms related to dysfunction of the involved organ(s). One third of the patients have ▶B symptoms. Approximately half of the patients have localized lymphoma, i.e. Ann Arbor stage I or II, and the remainder have disseminated disease. The morphological diagnosis is based on the World Health Organization Classification. Diffuse large B-cell lymphoma typically consists of a diffuse proliferation of medium-sized to large transformed B-lymphoid cells with a nucleus at least twice the size a normal lymphocyte. These large cells are a mixture of cells that resemble either the centroblasts or the immunoblasts that normally reside in reactive germinal centres. The diffuse large B-cell lymphoma entity is morphologically quite heterogeneous with several morphologic variants. The two most common variants are the centroblastic variant which is dominated by centroblasts and the immunoblastic variant with >90% immunoblasts. In the T-cell/histiocyte rich variant the majority of cells are small T-cells and histiocytes and less than 10% of the cells are neoplastic B-cells. An anaplastic variant of diffuse large B-cell lymphoma is recognized which has a similar morphology and ▶CD30 expression as the T-cell lymphoma anaplastic large cell lymphoma. However, anaplastic diffuse large B-cell lymphoma is clinically and genetically unrelated to anaplastic large cell lymphoma. Other rare variants include plasmablastic diffuse large B-cell lymphoma and diffuse large B-cell lymphoma with expression of full-length ALK. Diffuse large B-cell lymphoma cells usually express CD45 and B-lymphoid markers such as CD19, CD20, CD22, CD79a and PAX5. The proliferation rate is high with most cases expressing the proliferation associated marker ▶Ki-67 in >40% of the tumor cells. In some tumors >95% of the malignant cells express Ki-67. The prognosis is variable with a 5-year overall survival rate for all patients of 45–50%. The International Prognostic Index is widely used for prognostication

of diffuse large B-cell lymphoma. It consists of five clinical factors (age, stage, performance score, serum lactate dehydrogenase, number of extranodal sites involved) each with independent prognostic value regarding overall survival. The index allocates 35– 40% of the patients to the low risk group with a 5-year overall survival rate of >70%, while 15–20% of the patients have high risk lymphoma and a 5-year overall survival rate of 150) accomplish the common functions of recognition, incision, excision, degradation, polymerization and ligation by associating in different combinations and acting to remove the damage during a period of cell cycle arrest. ▶Carcinogen Macromolecular Adducts ▶Mismatch Repair in Genome Stability ▶Toxicological Carcinogenesis

DNA Photoproduct Definition Type of DNA damage formed after excitation of the DNA molecule by solar ▶ultraviolet light and kovalent

DNA Repair and Damage Processing ▶DNA Damage-Induced Apoptosis

DNA Vaccination

DNA Repair Capacity Definition

DRC; All living organisms have a ▶DNA repair system to protect the integrity of genomic DNA against constant assault from a plethora of endogenous and exogenous sources. DNA repair capacity (DRC) has often been used in epidemiologic studies as an indicator of the functionality of an individual’s DNA repair system. Reduced DRC is associated with increased cancer risk. ▶Mutagen Sensitivity

DNA Replication

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DNA Vaccination H OLGER N. LODE Charité University Medicine Berlin, Pediatrics, Berlin, Germany

Synonyms Genetic immunization

Definition Vaccination with deoxyribonucleic acid (DNA) against cancer is the most basic type of vaccination that, rather than consisting of the tumor-associated antigen itself, provides genes encoding for the antigen. Once produced in vivo following DNA delivery, the antigen is presented to the immune system inducing an antigen specific immune response. This response is augmented by the immunological properties of the DNA itself, mediated by unmethylated ▶CpG sequences. This essay reviews accomplishments and challenges in this area.

Definition Duplication of chromosomes by synthesis of DNA restricted to the S-phase of the ▶cell cycle. ▶Mitosis

DNA Topoisomerases II Definition Are the essential enzymes that play a role in virtually every cellular DNA process catalyzing the transient breaking and rejoining of DNA strands. They are able to cleave both DNA strands at the same time, allowing one DNA duplex to pass through another. ▶Nutraceuticals ▶Topoisomerases ▶Topoisomerases II

DNA Undermethylation ▶Hypomethylation of DNA

Characteristics DNA vaccination represents a young field in cancer immunotherapy. It started with the observation that injection of plasmid DNA into a mammal resulted in the synthesis of the encoded protein. The unformulated or “naked” plasmid DNA containing a simple expression cassette, consisting of a promoter functioning in mammalian cells and of a gene encoding for a protein antigen, was injected into the muscle of mice. The subsequent induction of antigen specific ▶CD8+ cytotoxic T-cells and antibodies was effective in protecting mice from challenges with the pathogenic agent expressing the antigen. This observation was surprising, given the low amount of antigen produced, the apparent lack of transfection of professional antigen presenting cells (APC) and the absence of any replicative step. The robustness of the technology was demonstrated for a variety of disease models. Mechanisms of Action The method of DNA delivery critically affects the mechanisms involved in the induction of an immune response. Intramuscular injection of plasmid DNA leads to in vivo transfection of myocytes. Mechanistic studies revealed that antigen specific immune responses following intramuscular injection of DNA is a result from ▶cross priming. This mechanism describes production of the antigen by the myocyte and subsequent uptake and presentation by professional APCs. This was clearly demonstrated in bone marrow chimeric mice and in experiments with transfected myoblasts. In both systems,

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the induction of an antigen specific immune response depended on the antigen presentation by APCs, and not by the myocyte. The transfer of the antigen from myocyte to APC follows different routes, ranging from uptake of secreted protein, processed peptide alone or with heatshock proteins or apoptotic bodies by APCs. The transfer of DNA into the myocyte and subsequent induction of an antigen specific immune response can be largely improved by in vivo ▶electroporation. The technique involves the application of an electric field around the DNA injection site. There are also needle free systems available, injecting DNA in solution using high pressure liquid jets. Clinical devices were developed by the pharmaceutical industry for application in humans, which are well tolerated. Bombardment of the epidermis with plasmid loaded onto gold particles using the ▶gene gun directly transfers DNA into APCs of the skin called ▶Langerhans cells. Once the protein antigen is expressed, professional antigen presentation is mediated by this cell type. Efficient induction of an immune response occurs after migration of these APCs from the skin into regional lymph nodes.

Gene transfer of plasmid DNA into APCs is also accomplished by the use of life attenuated bacteria such as salmonella typhimurium or listeria monocytogenes. In both cases, these micro organisms are infectious, but not pathogenic, and therefore serve as in vivo carrier systems for plasmid DNA vaccines. After in vivo application, ▶Peyer patches and the spleen become infected. Subsequently, the carrier microbes die due to distinct mutations in their genome and liberate multiple copies of the plasmid DNA vaccines in these ▶secondary lymphoid organs. There, the DNAvaccines are expressed by APCs leading to the induction of an antigen specific immune response. A central role for induction of an immune response by DNA vaccines is antigen expression by APCs and subsequent presentation to CD8+T-cells, ▶CD4+ T-cells and B-cells (Fig. 1). Adjuvant Activity of DNA Plasmid DNA derived from bacterial expression systems naturally contain unmethylated DNA sequences called CpG motifs. These sequences bind to Toll-like receptor 9 and are strong activators of ▶innate immunity. This

DNA Vaccination. Figure 1 Mechanisms involved in the generation of antigen specific humoral and cellular immune responses upon DNA vaccination. Antigen specific activation of cytolytic T lymphocytes (CD8+T-cells) occurs after proteasome dependent antigen processing of intracytoplasmic proteins into peptides associated with newly synthesized MHC class I molecules. MHC class I/peptide complexes are presented on the surface of APCs in conjunction with costimulatory molecules to CD8+T cells. The activation of ▶CD4+T-cells is primarily achieved by exogenous protein antigens taken up by the endolysosomal compartment. After degradation, peptides associate with MHC class II molecules which are then translocated to the cell surface. Specific CD4+ helper T cells recognize these MHC class II/peptide complexes and are activated to produce cytokines. These cytokines have multivarious activities helping B-cells to mature into antibody producing plasma cells and CD8+T-cells to transform into cytolytic effector cells. For antibody responses, B-cells recognize and respond to antigens that are either present extracellularly or exposed extracellularly by being transmembrane proteins.

DnaJ (Hsp40) Homolog Subfamily C Member 15

receptor is also expressed on APCs leading to improved antigen processing and presentation as well as the release of pro-inflammatory cytokines and chemokines that help to shift ▶adaptive immunity responses from ▶Th2 immune response to ▶Th1 immune response. Th1 responses are required for most effective anti-tumor immunity. Therefore, CpG motifs in the DNA vaccine backbone can be considered endogenous adjuvants linking innate immunity with ▶adaptive immunity, which provides for robust and long lasting antigen specific immune responses. Tailoring Immune Responses by DNA Vaccine Design In order to improve antigen specific immune responses, the versatility of DNA vaccine design allows for the simultaneous expression of antigen, co-stimulatory molecules and chemoattractants. These include cytokines, chemokines, molecules of the B7 family and CD40 ligand. The design of the protein antigen itself can be altered to be secreted for induction of B-cell responses or to be targeted into the endopasmatic reticulum or the proteasomal degradation pathway for the generation of T-cell epitopes. Protein antigens can be redesigned as mini genes only encoding for immunodominant peptide antigens. In summary the versatility of DNA vaccines allows for specific tailoring of an optimized immune response following a rational vaccine design. Formulation The formulation of DNA vaccines to improve antigen specific immune responses includes transfectionfacilitating lipid complexes, nanoparitcles and classical adjuvants. Lipid complexes are varying combinations of DNA with cationic lipids. Microparticles are generated with DNA entrapped in biodegradable poly-lactide-coglykolactide or complexed with non-ionic block copolymers or polycations. Among the classical adjuvants, aluminium phosphate is noteworthy for its effectiveness and simplicity of preparation. Microparticles appear to improve the trafficking of DNA to APCs by facilitating the transfer of DNA into regional lymphnodes. Mixed Modality Vaccines A very promising strategy that is entering clinical trials is to combine DNA vaccines with other gene delivery systems. This is based on observations that if DNA encoding an antigen is given as a prime followed by another gene-based vector system as a boost such as recombinant viruses encoding the same antigen, most optimal immune responses and protection are achieved. The responses are significantly greater than using DNA or the virus for both the prime and the boost or if the order of the administration is reversed.

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Results from Clinical Trials First generation DNA vaccines have been evaluated clinically as a therapeutic vaccine approach for cancer. These vaccines encoded for viral epitopes from transforming viruses, self-antigens expressed on tumors, and tumor specific antigens. In most trials so far, the plasmid DNA was injected intramuscularly, intradermaly or intranodaly. Antigen specific humoral and cellular responses were observed in human cancer trials. However, this did not translate into clinical responses in the trial patient populations characterized by large tumor burden and progressive disease. The clinical trials so far have proved the principle that immune responses can be generated in humans. They also highlight the need to apply strategies to increase the potency of the technology as outlined above and to generate second generation DNA vaccines for future application in cancer patients. In summary, DNA vaccines hold great potential as immunotherapeutic tools to prevent and treat human cancer. Their advantages include cost effectiveness, versatility, safety, stability, ease of construction and mass production and most importantly ability to induce robust humoral and cellular immune responses. Lack of success in early clinical trials so far is similar to early clinical results with treatments based on monoclonal antibodies which are now established cancer therapeutics. To push for success, the next generation of DNA vaccines will have to incorporate multiple strategies to enhance plasmid DNA immunogenicity. Additional avenues may involve exploring the possibilities of combining adoptive cell therapies with DNA vaccines such as ex vivo gene transfer into autologous dendritic cells.

References 1. Liu MA, Ulmer JB (2005) Human clinical trials of plasmid DNA vaccines. Adv Genet 55:25–40 2. Donnelly JJ, Wahren B, Liu MA (2005) DNA vaccines: progress and challenges. J Immunol 175:633–639 3. Fest S, Huebener N, Weixler S et al. (2006) Characterization of GD2 peptide mimotope DNA vaccines effective against spontaneous neuroblastoma metastases. Cancer Res 66:10567–10575 4. Lowe DB, Shearer MH, Jumper CA et al. (2007) Towards progress on DNA vaccines for cancer. Cell Mol Life Sci 64:2391–2403

DnaJ (Hsp40) Homolog Subfamily C Member 15 ▶Methylation-Controlled J Protein

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DNAJC15

DNAJC15 ▶Methylation-Controlled J Protein

DNAJD1 ▶Methylation-Controlled J Protein

DNF15S2 ▶Macrophage-Stimulating Protein

DNMTs Definition DNA methyltransferase enzymes; Responsible for maintenance of ▶methylation as well as de novo methylation. ▶Epigenetic Gene Silencing

Docetaxel R ICARDO H ITT 1 , C RISTINA R ODRI´ GUEZ 2 1

Medical Oncology Service, University Hospital 12 de Octubre, Madrid, Spain 2 Human Cancer Genetics Programme, Spanish National Cancer Center (CNTO), Madrid, Spain

Synonyms ▶Taxotere

Definition Docetaxel is a new class of anticancer agent that exerts the cytotoxic effects on microtubules. It is a semisynthetic drug with significant activity in a broad range

of tumor types that are generally refractory to conventional therapies, including chemotherapyresistant epithelial ▶ovarian cancer, ▶breast cancer, ▶non-small cell lung cancer, head and neck cancer, ▶bladder cancer, and ▶gastric cancer.

Characteristics Docetaxel is derived semisynthetically from 10-deacetylbaccatin III, is more water soluble than ▶paclitaxel and is more potent antimicrotubule agent in vitro. Mechanism of Action: Docetaxel induces polymerization of ▶tubulin, ▶microtubule bundling in cells, formation of numerous abnormal mitotic asters. The cytotoxic effects of docetaxel are also severalfold greater than paclitaxel in vitro and in tumor xenografts. Docetaxel inhibit proliferation of cells by inducing a sustained mitotis block at the metaphase–anaphase boundary at much lower concentrations than those required to increase microtubule polymer mass and microtubule bundle formation. These inhibitory effects at low drug concentrations are associated with the formation of an incomplete metaphase plate of chromosomes and an arrangement of spindle microtubules resembling the abnormal organization that occurs at low concentrations of the ▶vinca alkaloids. Docetaxel primarily block cell-cycle traverse in the mitotic phases and prevents the transition from Go to S phase. The inhibitory effects in the nonmitotic cellcycle phases include the disruption of tubulin in the cell membrane and direct inhibitory effects on the disassembly of the interphase cytoskeleton. These effects may result in the disruption of many vital cell functions such as locomotion, intracellular transport, and transmission of proliferative transmembrane signals. After disruption of microtubules and other processes by docetaxel, the precise means by which cell death occurs is not clear. Morphologic features and a DNA fragmentation pattern (▶nucleosomal DNA fragments) that are characteristic of programmed cell death, or apoptosis, in docetaxel-treated cells indicate that this taxane trigger apoptosis as do many other chemotherapeutic agents. Whether docetaxel-induced ▶apoptosis requires a functional ▶p53 pathway is unclear and probably depends on the cell line under study. The consensus seems to be that in most cell lines, disruption of p53 has little effect on drug sensitivity. Mechanism of Resistance: Selection of taxaneresistant cells in vitro is associated with changes in β-tubulin isotype expression. Six different isotypes of β-tubulin are expressed in nonmalignant tissues, with the class I isotype comprising 80–99% of cellular β-tubulin. The β III isotype increase the dynamic instability of microtubules, impairs rates of microtubule assembly, and increases resistance to taxanes.

Docetaxel

A second mechanism of acquired taxane resistance fits the general pattern of ▶MDR. The particular species of Pgp found in taxane-resistant murine ▶macrophages is similar, but not identical, to that found in ▶vinblastine- and ▶colchicine-resistant cells derived from the same parental line. These cells are cross-resistant with many other natural products, and resistance to docetaxel conferred by ▶mdr-1 can be reversed by many classes of drugs, including ▶tamoxifen, ▶cyclosporine A, antiarrhytmic agent. Other changes in tumor cells selected for drug resistance have included upregulation of ▶caveolin-1, a principal component of membrane-derived vesicles involved in transmembrane transport of small molecules and in intracellular signaling. Pharmacokinetics The single 1-hour infusion every 3 weeks is the most common administration of docetaxel. The ▶pharmacokinetics behavior on 1 or 2 h schedules is linear at doses of 115 mg/m2 or less and optimally fits a threecompartment model. Docetaxel binds rapidly and avidly to plasma proteins (>90%), especially to albumin, α1acid glycoprotein, and lipoproteins. In addition, peak plasma concentrations generally exceed levels required to induce relevant biologic effects in vitro. Limited information is available about the distribution of docetaxel in humans. Immediately after treatment, tissue uptake of radioactivity is highest in the liver, bile, and intestines, a finding that is consistent with substantial hepatobiliary extraction and excretion. High levels of radioactivity are also found in the stomach, which indicates the possibility of gastric excretion, as well as in the spleen, bone marrow, myocardium, and pancreas. Docetaxel has hepatic metabolism and biliary excretion and urinary excretion accounts only 2%. Approximately 80% of the administered dose of total radioactivity is excreted in the feces within 7 days after treatment, with the majority of excretion occurring in the first 48 h. In the hepatic ▶cytochrome P450-mixed function, oxidases are responsible for the bulk of drug metabolism, and CYP3A, CYP2B, and CYP1A isoforms may play major roles in biotransformation. The main metabolic pathway consists of oxidation of the tertiary butyl group on the side chain at the C-13 position of the taxane ring as well as cyclization of the side chain. Toxicity ▶Neutropenia is the principal toxicity of docetaxel. At dose of 100mg/m2, neutrophil count nadirs are 10% of estimated total natural production of carotenoids. Further, fucoxanthin is the characteristic pigments of brown seaweeds (phaeophyceae) which are the largest occurring group among seaweeds. In South East Asian countries, some brown seaweeds containing fucoxanthin are often used as a food source (Fig. 1).

Characteristics Effect on Cancer Cell Growth Cell proliferation is the key in promoting and further progression of carcinogenesis. In a study screening the antiproliferative activity of seaweed extracts on tumor cells, fucoxanthin from the brown seaweed,

a1-6 Fucosyltransferase Definition Fut8; A glycosyltransferase that transfers fucose onto the innermost N-acetylglucosamine in N-glycans via an

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Fucoxanthin. Figure 1 Structure of fucoxanthin.

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Undaria pinnatifida, is found to be the active principle. When several cancer cells are cultured with fucoxanthin, the cell viability decreases. The antiproliferative activity of fucoxanthin on human cancer cells is generally higher than other carotenoids. Fucoxanthin exhibits the higher activity than β-carotene and astaxanthin on human ▶colon cancer cells (Caco-2, HT-29, DLD-1) and human leukemia cell (HL-60). Treatment of Caco2 cells with fucoxanthin induces morphological changes such as a diminished size and rounded shape. Also, the cell membrane has shrunk with a condensed cytoplasm. The stronger inhibitory effect of fucoxanthin is found in human prostate cancer cells (PC-3, DU 145, LNCap). In this case, the effect of 15 kinds of carotenoids (phytoene, phytofluene, ξ-carotene, lycopene, α-carotene, β-carotene, β-cryptoxanthin, canthaxanthin, astaxanthin, capsanthin, lutein, zeaxanthin, vioaxanthin, neoxanthin, and fucoxanthin) present in foodstuffs is evaluated on the grown of the cancer cell lines. Among the carotenoids, neoxanthin and fucoxanthin cause a remarkable reduction in the growth of prostate cancer cells. Apoptosis There is a wealth of information pertaining to apoptosis in anticancer research. ▶Macrophages recognize the cells undergoing apoptosis and engulf them without adversely affecting or damaging the neighboring cells. Apoptosis-inducing activities provide a novel means of ▶chemoprevention and chemotherapy in the treating cancer. In an investigation on the apoptosis-inducing activity of fucoxanthin, a DNA ladder, which is a characteristic feature of apoptotic cells, is clearly visible in HL-60 cells treated with fucoxanthin. Similar results can be obtained with ▶camptothecin, which is known to be a strong apoptosis-inducing agent. The fragmented DNA content designated as the enrichment factor as estimated by sandwich ▶ELISA, increases with the concentration of fucoxanthin in the medium. DNA fragmentation, indicating by in situ ▶TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling), reveals that fucoxanthin reduces cancer cell viability by inducing apoptosis. Some apoptosisinducing agents are known to arrest a specific cell phase. Therefore, it can be presumed that fucoxanthin affects cell cycle. Mechanism Fucoxanthin suppresses expression of ▶Bcl-2 protein, which is responsible for suppression of programmed cell death as a survival factor. This is indicative of the fact that downregulation of Bcl-2 protein may contribute to fucoxanthin-induced apoptosis in cancer cells. DNA fragmentation induced by fucoxanthin is partially inhibited by a ▶caspase inhibitor Z-VAD-fmk. Further, fucoxanthin also regulates the redox signals,

and then facilitates the progression of apoptosis through Bcl-2 protein suppression, and caspase-dependent and-independent pathway. Combination with Troglitazone ▶Troglitazone is known to inhibit cell growth and induce apoptosis through the activation of ▶PPARγ. Oral administration of troglitazone inhibits the early stage of colon tumorigenesis. On the other hand, preincubation of cancer cells with fucoxanthin remarkably enhances the effect of troglitazone. Therefore, the combined action of PPARγ ligand such as troglitazone and fucoxanthin is more effective on chemoprevention of cancer than troglitazone, and possibly other agents, alone.

References 1. Hosokawa M, Wanezaki S, Miyauchi K et al. (1999) Apoptosis-inducing effect of fucoxanthin on human leukemia cell line HL-60. Food Sci Technol Res 5:243–246 2. Kotake-Nara E, Kushiro M, Zhang H et al. (2002) Carotenoids affect proliferation of human prostate cancer cells. J Nutr 131:3303–3306 3. Hosokawa M, Kudo M, Maeda H et al. (2005) Fucoxanthin induces apoptosis and enhances the antiproliferative effect of the PPARγ ligand, troglitazone, on colon cancer cells. Biochim Biophys Acta 1675:113–119

Fulvestrant A NTHONY H OWELL CRUK Department of Medical Oncology, University of Manchester, Christie Hospital NHS Trust, Manchester, UK

Synonyms Faslodex

Definition Was originally known as ICI 182,780 and is now marketed by AstraZeneca under the trade name Faslodex®. The chemical formula of fulvestrant is 7α-[9-(4,4,5,5,5-pentafluoro-pentylsulfinyl)nonyl]estra1,3,5(10)-triene-3,17β-diol. Fulvestrant, a steroidal 7α-alkylsulfinyl analog of 17β-▶estradiol, is an ▶estrogen receptor antagonist with no agonist effects. It is used as an endocrine treatment for postmenopausal women with hormonesensitive ▶advanced breast cancer.

Fulvestrant

Characteristics Mode of Action Currently, more than one million women worldwide are diagnosed with ▶breast cancer each year. In postmenopausal women, approximately 75% of breast tumors are hormone sensitive, expressing the estrogen receptor and/or progesterone receptor, and are stimulated to grow in the presence of estrogen. To understand the treatments for hormone-receptor positive breast cancer, we must first understand the role of ▶estrogen, a natural circulating hormone that has been shown to drive tumor growth. Once estrogen has bound to the ▶estrogen receptor, the receptors dimerize, before translocation to the nucleus, where the complex binds to specific DNA sequences (estrogen response elements) in target genes. Activating functions on the estrogen receptor (AF1 and AF2) recruit protein cofactors, allowing the transcription and expression of the target genes, resulting in increased cell division and tumor progression (Fig. 1). As an estrogen receptor antagonist, fulvestrant exhibits a high estrogen receptor binding affinity and produces a complete receptor blockade. Following binding of fulvestrant, dimerization of the estrogen receptor is impaired and the bound receptor is rapidly

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degraded, a process unique to fulvestrant amongst the ▶antiestrogen receptor agents. Nuclear localization is also disrupted, and both AF1 and AF2 are inactivated, leading to complete abrogation of estrogen signaling through the estrogen receptor (Fig. 1). This also means that fulvestrant has no estrogen agonist activity, which is important, since even partial agonist activity can lead to an increased incidence of endometrial abnormalities and cancer. As a steroidal analog of estradiol, fulvestrant is structurally distinct from the non-steroidal ▶tamoxifen, a selective estrogen receptor modulator, which is also used in the treatment of hormone receptor-positive breast cancer (Fig. 2). Although tamoxifen binds to the estrogen receptor, and permits dimerization and translocation, AF2 is not activated and so the transcription of estrogen-responsive genes is blocked (Fig. 1). However, this block is not complete, since AF1 continues to function, and therefore tamoxifen retains partial agonist activity, with the associated endometrial risks. Fulvestrant, with its unique mode of action, is fundamentally different from the third-generation ▶aromatase inhibitors such as anastrozole, letrozole or exemestane, which are used to treat breast cancer in an increasing proportion of postmenopausal women.

Fulvestrant. Figure 1 The mode of action of fulvestrant, tamoxifen and estradiol. (Reproduced from Dowsett et al. [2, Fig. 1]. Breast Cancer Res Treat 93:S11–S18 (2005). With kind permission of Springer Science and Business Media.)

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Fulvestrant. Figure 2 Chemical structure of fulvestrant.

Fulvestrant. Figure 3 Scheme showing how different endocrine therapies (fulvestrant, tamoxifen, aromatase inhibitors) work in breast cancer.

Following the menopause, most endogenous estrogen is produced via the conversion of adrenal androgens in peripheral tissues. Aromatase inhibitors work by blocking the aromatase enzyme, which catalyzes this conversion, thus reducing the levels of circulating estrogen available to bind to the estrogen receptor (Fig. 3). Current Utilization of Fulvestrant While treatment for hormone-sensitive early breast cancer aims to remove the tumor by surgical or radiological techniques followed by adjuvant ▶endocrine therapy, treatment for advanced disease is essentially palliative rather than curative, with the emphasis on extending life and preserving quality of life amongst patients. In postmenopausal women with hormone-sensitive advanced breast cancer, it is now standard practice to employ a sequence of endocrine agents, to slow the progression of the disease and delay for as long as possible the requirement for cytotoxic chemotherapy treatment. Consequently, novel endocrine agents that are both effective and lack crossresistance with existing therapies are required to extend the duration of the sequential treatment regimens. Fulvestrant is a new therapeutic option that can be added to the hormonal treatment sequence. Results from Phase III clinical trials showed that in postmenopausal

women with hormone-responsive advanced breast cancer who had progressed on previous antiestrogen therapy, fulvestrant was at least as effective as anastrozole, in terms of time to progression, objective response rates and survival. This evidence led to its regulatory approval and fulvestrant is currently licensed for use as a second-line endocrine treatment agent for advanced breast cancer after progression or recurrence on an antiestrogen. More recently, Phase III trial data have confirmed fulvestrant activity in the postaromatase inhibitor setting. Reflecting all these data, fulvestrant is considered in the National Comprehensive Cancer Network guidelines as an option after the failure of first-choice endocrine treatment (tamoxifen or an aromatase inhibitor). Thus, fulvestrant is a valuable addition to the endocrine armory. Importantly, due to its unique mechanism of action, analyses of patients progressing on fulvestrant have demonstrated continued sensitivity to subsequent endocrine therapies, indicating that fulvestrant lacks cross-reactivity with the ▶aromatase inhibitors and tamoxifen. Administration and Tolerability Instead of the daily oral dosing used with other endocrine therapies, fulvestrant is given as a monthly

Fumarase

250 mg/5 mL intramuscular injection, which provides slow release of the drug and sustained pharmacologic activity over the dosing interval (28 ± 3 days). The injection is well tolerated locally and may also help to assure treatment compliance. Once in the body, fulvestrant is predominantly bound to plasma proteins and metabolized by the liver, with negligible renal excretion, and it is not implicated in clinically significant drug–drug interactions, making it suitable for use in patients receiving polypharmacy for comorbid conditions. Fulvestrant is well tolerated, with most adverse events being mild to moderate in intensity. In clinical trials, the most commonly reported adverse events were nausea, asthenia and pain. Fulvestrant has potential tolerability benefits over some existing treatments, e.g. it is associated with less hot flashes than tamoxifen, and a lower incidence of joint disorders than anastrozole. Future Uses of Fulvestrant In recent years, the third-generation aromatase inhibitors, have been shown to be superior to tamoxifen for the treatment of both early and advanced breast cancer. Treatment guidelines currently recommend that an aromatase inhibitor should be used as either the primary endocrine therapy in postmenopausal women, or after 2–3 years of tamoxifen. However, even in this estrogen-deprived environment, some tumors will become resistant to treatment and begin to progress. Therefore, if cytotoxic chemotherapy is to be further delayed, an alternative endocrine therapy must be used. Indeed, as aromatase inhibitors continue to replace tamoxifen in the first-line setting, it is becoming increasingly important to identify agents that are effective after recurrence or progression on these drugs. As previously described, results from both Phase III and Phase II studies suggest that fulvestrant may be active after progression on aromatase inhibitors. Fulvestrant’s unique mode of action and lack of cross-reactivity also invites the possibility of potentially synergistic combinations with other treatment agents. Preclinical data suggest that the combination of fulvestrant with aromatase inhibitors will offer a more effective anti-tumor effect than either agent alone, and Phase III trials are underway to fully evaluate this treatment strategy. In addition, the epidermal growth factor (EGF) receptor-mediated pathway of gene transcription, which can provide an alternative growth stimulus for breast tumors in the absence of hormone receptors, has also been shown to cross-talk with the estrogen receptor-mediated pathway. This, in turn, has important implications for the development of resistance to endocrine therapy. As fulvestrant increases degradation

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of estrogen receptors, it may limit the potential for cross-talk with other pathways. Combination treatment with fulvestrant and an agent targeting growth factor receptors (such as gefitinib, lapatinib or trastuzumab) may therefore further limit cross-talk and potentially delay the time of onset of treatment resistance. Currently, several clinical trials are ongoing to investigate the activity of such combination therapy in patients with advanced breast cancer. Summary Fulvestrant, a steroidal analog of ▶estradiol, is an effective treatment for postmenopausal women with hormone-sensitive advanced breast cancer who have progressed on previous endocrine therapy. With its unique mode of action, fulvestrant provides a valuable addition to the endocrine treatment sequence, with significant benefits for patients. It is administered as a monthly intramuscular injection, and is well tolerated and associated with few adverse events. Fulvestrant may also have a potential use in combination treatment strategies, as the partner of choice with ▶EGF receptor inhibitors.

References 1. Howell A (2005) Fulvestrant (‘Faslodex’): current and future role in breast cancer management. Crit Rev Oncol Hematol 57:265–273 2. Dowsett M, Nicholson RI, Pietras RJ (2005) Biological characteristics of the pure antiestrogen fulvestrant: overcoming endocrine resistance. Breast Cancer Res Treat 93 (Suppl 1):S11–S18 3. Robertson JF, Osborne CK, Howell A et al. (2003) Fulvestrant versus anastrozole for the treatment of advanced breast carcinoma in postmenopausal women – a prospective combined analysis of two multicenter trials. Cancer 98:229–238 4. Chia S, Gradishar W, Mavriac L et al. (2008) Doubleblind, randomized, placebo-controlled trial of fulvestrant compared with exemestane after prior nonsteroidal aromatase inhibitor therapy in postmenopausal women with hormone receptor-positive, advanced breast cancer: Results from EFECT. J Clin Oncol Published online a head of print March 3, 2008 at http://jco.ascopubs.orglcgi/doi/ 10.1200/jco.2007.13.5822 5. Vergote I, Abram P (2006) Fulvestrant, a new treatment option for advanced breast cancer: tolerability versus existing agents. Ann Oncol 17:200–204

Fumarase ▶Fumarate Hydratase

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Fumarate Hydratase

Fumarate Hydratase S AKARI VANHARANTA , V IRPI L AUNONEN Department of Medical Genetics, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland

Synonyms FH; Fumarase

Definition Fumarate hydratase is an enzyme that functions in the mitochondrial citric acid cycle, catalyzing the reversible hydration/dehydration reaction in which fumarate is converted to malate.

Characteristics The human gene encoding fumarate hydratase is located in the chromosome segment 1q42.3-q43. It consists of ten exons that span over 20 kb of genomic DNA. The transcript is approximately 1.8 kb long and is predicted to encode a 510 amino acid polypeptide. The first exon of FH encodes a signal peptide that directs the protein to the mitochondrion. There the signal peptide is cleaved, and the remaining mature FH protein forms a functional homotetramer in the mitochondrial matrix. Some processed FH is also present in the cytosol, although the function of this cytosolic FH is unclear. In addition to the mitochondrial signal, the processed FH contains other domains such as alpha-helical and lyase domains. The alpha-helixes form a superhelical core for the tetramer. The functionally active FH enzyme converts fumarate to malate. This hydration reaction is a part of the citric acid cycle (also known as the tricarboxylic acid cycle or the Krebs cycle) which is an essential component of cellular carbohydrate metabolism. In the cytosol, fumarate is produced in the urea cycle and therefore FH is connected to protein metabolism as well. FH is well conserved, human FH sharing a 57% amino acid identity with the Escherichia Coli FumC protein, and it belongs to a protein superfamily which includes mostly other enzymes such as aspartase, adenylosuccinate lyase, and arginosuccinate lyase. The first clues as to the role of FH in human disease came from the identification of two siblings that presented with progressive encephalopathy, dystonia, ▶leucopenia, and ▶neutropenia. They had elevated levels of lactate in their cerebrospinal fluid and high fumarate excretion in their urine. A ▶homozygous mutation was discovered in a conserved region of the FH gene in both of these patients. Also, FH deficiency was shown to be present in all tissues studied in the patients, and their healthy parents were shown to carry

the mutation in a heterozygous form. Since then, about 20 families with FH deficiency have been reported in the literature. The symptoms are severe and the affected individuals usually die within a few months of birth. Evidence for yet another role of FH in human disease came from quite a different line of research. The genomic locus harboring the FH gene was independently mapped by genetic linkage analysis to segregate with inherited predisposition to ▶leiomyoma and ▶renal carcinoma; ▶Hereditary leiomyomatosis and renal cell cancer (HLRCC), and to ▶multiple cutaneous and uterine leiomyomatosis (MCUL). Soon after, these two conditions were shown to be variants of the same syndrome and the underlying gene was identified as FH. The tumors showed loss of heterozygosity (▶LOH) and retention of the mutated allele, therefore suggesting that the gene acted as a ▶tumor suppressor. Also, FH enzyme activity was shown to be reduced in the leukocytes and absent in the tumors of mutation carriers. Clinical Features of HLRCC/MCUL Since the first reports indicating the involvement of FH in tumorigenesis, more than 100 families with the HLRCC/MCUL phenotype have been reported in the literature (Fig. 1). Although no population-based studies have been carried out, it seems clear that the prevalence of HLRCC/MCUL is very low. There are reports of HLRCC/MCUL from all around the world, and there seem to be population differences in the phenotype. For example, HLRCC seems to be more common in Finland and North America, whereas in the UK, renal cell cancer is rarely detected in the families segregating heterozygous FH mutations. The most common manifestation of HLRCC/MCUL is cutaneous and/or ▶uterine leiomyomas. Early-onset renal cell cancer is significantly rarer and is typically of the papillary type II histology. Cutaneous leiomyomas are small benign tumors of the skin that show as multiple 0.5–2 cm skin-colored nodules, and their tissue of origin is thought to be the arrectores pili muscle of the hair follicle. They can manifest clinically as pain and paresthesias already in childhood, and the age of onset ranges from 10 to 50 in the HLRCC/MCUL families. Uterine leiomyomas (also known as ▶myomas or ▶fibroids) are smooth muscle cell tumors that arise within the smooth muscle lining of the uterus, the myometrium. They are some of the most common neoplastic tumors of women, and estimates of affected individuals range from 25% to up to 77% depending on the methods used for the diagnosis. Even though they are benign, they can cause severe morbidity such as aberrant bleeding, abdominal pain and even infertility. In families affected by HLRCC/MCUL, the onset of leiomyomas seems to be earlier than in the general

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F Fumarate Hydratase. Figure 1 Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant tumor predisposition syndrome caused by germline mutations in the fumarate hydratase gene. The most common features are cutaneous and/or uterine leiomyomas. Predisposition to renal cell carcinoma is present in a subset of families. Typically, HLRCC renal cell carcinomas display papillary type 2 histology.

population. In these families, leiomyomas are also more often symptomatic and therefore they more commonly result in hysterectomy (i.e. the surgical removal of the uterus). In addition to typical leiomyomas, some HLRCC/MCUL patients develop atypical leiomyomas. These are rare variants of leiomyomas which are sometimes hard to discern from uterine leiomyosarcomas. Whether HLRCC/MCUL predisposes to malignant leiomyosarcoma is still a somewhat open question, although some studies suggest that this might indeed be the case. Familial clustering of ▶renal cell cancer was the key finding in the identification of the syndrome HLRCC. In the first family reported, four patients aged 33–48 were identified. Since then, additional cases of renal cell cancer have been detected in some HLRCC/MCUL families, the median age of onset being around 40. The natural history of HLRCC/MCUL renal tumors is malignant with early metastasis often leading to the demise of the patient. In the early reports, all renal cell cancers related to HLRCC/MCUL displayed a distinctive papillary type II histology, although other types of renal tumors, such as collecting duct and clear-cell carcinoma, have been later associated with HLRCC/ MCUL as well. HLRCC/MCUL renal tumors are typically unilateral, which is in contrast to other inherited forms of renal cell cancer such as von ▶Hippel-Lindau Syndrome (VHL), Hereditary Papillary Renal Carcinoma, and ▶Birt-Hogg-Dubé Syndrome (BHD), in which tumors often affect both kidneys. FH-mutation carriers might be at risk of developing ▶Leydig cell tumors and ovarian cystadenomas. Incidental cases of other tumors, such as breast and prostate cancer and some hematological malignancies, have also been reported in HLRCC/MCUL families,

although it remains unclear whether any of these are true manifestations of the germline FH mutations. Mutations in the FH Gene The syndrome HLRCC/MCUL is transmitted in an ▶autosomal dominant manner, and germline FH mutations have been detected in 85% of all the families displaying the HLRCC/MCUL phenotype. Altogether, 60 different mutations have been identified. The vast majority (70%) are single base pair substitutions, of which ▶missense mutations comprise about 60%; the rest are non-sense mutations. Small deletions and insertions as well as ▶splice site changes have been reported. In addition, whole gene FH deletions have been detected in some families. Mutations occur throughout the gene. The mutation R58X has been detected in four families of diverse ethnic and geographical backgrounds in North America. ▶Haplotype analysis has suggested that the mutation has occurred independently in these families, indicating that this might represent a mutational hot spot. The same mutation has also been detected in families from the United Kingdom and Australia. Other mutations that have been detected in several families of different geographical backgrounds are, for example, N64T and R190H, and these may represent mutational hot spots as well. The mutations in families with the renal cell cancer phenotype do not differ from those seen in families without these malignant tumors and, in fact, the same mutations have been detected in families with either of the two phenotypes. This has raised the question of whether an additional genomic locus could act as a modifier together with FH mutations. A ▶founder effect has been detected at least in populations of the Finnish and Iranian origin. Two

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mutations, H153R and a 2-bp deletion in codon 181, have both been identified in three different families in Finland. Similarly, a splice site mutation IVS6-1G>A has been detected in a common haplotype in several families of Iranian origin. Most tumors arising in HLRCC/MCUL families have a second somatic inactivating hit in the FH gene. This is often acquired through the loss of the wild-type FH by a partial or whole genomic deletion of chromosome arm 1q. FH mutations are also rarely seen in sporadic tumors. Inactivation of the FH gene has been detected in three tumors from the Finnish population, one soft-tissue sarcoma of the lower limb, and two uterine leiomyomas, all showing loss of the wild-type FH. However, despite mutation screens comprising hundreds of tumor specimen, no other somatic changes in FH have been detected in various tumor types including prostate, breast, colorectal, lung, ovarian, thyroid, head and neck cancers, pheochromocytomas, gliomas, and melanomas. Therefore, it is safe to say that, in general, somatic inactivation of the FH gene is a very rare occurrence in human tumors. As determined by microarray based gene expression analysis, as well as by traditional immunohistochemical methods, ▶uterine fibroids carrying FH mutations have distinct biological properties which seem to require two hits in the FH gene. The molecular mechanisms through which mutations in FH lead to tumorigenesis are still far from being well understood. Some evidence suggests that the disruption of FH activity would lead to the stabilization of the ▶hypoxia inducible factor-1 (HIF1) under normoxic conditions, thus activating several growth-promoting signaling cascades. The mutations detected in the recessively inherited developmental disorder FH deficiency are also mostly missense mutations, and they occur throughout the FH gene. The mutational spectrum of FH deficiency does not seem to be different from that of HLRCC/MCUL and, indeed, a phenotype compatible with HLRCC/ MCUL has been reported in some of the parents of the children affected by FH deficiency.

References 1. Gottlieb E, Tomlinson IP (2005) Mitochondrial tumour suppressors: a genetic and biochemical update. Nat Rev Cancer 5:857–866 2. Kiuru M, Launonen V (2004) Hereditary leiomyomatosis and renal cell cancer (HLRCC). Curr Mol Med 4:869–875 3. Tomlinson IP, Alam NA, Rowan AJ et al. (2002) Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet 30:406–410 4. Vanharanta S, Pollard PJ, Lehtonen HJ et al. (2006) Distinct expression profile in fumarate-hydratase-deficient uterine fibroids. Hum Mol Genet 15:97–103

5. Wei MH, Toure O, Glenn GM et al. (2006) Novel mutations in FH and expansion of the spectrum of phenotypes expressed in families with hereditary leiomyomatosis and renal cell cancer. J Med Genet 43:18–27

Functional Foods ▶Nutraceuticals

Functional Vascular Stabilization ▶Vascular Stabilization

Fungus Definition Member of a class of relatively primitive vegetable organisms. Fungi include mushrooms, yeasts, rusts, molds, and smuts.

Funnel Factors G EMMA A RMENGOL , S ANTIAGO R AMON

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C AJAL

Department of Pathology, Vall d’Hebron University Hospital, Barcelona, Spain

Definition Is a molecule where several oncogenic signals converge and drive the proliferative signal downstream. This transformation-inducing signal inexorably passes and is canalized through the funnel factor. Funnel factors provide a clear reflection of the tumor’s transforming potential regardless of the triggering genetic alteration upstream. The level of expression of these factors should correlate with the degree of malignancy of the tumor and the most relevant clinical parameters, such as ▶metastasis and survival. Therefore, they may reflect the molecular information and transformation potential for each tumor.

Funnel Factors

Characteristics Background of Molecular Human Carcinogenesis Funnel factors are those final effectors that channel the malignant cellular growth signals, which are transduced through pathways or cascades that induce and mediate changes into the cell physiology. Several of these pathways or cascades are redundant, that is they can trigger a similar cellular effect. There are six acquired capabilities considered to be necessary for the malignant cellular growth: ▶self-sufficiency in growth signals, insensitivity to anti-growth signals, limitless replicative potential, resistance to ▶apoptosis, sustained ▶angiogenesis and, finally, the ability to infiltrate the surrounding tissue and metastasize. Each of these changes in cellular physiology can be brought about through dozens of signaling pathways or cascades, each implicating various genes or proteins. Many different oncogenic alterations may be involved in each biochemical route. This intricate molecular background and its biochemical consequences can help us to understand the great heterogeneity observed in tumors, with over 250 types of malignant human tumors with distinctive clinical and pathological characteristics and thousands of morphological and pathological tumor subtypes. So far, up to 300 mutated genes implicated in oncogenesis have been identified as human cancer genes. Many oncologists and pathologists ask whether all this information is really important for the management of individual cancer patients. The answer is unknown because only a few molecular targets have been identified in a small number of tumor types. For example, ▶amplification of ERBB2 is seen in 25–30% of ▶breast carcinomas, ▶EGFR mutations in less than 10% of ▶lung carcinomas, and ▶c-KIT in the rare ▶gastrointestinal stromal tumors; but in most carcinomas, there is no distinctive oncogenic target. In the near future, technological advances will allow us to study the complete genetic background, ▶mRNA profiling, and protein expression of individual tumors, and identify a myriad of genetic and biochemical alterations. But even then, attempts to inhibit or counteract single genetic alterations with the use of multiple specific agents would probably be chaotic. Nevertheless, dissection of the biochemical pathways is progressing. We now know which factors are the final growth signaling effectors that can control ▶transcription and protein synthesis. Then, it is logical to think that the level of expression of these final effectors, which channel the proliferation signal, can be associated with the real oncogenic role of a pathway in individual tumors. Cell Signaling in Human Tumors Among the acquired capabilities of tumor cells, a funnel factor has been described for self-sufficiency in growth signals. This essential oncogenic capability is one of the

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most extensively studied characteristics of tumor cells, and one that is constitutively activated in nearly all tumors. The process of converting extracellular signals into cellular responses, in this case cell growth and division, is called signal transduction. The growth signal transduction pathway is comprised of ▶growth factors, ▶growth factor receptors, factors transmitting the growth signal, and the final effector factors, some of which are located in the nucleus to activate ▶transcription factors and some in the ▶ribosomes to activate protein synthesis. The neoplastic cell, however, may be able to generate signals for survival or proliferation through various mechanisms without depending on exogenous signals. These mechanisms include alterations in the growth factors or receptors, or in the signaling pathways, themselves. Among the latter, the most highly recognized and important are the ▶RAS-▶RAF▶MAPK (ERK1/2) and ▶PI3K-▶AKT pathways, which regulate ▶mTOR. Specific molecular alterations are detected in these signaling cascades in the majority of tumors. Usually these are single alterations with an oncogenic impact, such as growth factor mutations or RAS mutations; other concomitant genetic alterations are not usually found in these biochemical pathways.

Searching for Funnel Factors: p-4E-BP1 In studies performed in various tumor types, the expression of key cell-signaling factors, including Her1 and Her2 growth factor receptors, as well as the RASRAF-MAPK and the PI3K-AKT-mTOR pathways were correlated with the associated clinico-pathological characteristics of these tumors. The downstream factors p70, S6, 4E-BP1, and EIF4E, which play a critical role in the control of protein synthesis, survival, and cell growth, were also analyzed. It was found that ▶phosphorylated ▶4E-BP1 (eukaryotic ▶translation initiation factor 4E binding protein 1) levels in breast, ovary, and prostate tumors were associated with malignant progression and an adverse prognosis, regardless of the upstream oncogenic alterations. Thus, p-4E-BP1 seems to act as a funnel factor for an essential oncogenic capability of tumor cells, self-sufficiency in growth signals, and could be a highly relevant molecular marker of malignant potential. The results showing that 4E-BP1 is associated with the prognosis in breast, ovary and prostate tumors, are supported by other data. In breast cancer, ▶phosphorylation of AKT, mTOR and 4E-BP1 has been associated with tumor development and progression; and in prostate cancer, one of the best biomarkers of the mTOR pathway is 4E-BP1, since overexpression of this factor has been highly associated with this type of tumor. Moreover, experimental studies have shown that 4E-BP1 is essential for cell transformation. Transfer of 4E-BP1 phosphorylation site mutants into breast carcinoma cells suppressed their tumorigenicity.

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4E-BP1 is a eukaryotic translation initiation factor 4E (EIF4E)-binding protein that plays a critical role in the control of protein synthesis, survival, and cell growth. During ▶cap-dependent translation, EIF4E binds to the mRNA ▶cap structure and promotes formation of the ▶translation initiation complex and ▶ribosome binding. When 4E-BP1 is active (non-phosphorylated 4E-BP1) it binds to EIF4E and impedes formation of the initiation complex; translation is then blocked, favoring apoptosis. However, when 4E-BP1 is phosphorylated, the affinity for EIF4E binding is reduced, EIF4E is released, and cap-dependent translation can initiate. 4E-BP1 has seven phosphorylation sites. It is likely that mTOR is the main phosphorylation pathway of 4E-BP1, although other ▶kinases may be implicated, such as ▶CDK1, ▶ATM, ▶PI3K-AKT, ▶ERK1/2, and perhaps other, still unidentified, kinases. Therefore, 4E-BP1 phosphorylation can be the consequence of many different oncogenic events occurring in several biochemical pathways, including amplification or mutation of growth factor receptors, loss of function or mutations in ▶PTEN, ▶ATM, ▶p53, ▶PI3K or ▶RAS, or other collateral mechanisms of cellular oncogenic activation (Fig. 1). Because of the elevated number

of genetic alterations that regulate 4E-BP1, we propose that the phosphorylated form of this protein can act as a “bottleneck” or funneling factor through which the transforming signals converge, channeling the oncogenic proliferative signal regardless of the upstream specific oncogenic alteration. The role of other 4E-BP ▶isoforms, such as 4E-BP2 and 4E-BP3, in human tumors is still unclear, and it is not known whether they can be activated in 4E-BP1negative tumors. Study of the EIF protein family will also be determinant when reliable antibodies allow us to analyze their expression in large series of tumors. Recent data have provided novel perspectives into the proliferative and oncogenic properties of EIF4E, since it has been shown to have an impact on nearly every stage of cell cycle progression. Earlier studies have shown that EIF4E levels are substantially elevated in several types of cancers. Funnel Factors in Other Oncogenic Pathways Extending the concept of funnel factor, there might be several funnel factors where the final biochemical effect converges for each of the oncogenic capabilities of tumor cells. For example, in the apoptosis pathways,

Funnel Factors. Figure 1 Schematic diagram showing how funneling factors channel the proliferation signal.

Furin

where the expression of certain proteins that inhibit apoptosis, such as survivin and livin, might be associated with resistance to apoptosis regardless of the activation of other antiapoptotic or proapoptotic genes that might be present. Study of the expression profiles of funnel factors from all the cell transformation pathways would allow us to obtain an individual functional-molecular signature for each tumor. This signature, combined with clinical and pathological data would help us to establish the malignant potential of each individual tumor and deduce its potential resistance to conventional chemotherapy and radiotherapy. Obviously, in addition to molecular characterization of tumors for prognostic purposes, it is necessary to study factors that might be potential therapeutic targets, currently one of the most promising areas in the field of cancer treatment. With this functional approach it seems worthwhile to investigate whether these funnel factors can be critical targets for cancer treatment.

References 1. Armengol G, Rojo F, Castellvi J et al. (2007) 4E-binding protein 1: a key molecular “funnel factor” in human cancer with clinical implications. Cancer Res 67:7551–7555 2. Avdulov S, Li S, Michalek V et al. (2004) Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells. Cancer Cell 5:553–563 3. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 4. Mamane Y, Petroulakis E, LeBacquer O et al. (2006) mTOR, translation initiation and cancer. Oncogene 25:6416–6422 5. Rojo F, Najera N, Lirola J et al. (2007) 4E-binding protein 1, a cell signaling hallmark in breast cancer that correlates with pathologic grade and prognosis. Clin Cancer Res 13:81–89

Furin R OBERT DAY 1 , A LEX Y. S TRONGIN 2 1

Department of Pharmacology, Institut de Pharmacologie, Faculté de Médecine, , Université de Sherbrooke, Sherbrooke, QC, Canada 2 Burnham Institute for Medical Research, La Jolla, CA, USA

Synonyms Furin; PACE; SPC1; PCSK3; Dibasic processing enzyme; Prohormone convertase; Paired basic amino acid cleaving enzyme

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Definition Furin (E.C. 3.4.21.75) is a highly specialized proteinase that cleaves the unique sequence motifs in a variety of proteins. Normally, following furin cleavage, the target protein is activated and, therefore, it can exhibit its biological activity. Because furin has been discovered first, currently it is the most studied enzyme of the proprotein convertase (PC) family of serine proteinases. Seven distinct proprotein convertases of this family (furin, PC2, PC1/3, PC4, PACE4, PC5/6, and PC7) have been identified in humans, some of which have ▶isoforms generated as the result of ▶alternative splicing. Structurally and functionally, furin resembles its evolutionary precursor: the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. The polypeptide sequence of the furin ▶catalytic domain is homologous to that of Bacillus subtilisin, an evolutionary precursor of PCs. Furin and related PCs are involved in the limited ▶endoproteolysis (▶protease activated receptor) of inactive precursor proteins which occurs at the sites marked by paired or multiple basic amino acids.

Characteristics A wide variety of proteins are initially synthesized as parts of higher molecular weight, but inactive, precursor proteins. Specific endoproteolytic processing of these ▶proproteins is required to generate the regulatory proteins in a mature and biologically active form. A large majority of these active proteins, including ▶matrix metalloproteinases, growth factors, and ▶adhesion molecules are essential in the processes of cellular transformation, acquisition of the tumorigenic phenotype, and metastases formation. The enzyme furin, which is encoded by the fur gene, was the first and can be considered the prototype of a mammalian subclass of subtilisin-like proteases. The localization of the gene immediately upstream from the FES ▶oncogene (V-FES feline sarcoma viral oncogene homolog) generated the name FUR (for FES upstream region). Furin is similar to other PCs in that it contains a signal peptide, a prodomain, a subtilisin-like catalytic domain, a middle P domain, a cysteine-rich region, a transmembrane anchor, and a cytoplasmic tail (Figs. 1 and 2). Furin and PCs are normally N-glycosylated ▶glycoproteins (▶glycosylation). Phosphorylation of the cytoplasmic tail is required for the trans-Golgi localization of furin which in vivo exists as di-, monoand non-phosphorylated forms. Propeptide cleavage is a prerequisite for the exit of furin molecules out of the ▶endoplasmic reticulum. The second cleavage in the propeptide occurs in the ▶trans-Golgi network, which is followed by the release of the propeptide bound to furin and the activation of furin. Furin is expressed in all examined tissues and cell lines and is mainly localized in the trans-Golgi

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Furin

Furin. Figure 1 A modular domain structure of furin and six related PCs. The A and B isoforms of PC5/6 are encoded by the same gene. The structure includes (i) the N-terminal signal peptide, which directs proteins into the secretory pathway, (ii) a pro-domain, which maintains the inactive zymogen state of PCs and which also acts as an intramolecular chaperone for the proper folding, (iii) a catalytic domain with the active site that exhibits an Asp (D)-His(H)-Ser(S) catalytic triad and an additional Asn(N), (iv) a barrel-like structured P domain that regulates enzyme stability, (v) a C-terminal domain that contains membrane attachment sequences, a Cys-rich region and intracellular sorting signals. Adapted from [4].

network. Some proportion of the furin molecules cycles between the trans-Golgi and the cell surface. Furin represents the ubiquitous endoprotease activity within constitutive secretory pathways and normally it is capable of cleaving the Arg-X-(Lys/Arg)-Arg consensus motif, where X is any amino acid type (Table 1). Furin and related PCs are activating proteases and normally they do not inactivate polypeptides. Because of the overlapping substrate preferences and cell/tissue expression, there is a substantive level of redundancy in the PC functionality, albeit certain distinct functions of the individual PCs have also been demonstrated. Furin knockout, however, is lethal in mice. Furin null embryos die because they fail to accomplish ventral closure successfully and to form a looping heart tube. These processes require cellular migration and proliferation, both of which are regulated by furin. Through regulation of cellular migration and proliferation, furin plays an important, albeit incompletely understood role, in cellular transformation, acquisition of the tumorigenic phenotype, cancer progression and metastasis. The expression of furin, however, discriminates sharply between small cell lung cancers, which have no expression, and non-small cell lung cancers, in which furin is overexpressed.

The roles of furin and other PCs in cancer have been characterized as the result of many studies of gene expression and enzyme inhibition. Because of the redundancy, it is not always clear if all PCs present in cancer cell/tissue are directly relevant to tumorigenicity. An enhanced expression of furin and related PCs in cancer is not necessarily an indicator of a poor clinical outcome. There is, however, evidence that high levels of furin-related PCs contribute to tumor growth and metastasis by controlling the activation of key cancer-associated proteins, including matrix metalloproteinases and growth factors such as ▶VEGF, ▶TGFβ and ▶PDGF. The multiple effects of PCs on cell proliferation, motility, adhesion and invasion have led to a concept that in the course of tumor development and progression PCs act as “master switches” of the key tumorigenic protein functionality. If this concept is valid, then PCs could be identified as important therapeutic targets in a number of cancer types. The challenge remains to identify the functionally-relevant, target PC in each cancer type, because it is unlikely that broad-range PC inhibitors would have significant clinically beneficial effects. No natural protein inhibitors of furin are known. D-Arg-based peptides, α1-anti-trypsin Portland and,

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Furin. Figure 2 Furin structure. The 794-residue pre-profurin sequence contains a 24-residue signal peptide, an 83-residue prodomain, a 330-residue subtilisin-like catalytic domain, a 140-residue middle P domain (also termed the “homo B,” a 115-residue cysteine-rich region, a 23-residue transmembrane anchor, and a 56-residue cytoplasmic tail. Adapted from [1].

especially the synthetic peptidic inhibitor decanoylArg-Val-Lys-Arg-chloromethylketone, are used to inhibit furin in the cleavage reactions in vitro and in cell-based tests. Inhibition of furin results in a significant reduction in tumor cell invasion. This reduction appears to be associated with a processing blockade of proteins directly involved in the mechanism of invasion including matrix metalloproteinases, growth factors and adhesion signaling receptors such as integrins. PCs including furin are implicated in many pathogenic states because they process to maturity membrane fusion proteins and pro-toxins of a wide variety of both naturally occurring and weaponized bacteria and viruses, including anthrax and botulinum toxins and H5N1 bird flu, Marburg and Ebola viruses. After processing by furin and the subsequent internalization inthe complex with the respective receptor followed by acidification of the ▶endosomal compartment (▶endocytosis), the processed, partially

denatured, infectious proteins expose their membranepenetrating peptide region and escape into the cytoplasm. The intact toxins and viral proteins, however, are incapable of accomplishing these processes. Normally, the low pathogenicity viral subtypes have mutations in the cleavage site sequence and thus a reduced sensitivity to furin. Accordingly, proteolytic processing by furin is an important determinant in the overall pathogenicity of viruses and bacterial toxins. Based on these data, PCs, including furin, are promising targets for drug design in a variety of acute and chronic diseases including cancer and infectious diseases.

References 1. Molloy SS, Anderson ED, Jean F et al. (1999) Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9:28–35

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Furin. Table 1

Furin targets and the sequence of the cleavage sites

Proteinases

P6 P4 P1↓P1′

FURIN

Furin, two autolytic cleavage sites

MMP-11 MMP-14 MMP-15 MMP-16 MMP-17 MMP-24 MMP-25 ADAM-9 ADAM-12 ADAM-19 ADAMTS1

Matrix metalloproteinase 11, stromelysin-3 Matrix metalloproteinase 14, MT1-MMP Matrix metalloproteinase 15, MT2-MMP Matrix metalloproteinase 16, MT3-MMP Matrix metalloproteinase 17, MT4-MMP Matrix metalloproteinase 24, MT5-MMP Matrix metalloproteinase 25, MT6-MMP A desintegrin and metallopeptidase domain 9 A desintegrin and metallopeptidase domain 12 A desintegrin and metallopeptidase domain 19 A desintegrin and metalloproteinase with thrombospondin type-1 motif, 1 A desintegrin and metalloproteinase with thrombospondin type-1 motif (aggrecanase-1), 4 A desintegrin and metalloproteinase with thrombospondin type-1 motif. 13 Bone morphogenetic protein 1 Bone morphogenetic protein 4 Meprin A alpha Beta-site APP-cleaving enzyme 1

ADAMTS4 ADAMTS13 BMP1 BMP4 Meprin-A BACE1 Serum proteins

Albumin VWF von Willebrand factor F9 Coagulation factor IX PROC Protein C Extracellular matrix FBN1 Fibrillin 1 ZPC3 Zona pellucida glycoprotein 3 Chaperone 7B2 Secretogranin V Receptors ITGA3 Integrin alpha chain, alpha 3 ITGA6 Integrin alpha chain, alpha 6 LRP1 Low density lipoprotein-related protein 1 NOTCH1 Notch1 INSR Insulin receptor DSG3 Desmoglein 3 MET Hepatocyte growth factor receptor c-met CUBN Cubilin/vitamin B-12 receptor SORL1 Sortilin-related receptor HGFR Hepatocyte growth factor/scatter factor receptor Growth factors and hormones IGF-1a Insulin-like growth factor 1a/somatomedin C NTF3 Neurotrophin 3 VEGFC Vascular endothelial growth factor C NPPB Natriuretic peptide B PTH Parathyroid hormone

RGVTKR↓SLSP KRRTKR↓DVYQ RNRQKR↓FVLS NVRRKR↓YAIQ RRRRKR↓YALT HIRRKR↓YALT QARRRR↓QAPA RRRNKR↓YALT VRRRRR↓YALS LLRRRR↓AVLP ARRHKR↓ETLK PRRMKR↓EDLN SIRKKR↓FVSS

75–76 107–108 97–98 111–112 131–132 119–120 125–126 155–156 107–108 205–206 207–208 105–206 252–253

PRRAKR↓FASL

212–213

RQRQRR↓AAGG 74–75 RSRSRR↓AATS RRRAKR↓SPKH PSRQKR↓SVEN GLRLPR↓ETDE

120–121 292–293 653–654 45–46

RGVFRR↓DAHK SHRSKR↓SLSC LNRPKR↓YNSG RSHLKR↓DTED

24–25 763–764 46–47 199–200

RGRKRR↓STNE ASRNRR↓HVTE

2731–2732 301–302

QRRKRR↓SVNP

181–182

PQRRRR↓QLDP NSRKKR↓EITE SNRHRR↓QIDR GGRRRR↓ELDP PSRKRR↓SLGD KRRQKR↓EWVK EKRKKR↓STKK LQRQKR↓SINL PLRRKR↓SAAL EKRKKR↓STKK

875–876 902–903 3943–3944 1665–1666 762–763 49–50 307–308 35–36 81–82 307–308

PAKSAR↓SVRA TSRRKR↓YAEH HSIIRR↓SLPA TLRAPR↓SPKM KSVKKR↓SVSE

119–120 138–139 227–228 102–103 31–32

Fusion Genes Furin. Table 1

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Furin targets and the sequence of the cleavage sites (Continued)

Proteinases

P6 P4 P1↓P1′

TGFB1 TNFSF12– TNFSF13 EDA-A2 NGFB

Transforming growth factor, beta 1 Tumor necrosis factor (ligand) member 12-member 13/proliferationinducing ligand APRIL Ectodysplasin a isoform β-Nerve growth factor Semaphorin 3A Viral envelope glycoproteins HO Hemagglutinin type H5 F Newcastle disease virus F fusion protein F Parainfluenza HPIV3 F fusion protein P130 Sindbis virus structural polyprotein p130 prM Flaviviral prM protein

SSRHRR↓ALDT RSRKRR↓AVLT

278–279 104–105

VRRNKR↓SKSN THRSKR↓SSSH KRRTRR↓QDIR

159–160 179–180 555–556

RRRKKR↓GLFG GRRQKR↓LIGA DPRTKR↓FFGG SGRSKR↓SVID SRRSRR↓SLTV

THRTKR↓STDG VQREKR↓AVGL SRRHKR↓FAGV TRRFRR↓SITE YFRRKR↓SILW GRRTRR↓EAIV LRRRRR↓DAGN

344–345 116–117 109–111 328–329 215–216, West Nile Virus 205–206, Dengue virus 460–461 498–499 115–116 537–538 435–436 501–502 432–433

RHRQPR↓GWEQ NSRKKR↓STSA KRRGKR↓SVDS GNRVRR↓SVGS KVRRAR↓SVDG ASRVAR↓MASD

304–305 196–197 398–399 218–219 455–456 273–274

HRREKR↓SVAL UL55 Cytomegalovirus/herpesvirus 5 protein UL55/glycoprotein B gp160 HIV-1 glycoprotein-160 Fo Measles virus fusion protein E2 Infectious bronchitis spike protein GP Marburg virus spike glycoprotein env Ebola envelope glycoprotein BALF4/GP110 Epstein-Barr virus/herpesvirus 4 Bacterial endotoxins ExoA Pseudomonas aeruginosa exotoxin A PA83 Anthrax protective antigen α-Toxin Clostridium alpha-toxin DT Diphtheria toxin Aerolysin Aeromonas aerolysin Shiga toxin Shigella shiga toxin I subunit A

2. Khatib AM, Siegfried G, Chretien M et al. (2002) Proprotein convertases in tumor progression and malignancy: novel targets in cancer therapy. Am J Pathol 160:1921–1935 3. Bassi DE, Fu J, Lopez de Cicco R et al. (2005) Proprotein convertases: ‘‘master switches’’ in the regulation of tumor growth and progression. Mol Carcinog 44:151–161 4. Thomas G (2002) Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 3:753–766 5. Fugere M, Day R (2005) Cutting back on pro-protein convertases: the latest approaches to pharmacological inhibition. Trends Pharmacol Sci 26:294–301

Fusin ▶Chemokine Receptor CXCR4

Fusion Genes PATRIZIA G ASPARINI Molecular-Cytogenetic Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Milano, Italy

Synonyms Fusion proteins; fusion oncogenes; chimeric genes; chimeric oncogenes; chimeric transcripts; hybrid genes

Definition A hybrid gene created by joining portions of two different genes (to produce a new protein) or by joining a gene to a different promoter (to alter or deregulate a gene transcription).

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Fusion Genes

Characteristics A wide variety of recurrent molecular alterations has been associated with cancer including ▶polymorphisms, changes in gene copy number (amplifications and deletions), point mutations, ▶epigenetic modifications and gene fusions due to structural ▶chromosomal rearrangements, such as translocations and less frequently inversions. As for these last ones, the genes located at the breakpoints of the rearrangement may be structurally changed with dramatic effects on their products. Molecularly, two events of structural aberrations

can be generated: “promoter swapping” (the exchange of promoter control regions) or fusion gene. In detail (Fig. 1a) “promoter swapping” occurs when the regulatory elements of a gene (promoter and/or enhancer) become aberrantly juxtaposed to a protooncogene, thus driving deregulated expression of an oncogene. Molecularly, the breakpoints of the rearrangements occur upstream from the coding region of the partner gene resulting in two chimeric genes which have exchanged their promoter regions, and less frequently noncoding exons. At the genomic level, the

Fusion Genes. Figure 1 A schematic representation of two events of stuctural aberations: (a) Promoter swapping and (b) Fusion genes and Fusion proteins.

Fusion Genes

3′ partner gene B is placed downstream of the 5′ gene A promoter region. The chimeric transcript contains 5′ ▶untranslated regions (UTR) from the A gene and a coding region B that is intact and encodes a normal protein B. This mechanism can be exemplified by the three translocations that characterize Burkitt lymphoma: t(8;14), t(8;22), and t(2;8). All these rearrangements lead to the activation of MYC, located on 8q24, by juxtaposing the coding sequences of the gene to one constitutively active immunoglobulin (Ig) genes promoter or regulatory regions (IgH at 14q32, IgK at 2p12, and IgL at 22q11). Fusion genes (Fig. 1b) arise when the coding regions of the two genes are juxtaposed, resulting in a chimeric transcript that produces a fusion protein with a new altered activity. In detail, in the majority of cases, fusion genes are formed when DNA breaks occur within two different genes mainly within the introns, A and B, and the gene fragments are joined in erroneous combinations. In most cases, the results are two fusion genes: A-B and B-A. On genomic level, the 3′partner gene B is placed under the 5′ gene A promoter control region which dominates the transcription control of the fusion gene. As a result, in the fusion protein the functional domains from the A and B proteins are brought together in a new abnormal combination. In cancer, the genes that are often interrupted by a chromosomal rearrangement are oncogenes , thus harboring fusion oncogenes. An appropriate example of a fusion oncogene is ▶BRC/ ABL characterizing chronic myelogenous leukemia which is driven by t(9;22)(q34;q11), also known as the ▶Philadelphia chromosome, the first translocation to be molecularly characterized. In particular, the translocation fuses the ABL gene normally located on 9q34, with the BCR gene at 22q11. The BCR/ABL fusion created on the derivative chromosome 22 encodes a chimeric protein with an increased ▶tyrosine kinase activity and abnormal localization. Table 1 enlists molecularly characterized recurrent chromosomal rearrangements found in cancers. Formation Of Fusion Genes Several factors influence the formation of fusion oncogenes and their role in tumorigenesis. Firstly, the rate at which fusion genes are formed is important. Literature suggests that at least some fusion genes are found in healthy individuals, implying that at least some gene fusions emerge at a notable rate. The mechanisms behind fusions are unknown but the occurrence of several double strand breaks that coincide in time and space are important. The proximity of damaged partner genes at the moments of repair is critical and the localization of chromatin and genomic regions in the interphase nuclei may be critical. Secondly, the presence of a fusion gene in a cell is not enough to cause cancer. Additional genetic or epigenetic changes are

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also needed and the risk for these additional events to occur affects the outcome. Thirdly, once the fusion is formed, its penetrance, i.e. the proportion of fusion carriers that develop tumors, is determined by selected mechanisms. Interestingly, many fusion oncogenes demonstrate a strict specificity for tumor type. The risk of getting a certain translocation could depend on cell type-specific processes that make the specific genes or DNA regions involved vulnerable to the translocation. It is clear that tumor development in different cell types and tissues locations involves many pathways, distinct genes, and also exogenous factors. A common mechanism for early genetic changes can however be distinguished in a number of different tumor types by specific chromosome rearrangements. Moreover, the transcriptional orientation of fusion partner genes is essential in order to harbor functional fusion genes. At times, the partner genes are not oriented in the correct direction with regards to their transcriptional orientation, and more complex rearrangements are needed to fuse the partner genes into functional fusion genes. For instance, the EWS-ERG fusion is found in about 10% of Ewing sarcomas and it is the result of a complex rearrangement, a ▶translocation and an ▶inversion, given that the genes involved are not transcribed in the same centromeric/telomeric direction. This requirement and the necessary presence of critical functional protein parts seem to influence how frequently variant fusion genes are present in tumors. Moreover, to produce a functional fusion gene is necessary that the exons flanking the breakpoints can give rise to splicing events that maintain their reading frames. Overall, the factors that generate double-strand breaks are largely unknown. Clinical Relevance Studies over the past decades have revealed that recurring chromosome rearrangements leading to fusion oncogenes are specific features not only of leukemias and lymphomas, but also of certain epithelial tumors. Presently, over 600 recurrent balanced tumor-associated chromosomal rearrangements have been molecularly characterized. However, the data are strongly biased in favor of hematologic malignancies and sarcomas. An important example of a recurrent rearrangement which leads to the development of a targeted therapy is the t(15;17)(q22;q21) in ▶acute promyelocytic leukemia which fuses the ▶PML gene (15q22) with RARα gene at 17q21. The PML protein contains a zinc-binding domain called a “ring” finger that may be involved in protein– protein interaction. RARα protein encodes the retinoic acid alpha-receptor protein (▶retinoic acid receptors a member of the nuclear steroid/thyroid hormone receptor superfamily. Although retinoic acid binding is retained in the fusion protein, the PML/RARα may confer altered DNA-binding specificity to the RARα ligand

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Fusion Genes. Table 1

Molecularly characterized recurrent chromosome rearrangements and fusion genes in cancer

Disease Hematopoietic tumor Lymphoid ▶Anaplastic Large Cell Lymphoma

Affected gene

NPM-ALK TPM3-ALK TFG-ALK ATIC-ALK MSN-ALK CLTCL-ALK ▶Burkitt Lymphoma, B-cell acute lymphoid leukemia MYC (relocation of IgH locus) MYC (relocation of IgK locus) MYC (relocation of IgL locus) B-cell precursor ALL E2A-PBX1 E2A-HLF TEL-AML1 BCR-ABL MLL-AF4 IL£-IgH ▶Diffuse large B-cell lymphoma BCL2-IgH BCL6- variant partners BCL8-IgH FCGR2-Igλ MUC1-IgH NFKB2-IgH Extranodal mucosa-associated MALT1-API2 lymphoid tissue MALT1-IgH BCL10-IgH BCL10-Igκ Plasma cells myeloma FGFR3-IgH and MMSET MAF-IgH MAF-Igλ CCND1-IgH MUM/IRF4-IgH Pre-T cell lymphoblastic leukemia, lymphoma MYC (Relocation to TCR α/δ locus) LYL1 (Relocation to TCRα/σlocus) TAL2 (Relocation TCRβ locus) SCL (Relocation to TCR α/δ locus) OLIG2 (Relocation to TCR α/δ) LMO1(RBTN1) (Relocation to TCR α/δ) LMO2 (RBTN2) (Relocation to TCR α/δ) HOX11 (Relocation to TCR α/δ) HOX1-1L2 CALM-AF10 NUP98-RAP1GDS1 Myeloid ▶Acute promyelocytic leukemia PML-RARα NPM-RARα PLZF-RARα

Rerrangement

t(2;5)(q23;q35) t(1;2)(q25;p23) t(2;3)(p23;q21) inv(2)(p23q35) t(X;2)(q11-12;p23) t(2;17)(p23;q23) t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11) t(1;19)(q23;p13) t(17;19)(q22;p13) t(12;21)(p12;q22) t(9;22)(q34;q11.2) t(4;11)(q21;q23) t(5;14)(q31;q32) t(14;18)(q32;q21) t(3;v)(q27;v) t(14;15)(q32;q11-13) t(1;22)(q22;q11) t(1;14)(q21;q32) t(10;14)(q24;q32) t(11;18)(q21;q21) t(14;18)(q32;q21) t(1;14)(p22;q32) t(1;2)(p22;p12) t(4;14)(p16;q32) t(14;16)(q32;q23) t(16;22)(q23;q11) t(11;14)(q13;q32) t(6;14)(p25;q32) t(8;14)(q24;q11) t(7;19)(q35;p13) t(1;14)(p32;q11) t(14;21)(q11;q22) t(11;14)(p15;q11) t(11;14)(p13;q11) t(10;14)(q24;q11) t(5;14)(q35;q32) t(10;11)(p13;q21) t(4;11)(q21;p15) t(15;17)(q21;q21) t(5;17)(q35;q21) t(11;17)(q23;q21)

Fusion Genes Fusion Genes. Table 1 (Continued)

1185

Molecularly characterized recurrent chromosome rearrangements and fusion genes in cancer

Disease

▶Acute myeloid leukemia Acute myeloid leukemia

Solid tumors Sarcomas Alveolar rhabdomyosarcoma

▶Alveolar soft-part sarcoma Angiomatoid fibrous histiocytoma Dermatofibrosarcoma protubeans Desmoplastic small round cell tumor Endometrial stromal sarcoma ▶Ewing sarcoma

Infantile fibrosarcoma Inflammatory myofibroblastic tumour

Low grade fibromyxoid sarcoma Myxoid chondrosarcoma

▶Myxoid liposarcoma ▶Synovial sarcoma

Soft-tissue clear cell sarcoma

Affected gene

Rerrangement

ETV6- variant partners NUP98-variant partners MLL-variant partners AML1-ETO CBFB-MYH11 FUS-ERG CEV14-PDGFRB P300-MOZ MOZ-TIF2 MOZ-CBP DEK-NUP214 RBM15-MKL MLF1-NPM1 AML1-EVI1

t(12;v)(p13;v) t(11;v)(p13;v) t(11;v)(q23;v) t(8;21)(q22;q22) inv(16)(p13q22) t(16;21)(p11;q22) t(5;14)(q33;q32) t(8;22)(q33;q32) inv(8)(p11q13)

PAX3-FKHR PAX7-FKHR TFE3-ASPL FUS-ATF1

t(2;13)(q3?;q14) t(1;13)(q36;q14) t(X;17)(p11;q25) t(12;16)(q13;p11)

COL1A1-PDGFB

t(17;22)(q13;q13)

EWS-WT1

t(11;22)(p13;q12)

JAZF1-JJAZ1 EWS-FLI EWS-ERG EWS-ETV1 EWS-E1AF EWS-FEV FUS-ERG ETV-NTRK3 TPM3-ALK TPM4-ALK CLTC-ALK FUS-CREB312 EWS-CHN TAF2N-CHN TCF12-CHN FUS-CHOP EWS-CHOP SYT-SSX1 SYT-SSX2 SYT-SSX4 EWS-ATF1

t(7;17)(p15;q21) t(11;22)(q24;q12) t(21;22)(q22;q12) t(7;22)(q22;q12) t(2;22)(q33;q12) t(17;22)(q12;q12) t(16;21)(p11;q22) t(12;15)(p13;q25) t(1;2)(q22;p23) t(2;19)(p23;p13) t(2;17)(p23;q23) t(7;16)(q33;p11) t(9;22)(q22;q12) t(9;17)(q22;q11) t(9;15)(q22;q21) t(12;16)(q13;p11) t(12;22)(q13;q12) t(X;18)(p11;q11)

t(6;9)(p23;q34) t(1;22)(p13;q13) t(3;5)(q25;q34) t(3;21)(q26;q22)

t(12;22)(q13;q13)

F

1186

Fusion Genes

Fusion Genes. Table 1 Molecularly characterized recurrent chromosome rearrangements and fusion genes in cancer (Continued) Disease Carcinomas ▶Follicular thyroid carcinoma ▶Papillary thyroid carcinoma

▶Prostate cancer ▶Renal-cell carcinoma

▶Salivary gland tumors (malignant) Secretory breast carcinoma ▶Non-small cell lung carcinoma

Affected gene PAX8-PPARγ H4-RET (PTC1) RIa-RET (PTC2) ELE1-RET (PTC3,4) RFG5-RET (PTC5) TPM3-NTRK1 (TRK) TPR-NTRK1 (TRK-T1) TFG-NTRK1 (TRK-T3) TMPRSS2-ERG TMPRSS2-ETV1 TMPRSS2-ETV4 PRCC-TFE3 ASPSCR1-TFE3 SFPQ-TFE3 NONO-TFE3 CTNNB1- PLAG1 TORC1-MAML2 ETV6-NTKR3 EMLH-ALK

complex. Leukemia patients with the PML/RARα gene fusion have an excellent response to the all-trans retinoic acid treatment, which stimulates the differentiation of promyelocytic leukemia cells. Similarly, the molecular characterization of the t(9;22)(q34;q11) in chronic myelogenous leukemia, which generates the fusion oncoprotein BCR/ABL, lead to the development of a successful targeted treatment of imatinib. In contrast to hematological neoplasia, our knowledge regarding fusion genes in solid tumors is very limited, due to the complexity and poor quality of their ▶cytogenetic karyotypes, yet they constitute only the 10% of known recurrent balanced chromosome rearrangements. However, fusion oncogenes may be more common in epithelial tumors than previously thought. Usually, translocations in solid tumors result in gene fusions that encode chimeric oncoproteins. The first chromosome abnormalities to be molecularly characterized in solid tumors were an inv(10)(q11.2; q21.2), as the more frequent alteration, and a t(10;17) (q11.2;q23), in ▶papillary thyroid carcinoma. These two abnormalities represent the cytogenetic mechanism which activate the proto-oncogene ▶RET on chromosome 10, by generating the fusion genes forming the oncogene RET/PTC1 and RET/PTC2, respectively. Moreover, other chromosomal rearrangements leading

Rerrangement t(2;3)(q13;p25) inv(10)(q11.2;q21) t(10;17)(q11.2;q23) inv(10)(q11q22) inv(1)(q21q22) inv(1)(q21q25) t(1;3)(q21;q11) inv(21)(q22.2;q22.3) t(7;21)(p21.2;q22.3) t(17;21)(q21;q22.3) t(X;1)(p11;q21) t(X;17)(p11;q25) t(X;1)(p11;p34) inv(X)(p11;q12) t(3;8)(p21;q12) t(11;19)(q21;p13) t(12;15)(p13;q25) inv(2)(p21;p23)

to RET activation were recently described and listed in Table 1. A great impact in the study of solid tumors is foreseen by the recent identification of a large subset of ▶prostate cancer harboring ▶TMPRSS2/ERG, fusions, TMPRSS2/ETV1 and TMPRSS2/ETV4, generated by inv(21)(q22.2;q22.3), t(7;21)(p21.2;q22.3) and t(17;21)(q21;q22.3) respectively. In particular, the gene fusion of the 5′ UTR of TMPRSS2 (a prostatespecific gene) to ERG or ETV1 (genes of the ▶ETS family), was identified in the majority of prostate cancer. Although the clinical significance of those fusions is unknown, recent investigations indicate that the expression of TMPRSS2/ERG among prostate cancer patients is a strong prognostic factor for disease progression. Although fusion proteins play an important role in oncogenesis, additional genetic alterations are essential in order to transform cells. Silencing the specific fusion genes that play fundamental roles for the corresponding tumor, blocking targets of fusion proteins and repressing the cooperating events are all promising therapeutic strategies that need to be further investigated. The detection of the intracellular targets of these fusions will harbor new and important insights into molecular pathways that underlie tumor development. Ultimately, a combination of these approaches with

FZD

conventional treatments may provide a powerful new approach to treat these fusion-positive tumors.

1187

FVC

References

Definition

1. Aman P (2005) Fusion oncogenes in tumor development. Semin Cancer Biol 15:236–243 2. Kuman-Sinha C, Tomlins SA, Chinnayan AM (2006) Evidence of recurrent gene fusions in common epithelial tumors. Trends Mol Med 12:529–536 3. Pierotti MA, Frattini M, Sozzi G et al. (2006) Oncogenes. In: Kufe DW, Bast RC, Hait WN, Hong DK, Pollock RE, Weichselbanum RR, Holland JF, Frej E (eds) Cancer Medicine. American Association for Cancer Research, London, pp 68–84 4. Xia SH, Barr FG (2005) Chromosome translocations in sarcomas and the emergence of oncogenic transcription factors. Eur J C Cancer 41:2513–2527 5. Vega F, Medeiros JL (2003) Chromosomal translocations involved in non-Hodgkin lymphomas. Arch Pathol Lab Med 127:1148–1160

Forced vital capacity. The volume of air that can be forcibly exhaled following maximal inspiration.

Fusion Oncogenes

▶Chronic Obstructive Pulmonary Disease and Lung Cancer

F FX Definition Human homologue of GDP-4-keto-6deoxymannose-3, 5-epimerase-4-reductase. This enzyme is rate-limiting in the GDP-fucose synthetic pathway. ▶Fucosylation

▶Fusion Genes

FZD Fusion Proteins ▶Fusion Genes

Definition Frizzled; seven-pass transmembrane Wnt receptors closely related to G protein-coupled receptors. ▶Wnt Signaling

G

Definition

They transduce an extracellular signal (through ligand binding) into an intracellular signal (G protein activation) and are involved in a wide variety of stimulusresponse pathways, from intercellular communication to physiological senses.

▶Myc Oncogene ▶Retinoblastoma Protein, Cellular Biochemistry

▶Endothelins ▶Protease Activated Receptor Family ▶Adrenomedullin ▶G-Proteins ▶Receptor Cross-Talk ▶Protease Activated Receptor ▶Chemoattraction ▶RHO Family Proteins

G1/S Transition Of the ▶cell cycle is when the cellular decision to start duplicating its DNA is initiated. Mediators regulating this process include ▶cyclins, ▶cyclin-dependent kinases (CDKs), and CDK-inhibitors together with the transcription factors ▶E2F and ▶pRb that control the ▶restriction point after which DNA replication is initiated.

G Antigen ▶GAGE Proteins

G2 Checkpoint Abrogation

G-Proteins T HOMAS WORZFELD, S TEFAN O FFERMANNS Institute of Pharmacology, University of Heidelberg, Heidelberg, Germany

Synonyms Heterotrimeric GTP-binding proteins; Heterotrimeric guanine nucleotide-binding proteins

Definition

Definition

A concept of anti-cancer therapy that relies on the inhibition of the G2 DNA damage ▶checkpoint by small molecules in the presence of ▶DNA damage. G2 checkpoint abrogation results in mitotic cell death.

G-proteins are named for their ability to bind and hydrolyze the guanine nucleotide ▶GTP. In the widest sense, the superfamily of guanine nucleotide-binding proteins comprises two structurally distinct classes, the monomeric GTP-binding proteins (also called ▶monomeric GTPases) which are involved in a variety of cellular processes, and the heterotrimeric GTP-binding proteins which are primarily involved in transmembrane ▶signal transduction by coupling membraneous receptors to various effector molecules. Traditionally, the term “G-protein” is only applied to the latter group, the heterotrimeric GTP-binding proteins.

▶UCN-01 Anticancer Drug

G-protein Couple Receptor Definition

Characteristics

GPCR; seven transmembrane receptor, 7TM receptor, is the largest protein family of transmembrane receptors.

Cellular functions in a living organism are regulated and coordinated by a huge variety of extracellular

1190

G-Proteins

signals including ▶hormones growth factors, paracrine factors, neurotransmitters, or sensory stimuli. Many of these signals are received by cells through receptors on the plasma membrane which convey the incoming information by coupling to G-proteins which are attached to the inner side of the plasma membrane. More than 1,000 genes coding for G-protein-coupled receptors (▶GPCR) have been identified making GPCRs one of the largest gene families of the mammalian genome. G-proteins activated through GPCRs regulate various effectors like enzymes and ion channels which produce intracellular signals resulting in specific cellular responses. G-protein functions are highly diverse due to their composition of different α-, β-, and γ-subunits, each of which are products of different genes. The α-subunit of the heterotrimeric Gprotein possesses structural and functional homologies to other members of the guanine nucleotide-binding protein superfamily. The β- and γ-subunits of heterotrimeric G-proteins form an undissociable complex and represent a functional unit. Some G-proteins are very specialized like those expressed only in sensory cells; others appear to have functions in a wide variety of cells and tissues. Many G-proteins seem to have overlapping distributions and functions indicating that complex functional relationships exist among different Gproteins.

Molecular and Cellular Regulation In order to convey a signal from an activated receptor to an effector, the heterotrimeric G-protein undergoes an activation–inactivation cycle which allows the G-protein to function as a regulatable molecular switch (Fig. 1). In the basal state, the βγ-complex as well as the GDP-bound α-subunit is associated. In this form, the G-protein can be recognized by an appropriate activated receptor which interacts with the G-protein heterotrimer. This interaction results in the dissociation of GDP from the α-subunit of the heterotrimeric G-protein. GDP is then replaced by GTP. Binding of GTP to the α-subunit induces a conformational change, which in turn leads to the dissociation of the α-subunit and the βγ-complex. The GTP-bound α-subunit as well as the βγ-complex is now able to interact with effector proteins. A ▶GTPase activity inherent to the G-protein α-subunit terminates the G-protein activation. The formed GDP remains bound to the α-subunit which now reassociates with the βγ-complex. The reassociation of the heterotrimeric G-protein induced by the hydrolysis of GTP represents the inactivation mechanism for the βγ-complex. Two bacterial toxins specifically interfere with the G-protein activation–inactivation cycle and have been useful tools in studying G-protein-mediated signaling. ▶Pertussis

G-Proteins. Figure 1 The G-protein cycle. Upon activation of a G-protein-coupled receptor by binding of an agonist, GDP is released from the α-subunit of the heterotrimeric G-protein and replaced by GTP. This in turn leads to the dissociation of the α-subunit and the βγ-complex which are now able to interact with effector proteins. A GTPase activity inherent to the G-protein α-subunit terminates the G-protein activation. The hydrolysis of GTP to GDP can be accelerated by regulators of G-protein signalling (RGS) proteins as well as by various effectors.

toxin blocks the interaction of activated receptors and various G-proteins whereas ▶cholera toxin leads to the constitutive activation of some G-proteins. A physiological regulation of the GTPase activity of the α-subunit occurs by several effector proteins which interact with the GTP-bound α-subunit and accelerate their GTPase activity leading to G-protein inactivation. In addition, a family of proteins called “regulators of G-protein signaling” (▶RGS proteins) are also able to increase the GTPase rate of the G-protein α-subunit. More than 20 G-protein α-subunits have been described in the mammalian system, and they can be divided into four subfamilies based on structural and functional homologies (Table 1). The main properties of individual G-proteins appear to be primarily determined by the identity of the α-subunit of the heterotrimeric G-protein. While some G-protein α-subunits show a very restricted expression pattern others are expressed in a wide variety of tissues and some G-protein α-subunits like Gαs, Gαq, Gα11, Gα12, and Gα13 appear

G-Proteins G-Proteins. Table 1

Mammalian G-protein α-subunits

Class

Subtype

Expression

Gαs

Gαsa Gαolf Gαi1 Gαi2 Gαi3 Gαoa Gαgust Gαt-r Gαt-c Gαz Gαq Gα11 Gα14 Gα15/16c Gα12 Gα13

Ubiquitous Brain, olfactory epithelium Widely distributed Ubiquitous Widely distributed Neuronal, neuroendocrine cells Taste cells, brush cells Retinal rods, taste cells Retinal cones Neuronal, platelets Ubiquitous Almost ubiquitous Kidney, lung, spleen, testis Hematopoietic cells Ubiquitous Ubiquitous

Gαi/o

Gαq

Gα12

1191

Effectors

▶Adenylyl cyclase ↑ Adenylyl cyclase ↑ Adenylyl cyclase ↓ Adenylyl cyclase ↓ Adenylyl cyclase ↓ VDCC ↓b, GIRK ↑b ▶Phosphodiesterase ↑ ? Phosphodiesterase ↑ Phosphodiesterase ↑ Adenylyl cyclase ↓ ▶Phospholipase C-β ↑ Phospholipase C-β ↑ Phospholipase C-β ↑ Phospholipase C-β ↑ PDZ-RhoGEF, LARG p115-RhoGEF, PDZ-RhoGEF, LARG

VDCC, voltage-dependent Ca2+ channel; GIRK, G-protein-regulated inward rectifier potassium channel. a Various splice variants. b Effector is regulated by βγ-subunits. c Species variants (Gα15, mouse; Gα16, human). ?, Regulation not shown directly.

to be expressed more or less ubiquitously. An individual cell expresses up to ten different G-protein α-subunits. Five G-protein β-subunits and 11 γ-subunits have been described in the mammalian system. With the exception of the β5-subunit which is expressed mainly in the central nervous system, the currently known β-subunits exhibit a high level of sequence homology (79–90%). In contrast, G-protein γ-subunits are much more heterogeneous. Similar to GTP-bound Gα, βγ-complexes can also regulate various effectors. The best examples of βγ-regulated effectors are particular isoforms of ▶adenylyl cyclase and ▶phospholipase C, as well as ion channels and ▶phosphoinositide-3kinase isoforms. With a few exceptions, there appear to be no major differences between different βγ-combinations with regard to their ability to regulate effector enzymes. Stimulatory regulation of adenylyl cyclases through GPCRs involves G-proteins of the Gs-family of which two main members are known, Gs and Golf. The Gαi/o-family members have been shown to mediate receptor-dependent inhibition of adenylyl cyclases. Since the cellular levels of these G-proteins are usually relatively high, they also represent an important source for βγ-complexes which can regulate a

variety of cellular effectors. The G-protein Go is the most abundant G-protein in the mammalian nervous system. Go is involved in the inhibitory regulation of voltage-dependent Ca2+ channels, a process which appears to be mediated by the βγ-complex of Go. Several G-protein α-subunits are primarily expressed in sensory cells and have been involved in the signal transduction of sensory stimuli. Rod-transducin (Gt-r) and cone-transducin (Gt-c) play well established roles in the phototransduction cascade in the outer segments of retinal rods and cones where they couple light receptors to downstream signaling components of the retinal phototransduction cascade. In contrast to the transducins, the function of gustducin (Gαgust) in taste cells is less well understood. Among the five taste qualities (sweet, umami, bitter, sour, and salty), sweet, umami, and bitter tastes appear to be transduced through heterotrimeric G-proteins. Gαq-family members mediate the pertussis toxin insensitive regulation of phospholipase C β-isoforms. The Gq-family consists of four members whose α-subunits are expressed from individual genes with different expression patterns. Gαq and Gα11 appear to be expressed more or less ubiquitously and are primarily responsible for coupling of receptors to phospholipase C β-isoforms. In contrast, the murine G-protein α-subunit Gα15 and its

G

1192

GA733-2

human counterpart Gα16 are only expressed in a subset of hematopoietic cells, and the expression of Gα14 is restricted to several organs, e.g., kidney, testis, and lung. Receptors activating Gq-family members in mammalian systems do not discriminate between Gq and G11. The G-proteins G12 and G13 constitute the G12family and appear to be expressed ubiquitously. G12/13 regulate the actin cytoskeleton through the activation of the monomeric GTPase Rho (▶Rho-family proteins). This activation is mediated by a subgroup of guanine nucleotide exchange factors (▶GEFs) for Rho including p115-RhoGEF, PDZ-RhoGEF, and LARG. In recent years, genes of almost all G-protein α-subunits have been inactivated in mice. The resulting phenotypes of Gα-deficient animals have provided insights into the biological role of G-proteins demonstrating that G-protein-mediated signaling processes are crucially involved in multiple processes during development as well as in the adult organism.

Clinical Relevance G-protein-mediated signaling processes are operating in all cells of the human organism. They are involved in many physiological and pathological processes. Many clinically relevant drugs function as agonists or antagonists of GPCRs and exert their effects through G-protein-mediated signaling pathways. Some diseases have been found to be caused by distinct defects in single G-protein α-subunits. Emerging experimental data indicate that GPCRs, e.g., the receptors for ▶thrombin (▶Protease-activated receptor), ▶prostaglandin E2 (PGE2) (▶Prostaglandins), lysophosphatidic acid (LPA), and sphingosine-1-phosphate (S1P), are crucially involved in tumor growth and ▶metastasis. ▶Gain-of-function mutations of the gene encoding Gαs (GNAS) give rise to the gsp oncogene (▶Oncogene) which has been found in almost 30% of thyroid toxic adenomas as well as in some thyroid carcinomas (▶Thyroid carcinogenesis) and growth hormone producing pituitary adenomas. The sporadic somatic mutation leads to the substitution of Arg201, the same residue which is ADP-ribosylated by cholera toxin, and results in a constitutively active form of Gαs by blocking its GTPase activity. This leads to activation of adenylyl cyclase independent of receptor agonists. The same sporadic mutation occurring early in embryogenesis results in a ▶mosaicism which is responsible for the ▶McCune-Albright syndrome characterized by polyostotic fibrous dysplasia of the bone, precocious puberty, and café-au-lait pigmentation of the skin. An analogous mutation of the gene encoding Gαi2 (GNAi2) (the gip2 oncogene) has been found in human

ovarian sex cord stromal tumors and adrenal cortical tumors (▶Adrenocortical cancer). In vitro studies indicate that expression of constitutively active forms Gαq and Gα11 can lead to transformation of fibroblasts when expressed at low levels. No transforming mutants of Gαq and Gα11 have been detected in human tumors, but various Gq/G11coupled receptors have been shown to be involved in the stimulation of proliferation in small lung cancer cells. The ▶Kaposi sarcoma-associated herpesvirus (KSHV/ HHV8) is suppose to encode a Gq/G11-coupled receptor and signaling via this receptor has been shown to lead to cell transformation, tumorigenicity, and angiogenesis, and thus to critically contribute to KSHV-mediated oncogenesis. GNA12 the gene encoding Gα12 was identified as an oncogene (the gep oncogene) in soft tissue sarcoma. Constitutively active mutants of Gα12 and Gα13 have been shown to exhibit potent transforming activity in different systems. No activating mutation of Gα12 or Gα13 has been found in human tumors so far, however, increased expression levels of both proteins have been detected in various human cancers. In addition to their role in the regulation of cellular growth and their transforming ability, signaling via Gα12/13-proteins has recently been implicated in the control of ▶invasion and metastasis of breast and prostate cancer cell lines. By regulating a subgroup of RhoGEF-proteins, both Gα12 and Gα13 can activate the small GTPase RhoA which has been suggested to play an important role in cancer ▶progression and invasion.

References 1. Wettschureck N, Offermanns S (2005) Mammalian G proteins and their cell type specific functions. Physiol Rev 85(4):1159–1204 2. Spiegel AM, Weinstein LS (2004) Inherited diseases involving g proteins and g protein-coupled receptors. Annu Rev Med 55:27–39 3. Kelly P, Moeller BJ, Juneja J et al. (2006) The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis. PNAS 103(21):8173–8178 4. Dorsam RT, GutkindJS, (2007) G-protein-coupled receptors and cancer. Nat Rev Cancer 7(2):79–94 5. Malliri A, Collard JG (2003) Role of Rho-family proteins in cell adhesion and cancer. Curr Opin Cell Biol 15 (5):583–589

GA733-2 ▶EpCAM

GAGE Proteins

GABA Definition

▶Gamma-Aminobutyric Acid ▶Photodynamic Therapy

1193

GAGE Proteins H ENRIK J. D ITZEL 1,2 , M ORTEN F. G JERSTORFF 1 1

Medical Biotechnology Center, Institute of Medical Biology, University of Southern Denmark, Odense C, Denmark 2 Department of Oncology, Odense University Hospital, Odense C, Denmark

Synonyms

GADD45

CT4; G antigen

Definition Definition

Growth arrest and DNA damage, a ▶p53-responsive protein that induces a ▶G2/M ▶checkpoint of the cell cycle. ▶Daxx ▶G2/M Transition

GADD153 Definition Growth Arrest DNA Damage 153 is a small nuclear protein that is capable of dimerizing with various ▶transcription factors. Under normal cellular conditions this protein is not expressed in detectable levels, but is highly upregulated during times of cellular stress such as ▶anoxia.

Gadolinium Definition A rare earth metal (lanthanide), gadolinium ions are chelated to form paramagnetic contrast agents for use in ▶magnetic resonance imaging (MRI). ▶Dynamic Contrast-Enhanced Magnetic Resonance Imaging

Belong to the ▶cancer testis antigen (CTA) family and consist of at least 16 highly homologous proteins (GAGE 1, GAGE2A-E, GAGE10, GAGE12B-J, GAGE13).

Characteristics The 16 genes that encode GAGE proteins are located in an equal number of tandem repeats on chromosome X (p11.2-p11.4 region). GAGE 1 is unique among the GAGE proteins because of an exclusive C-terminal encoded by an exon that is interrupted in the other GAGE genes. The remaining GAGE members are composed of five exons encoding 116–117 amino acids with 98% identity, while GAGE1 consist of 138 amino acids. The molecular size of GAGE proteins is 26–29 kDa. In contrast to other CTAs, the expression of which in adult normal tissues is restricted to germ cells of testis, GAGE is also expressed in a subset of oocytes of resting primordial follicles and in maturing oocytes of ovaries. In the testicular seminiferous tubuli, GAGE expression is restricted to the spermatogonia and, to a lesser degree, the primary spermatocytes. GAGE gene transcripts have been found in numerous cancers, most frequently ▶malignant melanoma (24–42%), ▶lung cancer (19–54%) ▶thyroid cancer (30%) ▶breast cancer (26%) ▶hepatocellular cancer (38%) and ▶ovarian cancer (30%) sarcomas (19%) (▶Osteosarcoma, ▶rhabdomyosarcoma), and ▶bladder cancer (12%). Immunohistochemical studies have also identified GAGE in many cancers, but generally at a lower frequency than that observed by ▶RT-PCR, including bladder cancer (40%) [00187], ▶malignant melanoma (17%), ▶lung cancer (16%) ▶breast cancer (12%), and ▶thyroid cancer (10%). The staining pattern varied significantly among and within specimens, and most GAGE-positive tumors also contained cancer cells lacking GAGE expression. GAGE has been correlated to poor prognosis in ▶gastric cancer, ▶esophageal carcinoma and ▶neuroblastoma.

G

1194

Gain of Function p53

In vitro studies have shown that GAGE expression in tumors can be induced by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (▶Methylation), but the transcription of individual GAGE genes seems to be differently regulated, since the GAGE members are not always co-expressed. GAGE proteins are immunogenic and lead to antiGAGE antibody and ▶cytotoxic T lymphocyte (CTL) responses in some cancer patients. The GAGE 1 gene was originally identified as encoding an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by the MHC class I molecules HLA-Cw6 and recognized by a CTL clone derived from the melanoma patient. From the same patient, another CTL clone recognizing the peptide YYWPRPRRY, which is encoded by GAGE2A-E and presented by HLA-A29 molecules, was also isolated. Anti-GAGE antibodies are present in 6% of melanoma patients, but not in pancreas cancer patients. Subcellular localization of GAGE in normal cells (e.g. germ cells) and cancer cells is similar; exhibiting weak cytoplasmic staining and variable nuclear staining. This suggests that GAGE, when expressed in cancer cells, is expressed in the natural context and thus may play a functional role therein. The nuclear localization of GAGE in spermatogonia and cancer cells suggests that GAGE may be a regulator of germline gene expression. Due to the restriction of GAGE expression to immunoprivileged normal tissues and their relatively frequent expression in different types of cancer, GAGE proteins are considered attractive candidates for T cellmediated, cancer-specific ▶immunotherapy. However, the lack of GAGE expression in subsets of cancer cells within GAGE-positive tumors, as also observed for other CTAs, may limit its value as a therapeutic target. Combining different CTA targets may compensate for the heterogeous expression of each CTA within cancers and improve the therapeutic potential. The function of GAGE members remains largely unknown, although one study has reported antiapoptotic properties of GAGE12 (formerly known as GAGE7). GAGE12-transfected cells were shown to be resistant to ▶apoptosis induced by ▶interferon-gamma or by the death receptor ▶Fas/APO-1/CD95. In the Fas pathway, the anti-apoptotic activity of GAGE12 maps downstream of ▶caspase-8 activation, and upstream of poly (ADP-ribose) polymerase (PARP) cleavage. Furthermore, GAGE12 renders the cells resistant to the chemo- and radio-therapeutic agents (▶Chemotherapy of cancer, progress and perspectives, ▶radioimmunotherapy), ▶Taxol and gamma-irradiation.

References 1. Van den Eynde B, Peeters O, De Backer O et al. (1995) A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J Exp Med 182(3):689–698

2. Simpson AJ, Caballero OL, Jungbluth A et al. (2005) Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 5(8):615–625 (Review) 3. Gjerstorff MF, Johansen LE, Nielsen O et al. (2006) Restriction of GAGE protein expression to subpopulations of cancer cells is independent of genotype and may limit the use of GAGE proteins as targets for cancer immunotherapy. Br J Cancer 94(12):1864–1873

Gain of Function p53 G IOVANNI B LANDINO Rome Oncogenomic Center, Department of Experimental Oncology, Regina Elena Cancer Institute, Rome, Italy

Definition Gain of function is the property of some proteins whose alterations determine the acquisition of novel functions mainly opposite to those exerted by the ▶wild-type counterparts.

Characteristics

The gene product of the tumor suppressor ▶p53 represents a paradigm of a protein whose alterations, mainly missense mutations, cause the acquisition of novel functions that contribute to the insurgence, the maintenance, the spreading and the ▶chemoresistance of certain tumors. p53 is a ▶transcription factor that can be roughly divided in three functional domains: (i) the N-terminus where resides the transcriptional activity; (ii) the central DNA binding domain that is responsible for the specific recognition of p53 binding sites; and (iii) the C-terminus domain that exerts oligomeric and autoregulatory activities. Half of human cancers bear p53 mutations that mainly occur within the specific DNA binding domain. The resulting proteins are characterized by the loss of the antitumoral activities of wild-type p53 (wt-p53) and, at least, for some of them by the gain of novel oncogenic activities. Many in vitro and, very recently, in vivo studies have shown that, at least, some of these mutant p53 proteins gain novel oncogenic functions that range from increased proliferation to enhanced chemoresistance to anticancer treatments. To date, the diverse mutant p53 proteins have been classified accordingly to their structural features. Indeed, they can be divided in two large classes: (i) DNA contact defective mutants whose missense mutation impacts on the region of the protein that contacts the DNA. The prototypes of this class of mutants are p53His273 and p53Trp248 that are also the


most frequent p53 mutations found in human cancers. (ii) Defective structure mutants whose missense mutation resides within the region of the stabilization of the internal loops (L2 and L3) of p53 and consequently their overall structure is quite different than that of wt-p53. Despite many ongoing attempts, a functional classification of p53 mutations that resembles their structures features has not been identified yet. By comparing wild-type versus mutant p53 two peculiar differences can be revealed. While wt-p53 is a shortliving protein capable to activate target genes transcriptionally through the recognition of specific binding sites on their regulatory regions, mutant p53 is a very stable protein that is unable to bind wt-p53 consensus. There is scarce evidence on the molecular basis of the prolonged half-life of mutant p53 compared with that of wt-p53, but this peculiar feature might play a key role in gain of function. Molecular Mechanisms The molecular mechanisms underlying gain of function of mutant p53 proteins need to be clarified yet. To date, two scenarios can be proposed as molecular basis for mutant p53 gain of function. The first one relies on the prolonged half-life of mutant p53 proteins that are consequently very abundant in tumor cells. Thus,

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mutant p53 proteins can be engaged in many and multiple physical protein–protein interactions. One of the most studied is that with the newly discovered ▶p53 family members, p73 and p63 (Fig. 1a). The latter, despite their critical roles in development and differentiation, have been shown to recapitulate most of the wt-p53 tumor suppressor activities when exogenously expressed in p53+/+ and p53 null cells. Floating protein complexes involving mutant p53 and p73 or p63 have clearly evidenced in diverse tumor cell lines. The net result of these protein complexes is the severe impairment of p73/p63-mediated growth suppression and ▶apoptosis. Chromatin immunoprecipitation experiments have clearly shown that mutant p53 severely impairs the in vivo recruitment of both p73 and p63 to the regulatory regions of their target genes. The polymorphism at position 72 of mutant p53 has been shown to be a critical determinant of the strength of the protein complex mutant p53/p73. Indeed, the 72R (Arg) forms of p53Ala143 and p53His175 mutants bind to p73 and impairs p73-mediated gene target transcriptional activation more efficiently than the equivalent 72P (Pro) mutants. The biochemical analysis of the protein complex mutant p53/p73 has revealed that the interaction surface is composed by the core domain of mutant p53 and the specific DNA binding

Gain of Function p53. Figure 1 Schematic representation of two molecular scenarios underlying gain of function of mutant p53. (a) Mutant p53 proteins form protein complexes with the newly discovered p53 family members, p73 and p63 (▶p53 protein biological and clinical aspects). This results in the impairment of p73 or p63 recruitment on the regulatory regions of their target genes and of p73/p63-mediated apoptosis in response to DNA-damaging agents. (b-upper part) Mutant p53 binds directly to DNA through a consensus that remains to be identified yet; (b-lower part) Mutant p53 takes part to large protein complex facilitating the recruitment of the acetylase, p300. The final outcome is the transcriptional modulation of target genes responsible for the indicated biological effects.

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domain of p73. These findings have discovered a novel function for the core domain of mutant p53, such as, its ability to function as a protein–protein interaction platform. Is there any link between the selective pressure for p53 missense mutations in the core domain and gain of function activity of mutant p53 proteins? There is no sufficient evidence to make a conclusion. The vast majority of p53 mutant proteins are unable to bind wt-p53 binding sites present on the regulatory regions of its target genes and consequently are inefficient in the activation of wt-p53 mediated antitumoral effects. Opposite to such loss of function, mutant p53 proteins may acquire, through its mutated core domain, new properties that contribute to their gain of function. Despite many efforts, the presence of wt-p53/p73 protein complexes has not been identified in tumor cells. This might suggest that mutant p53 binds to a pool of selected proteins diverse than those bound by wt-p53. The presence of mutant p53 or wt-p53 in large and multiple protein complexes might represent a key determinant of their biological outputs. A paradigmatic example has been recently provided by the analysis of the transcriptional cross-talk between mutant p53 and the transcription factor NF-Y. The latter binds to both wt- and mutant-p53 but the transcriptional effects on NF-Y target genes are repression or activation, respectively. Is mutant p53 a bona fide transcription factor? (Fig. 1b) There is growing evidence accounting for transcriptional activity of mutant-p53 as molecular mechanism underlying its gain of function activity. This possibility mainly resides on the assumption that the N-terminus of mutant p53 in functionally intact and might exert specific transcriptional activity. To date, one of the key features of a bona fide transcription factor such as a specific DNA binding site for mutant p53 proteins has not been identified yet. This might suggest that mutant p53 can exert its transcriptional activity by engaging with DNA binding proteins, acetylases, deacetylases, and other proteins in the context of large protein complexes. If this hypothesis will be further demonstrated, as occurred for the transcriptional crosstalk mutant p53/NF-Y, it will mean that the plethora of the putative mutant p53 target genes is rather large, thereby providing the molecular basis underlying the diverse mutant p53-mediated oncogenic effects. There is scarce evidence on the role of mutant in these large protein complexes and specifically the contribution of its N-terminus transcriptional activation domain. It was originally shown that mutant p53 proteins, whose residues 22 and 23 were mutated, lost their oncogenic activity as well as their ability to transactivate specific target genes. Despite the molecular details need to be clarified yet, these findings strongly support a direct involvement of the N-terminus of mutant p53 in the activity of the large transcriptional competent protein

complexes. The biochemical analysis of the spatial and temporal events regulating the cross-talk between mutant p53 and NF-Y has revealed a different transcriptional contribution of mutant p53 proteins (p53 protein biological and clinical aspects). Indeed, it was found that mutant p53 facilitates the recruitment of p300 acetylase, thereby indicating that it might serve as scaffold protein whose contribution results in the stabilization and proper activation of the transcriptional competent protein complex. Recent evidence has shown an additional molecular mechanism for mutant p53 gain of function activity. It is based on the tight association of mutant p53 with the nuclear matrix in vivo, and with high affinity to nuclear matrix attachment region (MAR) DNA in vitro. These findings suggest that mutant p53 interacting with key structural components of the nucleus, exerts its gain of function activity through the perturbation of the nuclear structure and function. As described earlier, the molecular scenario(s) underlying gain of function of mutant p53 are rather complex. It might be reasonable that a combination of specific protein–protein interaction and direct transcriptional activity takes place in driving gain of function of mutant p53. Many questions regarding the contribution of cell context, type of p53 mutations, posttranslational modifications are still unanswered and might be determinant in the final outcome of mutant p53 activities.

Clinical Aspects Many in vitro and in vivo evidences have shown that the status of p53 is a key determinant of tumor aggressiveness and chemosensibility to common anticancer treatments. Tumors carrying p53 mutations are more resistant to the killing of anticancer agents and relapse more frequently than those bearing wt-p53. As a consequence of it, the overriding of mutant p53 gain of function might be extremely useful for treating mutant p53 tumors. Diverse approaches ranging from reactivation of mutant p53 to its wt-conformation as well as the elimination of mutant p53 have been undertaken in the last few years. In addition to them, the use of short peptides capable to specifically disassemble oncogenic protein complexes involving mutant p53 protein might be attempted to increase the chemosensibility of tumor cells. The well-established mutant p53/p73 protein complex could be a preferential target to be tackle with such approach. The amount of free and available p73 to be recruited in activating proapoptotic pathways in response to different anticancer treatments might be significantly increased by pretreating mutant p53 cells with short interfering peptides capable to disrupt the protein complex mutant p53/p73. These effects might render mutant p53 cells more prone to the killing of anticancer treatments.

Gallbladder Cancer

References 1. Strano S, Dell’Orso S, Di Agostino S et al. (2007) Mutant p53: an oncogenic transcription factor. Oncogene 26:2212–2219 2. Di Agostino S, Strano S, Emiliozzi V et al. (2006) Gain of function of mutant p53: the mutant p53/NFY protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation. Cancer Cell 3:191–202 3. Soussi T, Beroud C (2001) Assessing TP53 status in human tumours to evaluate clinical outcome. Nat Rev Cancer 1:233–240 4. Sigal A, Rotter V (2000) Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome. Cancer Res 60:6788–6793

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types and binds glycoconjugates on the cell surfaces and ▶extracellular matrix. ▶CEA Gene Family

Gallbladder Cancer G IORGIA R ANDI Department of Epidemiology, Institute for Farmacological Research Mario Negri, Milan, Italy

Gain-of-Function Mutation Definition Is any mutation of a gene that causes increased function and/or activity of its encoded protein or of a protein that is directly or indirectly regulated by the mutated gene. ▶Gain of Function P53 ▶Gastrointestinal Stromal Tumor

GAK ▶Cyclin G-Associated Kinase

Galactorrhea Definition Breast milk secretion at a time other than normal lactation. ▶Prolactin

Galectin-3 Definition Protein with an amino-terminal non-lectin domain and a carboxy-terminal lectin domain. Expressed in many cell

Definition The biliary tract consists of an interconnected system of intra- and extrahepatic ducts that transport ▶bile secreted from the liver to the digestive tract. The gallbladder is an important organ of the biliary system lying just under the liver, receiving, storing and then releasing the bile through bile ducts into the duodenum to help digesting fat. Gallbladder cancer (GC) is the most common type of cancer of the biliary tract.

Characteristics GC is a relatively rare neoplasm and despite being a non sex-related cancer is several-fold more frequent among women than among men. Detection of GC is quite difficult because symptoms and signs of GC are not specific and often appear late in the clinical course of the disease. For this reason, diagnosis is generally made when the cancer is already in advanced stages, and prognosis for survival is less than 5 years in 90% of cases. Descriptive Epidemiology GC incidence is characterized by a wide worldwide variation (Fig. 1) being low in several European countries and the United States of America (USA), relatively high in selected central European countries, and very high in some countries of Latin America and Asia. GC has been shown to be the first cause of cancer death among women in some areas of Chile. According to ▶incidence rates recorded by cancer registries in mid-1990s, the highest incidence rate worldwide was shown by women from Delhi, India (21.5/100,000), followed by South Karachi, Pakistan (13.8/100,000) and Quito, Ecuador (12.9/100,000). Cancer registries reporting high GC incidence rates were in Far East Asia (Korea and Japan), Eastern Europe (Slovakia, Poland, Czech Republic and Yugoslavia), and South America (Colombia). In Western Europe, elevated incidence rates were shown in Granada, Spain. Although

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Gallbladder Cancer

Gallbladder Cancer. Figure 1 Worldwide incidence of gallbladder cancer among women. Geographic areas according to incidence rates of mid-1990s from 40 selected cancer registries: areas in red are characterized by very high incidence rates (age-standardized rates ≥ 10 per 100,000 women); areas in yellow are characterized by high incidence (age-standardized rates between 6 and 10 per 100,000 women); areas in green are characterized by medium incidence rates (age-standardized rates between 2 and 6 per 100,000); areas in blue are characterized by low incidence rates (age-standardized rates < 2 per 100,000 women). *Countries in Europe were not specified because of space reasons: Germany, Italy, France, Slovenia, Croatia, Lithuania, Russia, Estonia shown medium incidence rates; Latvia shown low incidence rate. **No official incidence rate was available for Chile, but GC has been shown to be the first cause of cancer death among women in some areas of the country.

systematically lower than in women, high incidence rates among men (ranging between 4.4 and 8.0/100,000) were found in some areas of Asia and Eastern Europe. Low incidence rates (below 3/100,000 women and 1.5/100,000 men) emerged for most registries from Northern Europe, with the partial exception of Sweden, and from the USA (SEER), and Canada. The female to male (F/M) incidence ratio of GC incidence rates varied greatly: it was more than 5 in several high-risk areas (e.g., Pakistan, India, Colombia and Spain) as well as in low-risk areas (e.g., Denmark), but was typically between 2 and 3 in the majority of countries. F/M ratio was close to 1 in Korea, Japan and some part of China. Incidence rates of GC in various ethnic groups from selected cancer registries in the USA confirmed the worldwide pattern, as GC was substantially more

frequent among Hispanic than non-Hispanic white women and remarkably elevated among Korean and Chinese men. Very high incidence was also reported by men and women among American Indians in New Mexico. Also the F/M ratio reflected the worldwide situation being high among Hispanic whites, and close to 1 among Koreans, Filipinos, Japanese and Chinese. Risk Factors ▶Carcinogenesis of gallbladder is still poorly understood, and limited number of ▶epidemiologic studies have been published on this issue because of (i) the rarity of GC in countries where most medical research is funded and performed, (ii) the difficulties of histological identification of GC, (iii) the lack of relevant animal models and tumor cell lines for GC, (iv) the lack of comprehensive national or international registries

Gallbladder Cancer

for information on GC cases. ▶Risk factors for GC include genetic predisposition, geographic variation and ethnicity, increasing age, female gender, chronic inflammation, congenital abnormalities, low socio-economic status, low frequency of ▶cholecystectomy for gallbladder diseases and exposure to certain chemicals. History of ▶gallstones and ▶cholecystitis are considered the major risk factors for GC. Several cohort and case-control studies found strong association between history of benign gallbladder diseases (mainly gallstones) and GC risk. Cholesterol and mixed gallstones (containing more than 50% of cholesterol) account for 80% of the all gallstones found, and pigment stones (composed largely of calcium bilirubinate) account for the remaining fraction. The etiology of cholesterol gallstones is thought to involve the interaction of genetic factors (e.g. modification of MDR3 and CYP7A1 genes, and numerous lithogenic genes) and several environmental factors (age, female gender, ▶obesity, multiple pregnancies, a family history of gallstones and low levels of physical activity). The worldwide distribution of gallstone prevalence shows a strong geographic and ethnic variation, and a positive correlation with the incidence rates of GC. High gallstone prevalence (≥50%) among women was found among American Indians in the USA, and among Mapuche Indians in Chile, both populations presenting very high GC incidence rates. Other areas with high or medium prevalence of gallstones were identified in South America, in Eastern and Western Europe. Very few is known about some regions of the world like India where high incidence of symptomatic gallstones has been observed, but results from ultrasound-based studies are not available. Low-risk areas for gallstones (i.e., prevalence 100 ng/l), abnormal secretin stimulation test (increment in fasting serum level of gastrin >200 ng/l after intravenous secretin), and an elevated basal level of gastric acid output (>10 mEq/h). Gastrinomas are thought to originate from gastrinproducing (G) cells that are normally dispersed in gastric antral and upper duodenal mucosa (▶Gastrointestinal tumors; ▶Gastrointestinal hormones). However, there is no direct correlation between the distribution of G cells in the digestive system and frequency of gastrinoma location. The tumors commonly arise in ectopic sites such as pancreas (the second most common location) that is normally devoid of G cells. More than 50% of gastrinomas arise in the duodenum. Ninety percent of all gastrinomas are located in the “gastrinoma triangle” (the anatomic junction of cystic and common bile ducts, second and third portion of the duodenum, and neck and body of the pancreas). Solitary pancreatic or duodenal gastrinomas are characteristic for sporadic ZES cases. Location of the tumors in proximal duodenum and their multiplicity are common in ZES-MEN1 patients. Cases of gastrinoma located solely in periduodenal and peripancreatic lymph nodes have been reported, and clinical cure has been achieved following surgical excision of regional lymph nodes in some of such cases. Microscopically, gastrinomas are composed of small, uniform cells with abundant eosinophilic cytoplasm, and small uniform nuclei with inconspicuous nucleoli. Correlation between histologic features and malignant behavior is poor. Mitotic activity and nuclear pleomorphism, when rarely present, are unreliable predictors of prognosis in gastrinoma. Despite their small size and lack of invasion into adjacent organs, duodenal gastrinomas demonstrate high malignant potential with propensity for regional lymph node and distant metastases. Because gastrinoma may be an initial manifestation of ▶MEN1 in up to one third of familial ZES cases, it is currently recommended to evaluate all ZES patients for medical and family history of the endocrine neoplasms and assess their parathyroid and pituitary function. The differentiation of patients with familial ZES-MEN1 from patients with sporadic ZES is important because of the differences in natural history of ZES, need for family screening, difficulty in controlling acid hypersecretion, and need for exploratory laparotomy for cure. Gastrinomas are reported to be malignant in 60–90% of cases. About 34% of patients with ZES have metastatic disease at the time of diagnosis. Periduodenal and peripancreatic lymph nodes, liver, and bone are the most common sites of metastases. Gastrinomas in

MEN1-ZES and sporadic ZES patients have similar rates of metastases. Therapy Since ZES can be cured only by excision of a gastrinoma in the early stage of the disease, the exact tumor localization may be crucial in successful management of ZES patients. Duodenal gastrinomas are frequently small in size (less than 1.0 cm) and are difficult to find preoperatively. Tumor localization studies (▶CT, ▶MRI, and ▶octreotide scanning) are necessary for preoperative localization. Exploratory ▶laparotomy with intraoperative ultrasonography, transduodenal endoscopic illumination, duodenotomy, and surgical resection of the tumor is currently recommended for patients with sporadic ZES patients. The usefulness of surgery varies for patients with ZESMEN1 who usually have multiple tumors. Treatment of patients with ZES is also directed at reducing gastric acid hypersecretion. Total gastrectomy or vagotomy has been used. However, over the past 15 years a highly potent antisecretory proton pump inhibitor, ▶omeprazole, has been effective in suppression of gastric acid output and has significantly decreased the early mortality of patients due to complications of ulcer disease. With the increased ability to control hyperchlorhydria, the progression of gastrinoma and metastases are becoming the primary factor in long-term survival of patients with ZES. The clinical course of gastrinoma is indolent and the prognosis is directly related to the spread of the tumor. Patients with liver metastases have only a 20–30% chance of surviving for 5 years, whereas patients without liver metastases have an excellent long-term prognosis (>90% 5-year survival rate). Genetics The role of the MEN1 gene as an early event in gastrinoma tumorigenesis has been established in MEN1associated gastrinomas as well as in 33% of sporadic gastrinomas regardless of metastases. In both familial and sporadic gastrinomas the MEN1 gene, located on chromosome 11q13, is thought to act as a tumor suppressor based on the presence of inactivating mutations in the normal tissue/blood DNA, accompanied by the loss of the wild type allele in the tumor. Either ▶homozygous deletion or ▶hypermethylation at the 5′ region of the p16/MTS1 or ▶p16INK4a tumor suppressor gene on chromosome 9p21 was demonstrated exclusively in sporadic gastrinomas and not in other pancreatic neuroendocrine tumors. The finding suggests that p16/MTS1 or p 16INK4adefect is restricted to gastrinproducing tumors. Somatic genetic changes associated with the development of most sporadic gastrinomas and as well as with the progression to malignancy are currently unknown.

Gastrointestinal Stromal Tumor

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References

Definition

1. Jensen RT, Fraker DL (1994) Zollinger-Ellison syndrome: advances in treatment of gastric hypersecretion and gastrinoma. JAMA 18:1429–1435 2. Norton JA, Fraker DL, Alexander HR et al. (1999) Surgery to cure the Zollinger-Ellison syndrome [see comments]. N Engl J Med 341:635–644 3. Debelenko L, Zhuang Z, Emmert-Buck MR et al. (1997) Allelic deletions on chromosome 11q13 in multiple endocrine neoplasia type 1-associated and sporadic gastrinomas and pancreatic endocrine tumors. Cancer Res 57:2238–2243 4. Zhuang Z, Vortmeyer A, Pack S et al. (1997) Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res 57:4682–4686 5. Muscarella P, Melvin WS, Fisher WE et al. (1998) Genetic alterations in gastrinomas and nonfunctioning pancreatic neuroendocrine tumors: an analysis of p16MTS1 tumor suppressor gene inactivation. Cancer Res 58:237–240

GastroIntestinal Stromal Tumor (GIST) is a type of sarcoma (i.e., a connective tissue neoplasia). GIST is a rare cancer affecting the digestive tract or nearby structures within the abdomen.

Gastroesophageal Reflux Disease Definition GERD; The reflux from acidic gastric content into the esophagus mostly due to a gastric hernia results in esophagitis, favoring cancer risk in the lower esophagus. ▶Alcohol Consumption ▶Esophageal Cancer

Gastrointestinal Hormones ▶Gut Peptides

Gastrointestinal Stromal Tumor C HI TARN , A NDREW K. G ODWIN Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA

Synonyms GIST

Characteristics Epidemiology and Clinical-Pathological Features of GIST GISTs are believed to arise from the ▶Interstitial Cells of Cajal (ICCs), the pacemaker cells for the autonomous movement of the GI tract. Other studies have suggested that GISTs arise from interstitial mesenchymal precursor ▶stem cells; however, pinpointing the progenitor cell has been difficult. Although it is considered a rare tumor, nearly 4,500–6,000 new cases are diagnosed annually in the United States. The peak incidence of GIST occurs later in life with a median age of 58 years; however, there are also reports of pediatric GISTs. The most common sites of origin for GIST are the stomach (39–70%) and small intestine (31–45%). Other primary sites include the large bowel, rectum, appendix, and rarely the esophagus. A small percentage of GISTs arise outside the tubal gut, i.e., within the mesentery, gallbladder, and omentum. These tumors are known as extra-gastrointestinal GIST. The symptoms of GIST include bloating, gastrointestinal bleeding, or fatigue related to anemia. The common metastatic sites for GIST include the liver and omentum, and less frequently, the lung and bone. Diagnosis Approximately 95% of GISTs express the antigen CD117 (better known as ▶KIT) when examined by ▶immunohistochemistry (IHC) (Fig. 1). The c-KIT gene is the normal cellular homologue of a viral oncogene (v-Kit, Hardy Zuckerman 4 feline sarcoma viral oncogene homologue). There is a small subset of GISTs that lack KIT expression. Oncogenic Signaling Pathways in GIST KIT is a 145-kDa transmembrane glycoprotein and is a member of the tyrosine kinase family of receptors. Other members in this family include PDGFRα and β (Fig. 2a). KIT is normally expressed in hematopoietic stem cells, ▶mast cells, melanocytic cells, germ cells, and the ICC. The normal function of KIT is dependent on binding to its ligand, ▶Stem Cell Factor (SCF), and is essential in embryonic development. Acquired mutations in the c-KIT gene are referred to as “▶gain-offunction” or “activating” mutations, which lead to constitutive ligand-independent activation of the tyrosine kinase activity of the receptor. Molecular genetic studies have shown that the vast majority of GISTs (70– 80%) possess a c-KIT mutation in either exon 9, 11, 13,

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Gastrointestinal Stromal Tumor. Figure 1 GIST. Left: Surgically resected gastric GIST; right: Immunohistochemical staining of a paraffin-embedded GIST tissue section for KIT protein expression.

or 17, and that a subset of GIST (10%) possesses a PDGFRα mutation in either exon 12, 14, or 18. Even though c-KIT or PDGFRα mutations are detected in GIST, approximately 10% of GISTs lack mutations in either of these genes. Therefore, other yet to be discovered genetic and potentially epigenetic mechanisms, independent of c-KIT and PDGFRα activation, may contribute to the pathogenesis of GIST. KIT gain-of-function mutations result in autoactivation of the receptor, and consequently transmit the KIT oncogenic signal to downstream targets such as phosphatidylinositol 3-Kinase (▶PI3K), ▶AKT (as known as protein kinase B), and mitogen-activated protein kinases (▶MAPK). These signaling proteins influence proliferation, ▶apoptosis, differentiation and/or cell ▶adhesion (Fig. 2b). Clinical observations and laboratory research have shown that different gain-offunction mutations within the same receptor can affect different down-stream pathways and clinical response to therapy. Cytogenetics and Molecular Cytogenetic Alterations Even though mutations in c-KIT or PDGFRα appear to be the primary driving force in the pathogenesis of GIST, several other genetic and genomic changes have been documented which contribute to the development of this disease. ▶Monosomy for chromosome 14 is one of the most frequent (>60%) genomic alterations detected in GISTs. Other common changes in metastatic GISTs include loss of chromosome arms 1p, 9p, 10, 14q, 15q, and 22, and gains involving 5p and 20q. Therapy ▶Cytoreductive surgery is the standard of care for patient with primary GIST. This surgery seeks to remove the entire gross tumor, and may require total or subtotal organ resection, depending on tumor location

and size. However, surgery has limited success for locally recurrent or metastatic GIST and clinical response of patients to systemic therapy using conventional chemotherapies is abysmal. ▶Chemotherapy response rates in patients with metastatic disease are less than 5% for all tested cytotoxic agents with a median survival of 10–20 months. ▶Imatinib mesylate (STI571 or Gleevec™), is an oral 2-phenylaminopyrimidine derivative that acts as a selective inhibitor against several ▶receptor tyrosine kinases including KIT, PDGFRα, and ▶BCR-ABL (which is the causative ▶chromosomal translocation in ▶chronic myelogenous leukemia). Based on a high percentage of metastatic GIST patients demonstrating clinical response to imatinib in phase I/II ▶clinical trials, the FDA granted Novartis approval of imatinib for the treatment of advanced GIST in 2001. Clinically, it has been reported that patients with metastatic or localized GIST possessing a mutation in exon 11 (i.e., involving the juxtamembrane domain of the receptor) of c-KIT mutations have a longer ▶progression free survival and overall survival when treated with imatinib as compared to patients with tumors with other types of c-KIT mutations. Likewise, mutations in exon 12 (juxtamembrane domain) of PDGFRα are more sensitive to imatinib treatment than patients with other types of mutations in PGDFRα. In summary, GISTs are the most common mesenchymal tumors of the digestive tract and possess mutations in either c-KIT or PDGFRα. For patients with localized resectable GIST, surgery remains the treatment of choice, whereas, for patients with metastatic disease, imatinib-based therapy is providing benefit for longterm disease control.

References 1. Hirota S, Isozaki K, Moriyama Y et al. (1998) Gain-offunction mutations of c-kit in human gastrointestinal stromal tumors. Science 279:577–580


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Gastrointestinal Stromal Tumor. Figure 2 Structure and signaling pathways of KIT and PDGFRα. (a) Schematic structure of KIT and PDGFRα. (b) Signaling pathways down-stream of KIT activated by stem cell factor or by oncogenic mutations. Shown are some of the most studied signaling events that are set in motion in response to KIT activation. Red circles with a “P” indicate phosphorylation of the protein. Phosphorylation of proteins by kinases is a means of controlling the activity of the recipient protein.

2. Hirota S, Isozaki K, Nishida T et al. (2000) Effects of lossof-function and gain-of-function mutations of c-kit on the gastrointestinal tract. J Gastroenterol 35(Suppl 12):75–79 3. Tarn C, Godwin AK (2005) Molecular research directions in the management of gastrointestinal stromal tumors. Curr Treat Options Oncol 6:473–486

4. Tarn C, Godwin AK (2006) The molecular pathogenesis of gastrointestinal stromal tumors. Clin Colorectal Cancer 6(Suppl 1):S7–S17 5. Demetri GD, von Mehren M, Blanke CD et al. (2002) Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 347:472–480

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Gastrulation

Gastrulation Definition Is the formation of a gastrula from a blastula during embryogenesis. ▶Homeobox Genes

Gaucher Disease Definition

An inborn genetic disease due to a defective βglucosidase that normally hydrolyzes glucosylceramide. In another form of the disease, the protein (saposin C) that activates the glucosidase is absent. ▶Sphingolipid Metabolism

Gatekeeper Definition Type of tumor suppressor gene; Refers to a subgroup of gene products, whose major cellular function is in the control of cell division, death or lifespan. Inactivation of gatekeeper genes favors, through a variety of mechanisms, the unrestrained growth typical of cancer cells. Multiple gatekeeper mutations may be required for the neoplastic transformation of a cell. Gatekeeper gene products often participate in the control of ▶cell cycle (for example, the Rb gene product [▶retinoblastoma protein, cellular biochemistry]) or in the signals that regulate proliferation (for example, the ▶APC gene product). It is possible that large proteins with several functional domains (pleiotropic genes), such as ▶BRCA1 and ▶BRCA2 proteins originally described as gatekeepers may also have caretaker functions. ▶von Hippel-Lindau Tumor Suppressor Gene ▶Breast Cancer Genes BRCA1 and BRCA2 ▶Caretakers Genes

GBM ▶Glioblastoma Multiforme

GBP28 ▶Adiponectin

GCL Definition

Glutamate cysteine ligase, also known as γ-glutamylcysteine synthetase (GCS), comprises a catalytic subunit and a regulatory subunit. It is the rate-limiting enzyme in ▶glutathione biosynthesis. ▶Phase 2 Enzymes

Gatekeeper Position Definition Is a protein residue located at the back of the ATP binding site, whose properties (size, charge and hydrophobicity) regulate the binding of inhibitors. ▶Nilotinib

GC-MS Definition Gas chromatography-mass spectroscopy (GC-MS) has the advantage of unequivocally identifying the analytes in a mixture. It is used to identify ▶adducts to DNA or

GEF

altered DNA bases, which result for instance from ▶oxidative DNA damage.

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GDF Definition

GC/Post-GC Type B-Cell Tumors

Synonym growth differentiation factor-11; ▶BMP-11 and member of the ▶TGF-β superfamily.

Definition Since somatically mutated V genes (▶immunoglobulin genes) are a hallmark of ▶B cells that have left the ▶germinal center (GC), studies of V genes in B-cell tumors provide information on their stage of maturation; post-GC type tumors that carry mutated V genes include ▶DLBCL, ▶Burkitt lymphoma, ▶marginal zone lymphoma and classical ▶Hodgkin lymphoma. The pattern of mutations in ▶follicular lymphoma reveals intraclonal heterogeneity, indicating ongoing mutation (GCtype); ▶chronic lymphocytic leukemia and ▶mantle cell lymphoma cells harbor unmutated V genes (pre-GC type). ▶BCL6 Translocations in B-cell Tumors

GDF15 ▶MIC-1

GDP Definition Guanosine diphosphate. ▶RAS

GCSF GDP-Fucose Definition

▶Granulocyte Colony-Stimulating Factor

Definition A common donor substrate for fucosyltransferases. ▶Fucosylation

GCV Definition

▶Ganciclovir

GEF Definition

GDEPT ▶HSV-TK/Ganciclovir Mediated Toxicity

Guanine nucleotide exchange factors (GEFs) are proteins that stimulate the exchange (replacement) of guanine nucleoside diphosphates for guanine nucleoside triphosphates bound to ▶G proteins. ▶RAS Activation

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Gefitinib

Gefitinib Definition An orally available tyrosine kinase inhibitor that has the chemical structure of 4-(3-chloro-4-fluoroanilino)-7methoxy-6-(3-morpholinopropoxy)quinazoline and is capable of selectively inhibiting the epidermal growth factor receptor kinase. Gefitinib is used to treat patients with advanced ▶non-small cell lung carcer, and it is most effective for patients who have specific mutations within the catalytic domain of the epidermal growth factor receptor kinase drug target.

and subsequently degraded upon treatment with Geldanamycin or its clinically more relevant derivatives 17-AAG (17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-(Dimethylaminoethylamino)-17demethoxygeldanamycin). ▶Ansamycin Class of Natural Product Hsp90 Inhibitors ▶B-Raf Signaling ▶Hsp90

▶Drug Design ▶Tyrosine Kinase Inhibitors

Gelsolin C HRISTINE C HAPONNIER 1 , H ELEN L. Y IN 2 1

Gelatin-Binding Protein 28 ▶Adiponectin

Gelatinase B

Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland 2 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

Definition

Is a widely expressed 82–84 kDa ▶actin-binding protein that is found inside the cells and also in a secreted form in extracellular fluid. Gelsolin severs actin filaments ▶actin filament severing by breaking the ionic interactions between actin molecules within the filament and it caps the fast growing (+) end of actin filaments. Filament severing and capping are regulated by Ca2+, the ▶phosphoinositide phosphatidylinositol 4,5 bisphosphate (PIP2) and pH.

▶Serum Biomarkers

Characteristics

Geldanamycin Definition GM; A benzoquinone ansamycin antibiotic from Streptomyces hygroscopicus var. Geldanus that binds to Heat Shock Protein 90 (▶Hsp90) and thereby blocks its function as an important (▶Ansamycin Class of Natural Product Hsp90 Inhibitors) chaperone. From a tumor biology perspective, the more than 50 ▶HSP90 client proteins include ▶TP53, the kinases ▶SRC, Raf-1, B-Raf, HER-2/neu/ErbB2 and ▶BcrAbl as well as several members of the ▶steroid hormone receptor family, which become de-stabilized

Cytoplasmic gelsolin was discovered in 1979 and named for its ability to activate gel–sol transformation of actin filaments in a Ca2+-dependant manner. Gelsolin is the founding member of a ▶gelsolin family of ▶actin filament severing and/or capping proteins. Other members include villin, scinderin, adseverin, CapG, and flightless I. Among these, gelsolin and CapG have been implicated as ▶tumor suppressor. The ▶actin cytoskeleton is remodeled dynamically during cell movements. Gelsolin contributes to dynamic remodeling by promoting the disassembly and subsequent reassembly of the actin cytoskeleton in response to changes in intracellular signals. Disassembly is mediated through filament severing and reassembly is mediated by filament uncapping. Gelsolin is activated by micromolar Ca2+ to bind to the side of actin filaments and to sever them. After severing, gelsolin remains attached to the (+) ends of these short filaments

Gelsolin

even as Ca2+ level decreases to submicromolar level. Gelsolin is detached from the (+) ends (uncapping) by PIP2. It is hypothesized that the combination of severing to generate large number of short capped actin filaments and subsequent uncapping can account for explosive actin filament growth. The importance of gelsolin for cell motility has been demonstrated by gelsolin overexpression and depletion by RNA interference in tissue culture cells and by gelsolin gene knockout. For example, although the gelsolin ▶knockout mice are viable, their ▶neutrophils and ▶fibroblasts have decreased motility. Gelsolin has also been implicated in ▶apoptosis, either as an enabler or a protector. Gelsolin is cleaved by ▶caspase-3 into two halves and the resulting amino terminal half severs actin filament in a Ca2+independent manner. This may contribute in part to the morphologic changes associated with apoptosis in some type of cells. However, a protective role of gelsolin has been suggested in Jurkat cells and in neuronal cells. Thus, blockage or enhancement of gelsolin cleavage might retard or enhance apoptosis depending on the cellular context. The relation between gelsolin, apoptosis and tumorigenesis probably reflects a complex balance between the multiple effector functions of gelsolin.

Gelsolin and Cancer The role of gelsolin in reorganizing the actin cytoskeleton suggests that it may be involved in promoting tumor cell ▶invasion and dissemination. The first study investigating the distribution of gelsolin in ▶breast cancer showed that, in ▶ductal carcinoma, it is notably downregulated in epithelial cells compared with normal breast tissue. However, gelsolin is expressed at a high level in stromal ▶myofibroblasts. Therefore, cytoplasmic gelsolin is expressed in normal epithelial cells, in some tumor cells and importantly in intratumoral and peritumoral stromal myofibroblasts, and in vessel smooth muscle cells. The function of gelsolin in these highly contractile cells is not yet understood. Since the initial study, there have been many other investigations to determine if gelsolin has a role in tumorigenesis. These studies suggest that depending on the type of tissue and the stage of cancer growth, gelsolin may act as a tumor suppressor or a tumor enhancer. In some cases, gelsolin is a reliable prognostic marker. In this general and short chapter, it is not possible to review of all reports in the field. We therefore took the liberty of selecting only studies that investigate both in vitro and in vivo conditions; these studies should be most relevant for understanding the role of gelsolin in tumor development.

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Gelsolin Considered as a Tumor Suppressor ▶Immunohistochemistry studies showed that gelsolin expression is downregulated in many types of cancer including breast, stomach, colon, bladder, prostate, lung, kidney, and ovary. For example, 78% of ▶bladder tumor tissues and six bladder cancer cell lines displayed low or undetectable gelsolin compared with their normal counterparts. Furthermore, transfection of exogeneous gelsolin cDNA into a bladder cancer cell line reduced colony-forming ability and tumorigenicity in vivo. Loss of gelsolin was also observed in human ▶ovarian cancer cell lines and in ovarian carcinoma compared with normal ovaries and benign adenoma. As in the case of bladder cancer cells, reexpression of gelsolin in ovarian tumor cell lines resulted in a reduction in colony formation. Knockdown of gelsolin using small interfering (si) RNA has given contradictory results. For example, gelsolin depletion in the human immortalized mammary epithelial cell line MCF10A unexpectedly increased cell motility and induced overexpression of the small ▶GTPase ▶Rac, which has been implicated in dynamic ▶lamellipodia formation. Gelsolin depletion also converted ▶cadherin from E- to N-type via the induction of the ▶transcription factor Snail, whose expression is inversely correlated with E-cadherin mRNA levels in several epithelial tumor cell lines. These unexpected results suggest how a decrease in gelsolin expression can nevertheless lead to enhanced tumor ▶cell motility. The molecular basis for the decrease in gelsolin expression in tumor cells is not known. There are suggestions that gelsolin mRNA level may decrease due to ▶epigenetic modifications resulting from DNA ▶methylation and ▶histone deacetylation, in ▶ovarian cancer and in ▶breast cancer cell lines. Paradoxically, although gelsolin can suppress tumor progression, it was also shown that overexpression of gelsolin in malignant tumors was associated with poor prognosis. Gelsolin has been suggested to be a motility marker for tumor cells, although its prognostic value has so far only been examined in a small number of malignant tumors. Furthermore, there is surprisingly little information about the possibility that other members of the ▶gelsolin family may substitute for the depleted gelsolin in cancer cells. Gelsolin Considered as a Tumor Enhancer Besides acting as a tumor suppressor, gelsolin has also been implicated as a tumor enhancer, particularly in lung and breast carcinomas. For example, ▶nonsmall cell lung carcinoma (NSCLC), which comprises 70–80% of all lung carcinomas and has a 5-year survival rate of about 15%, is usually associated with decreased gelsolin level in about 70% of the cases

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Gelsolin Family

examined. However, the decrease in gelsolin is not associated with adverse prognostics. Instead, the small percent of cells with high focal gelsolin expression was correlated with the highest risk of cancer recurrence compared with tumors that had no or low gelsolin expression. Likewise, the expression of gelsolin in breast cancer is variable and may involve only a few cells in the issue. Although gelsolin is absent in many breast cancers, about one third of cases have detectable gelsolin. In some cases, gelsolin expression was focally localized and accentuated in cell clusters or in single invasive cells that show transendothelial migration or are embedded in the stroma. Interestingly, an overexpression of gelsolin, when associated with ERBB2 and EGFR expression, resulted in a more agressive tumor phenotype. Altogether, these results suggest that although gelsolin can be extinguished during cell transformation, its focal high expression in a subpopulation of tumor cells can facilitate tumor dissemination and metastasis by promoting tumor cell locomotion. In summary, the current findings are consistent with the hypothesis that gelsolin functions in a complex manner in the development and progression of tumors.

References 1. Chaponnier C, Gabbiani G (1989) Gelsolin modulation in epithelial and stromal cells of mammary carcinoma. Am J Pathol 134:597–603 2. Noske A, Denkert C, Schober H et al. (2005) Loss of gelsolin expression in human ovarian carcinomas. Eur J Cancer 41:461–469 3. Silacci P, Mazzolai L, Gauci C et al. (2004) Gelsolin superfamily proteins: key regulators of cellular functions. Cell Mol Life Sci 61:2614–2623 4. Thor AD, Edgerton SM, Liu S et al. (2001) Gelsolin as a negative prognostic factor and effector of motility in erbB2-positive epidermal growth factor receptor-positive breast cancers. Clin Cancer Res 7:2415–2424 5. Yang J, Tan D, Asch HL et al. (2004) Prognostic significance of gelsolin expression level and variability in non-small cell lung cancer. Lung Cancer 46:29–42

Gelsolin Family Definition

A superfamily of ▶gelsolin-like proteins that has a conserved modular motif. Gelsolin is the founding member; other members include villin, scinderin, adseverin, supervillin, CapG, and flightless I. With

the exception of flightless I, all are shown to be Ca2+-activated ▶actin severing and/or binding proteins. Some of these proteins have also been implicated in transcriptional regulation.

Gemcitabine G RAZIELLA P RATESI Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Synonyms dFdC; 2′,2′-Difluoro-2′-deoxycytidine; GEMZAR

Definition Is an antitumor drug belonging to the class of antimetabolites. Antimetabolites are agents that interfere with normal metabolism due to their structural similarity to normal intermediates in the synthesis of RNA and DNA precursors. Due to differences in metabolism between normal and cancer cells, antimetabolites have the potential to act with a certain degree of specificity in cancer cells. Antimetabolites target the synthetic pathway of pyrimidines (uracil, cytosine, thymine) and purines (guanine, adenosine) nucleotides, where they can serve as substrates for enzymes, or can inhibit enzymes, or both. As a consequence of their interference, either by incorporation or inhibition, antimetabolites induce cell damage leading to apoptotic cell death. Several cytidine and deoxycytidine analogs have been synthesized in the past and tested for antitumor efficacy. The first successful agent in clinical practice was ara-C (1-β-D-arabinofuranosylcytosine) which is currently used as first-line treatment in acute myeloid leukemia and ▶non-Hodgkin lymphoma.

Characteristics Mechanism of Action and Resistance Is a potent and specific fluorine substituted ara-C analog with pronounced activity against solid tumors. Gemcitabine presents advantages in ▶pharmacokinetic and ▶pharmacodynamic properties over ara-C. Its mechanism of action is well characterized. Gemcitabine exhibits cell-phase specificity, primarily killing cells undergoing DNA synthesis (S-phase) and also blocking the progression of cells through the ▶G1/S transition. Membrane transport is mediated by facilitated diffusion catalyzed by human equilibrative nucleotide transporter 1, a carrier for physiologic nucleosides. The molecule itself is inactive and needs to be metabolized

Gemcitabine

intracellularly to its mononucleotide by deoxycytidine kinase, and then by nucleotide kinases to the active diphosphate (dFdCDP, gemcitabine-DP) and triphosphate (dFdCTP, gemcitabine-TP) nucleotides. The activity of gemcitabine is strongly correlated with the extent of gemcitabine-TP. Once incorporated into the DNA strand, an additional natural nucleotide is added, masking gemcitabine, preventing ▶DNA repair by base pair excision, and eventually resulting in chain termination. Such process is self-potentiated by the inhibitory effect of gemcitabine-DP on the reaction generating normal deoxycitidine-TP, thus favoring the incorporation of gemcitabine-TP into DNA. Gemcitabine is also incorporated into RNA and induces cell ▶apoptosis. Ribonucleotide reductase can be inhibited by gemcitabine-DP, resulting in depletion of deoxycitidine-TP and deoxiadenosine-TP. Deoxycitidine-TP is a feedback regulator of nucleotide kinase, thus favoring the activation of gemcitabine. The depletion of deoxiadenosine-TP will inhibit DNA repair. Collectively, the various effects of gemcitabine result in a unique pattern of self-potentiation. Inactivation of gemcitabine can occur by deamination into its inactive metabolite difluoro-deoxyuridine by cytidine deaminase (CDD) or dephosphorylation of nucleotide-MP by 5′-nucleotidase (5′-NU), which exerts the opposite function of deoxycitidine kinase. Three general mechanisms of resistance to nucleotide analogs have been described. The first one arises from insufficient intracellular concentration of nucleotidesTP, which may result from inefficient cellular uptake, reduced levels of activating enzymes, increased degradation by increased 5′-NU or CDD activity, or expansion of the natural deoxynucleotide-TP pool. The second mechanism may be due to inability to achieve sufficient alterations in DNA strands or deoxynucleotide-TP pool. The third mechanism may be a consequence of defective apoptotic pathways, which can impair the activity of antitumor drugs of various mechanistic classes. Clinical Profile For clinical use gemcitabine is administered intravenously. The standard administration of gemcitabine is a 30 min infusion of 1,000 mg/m2/week up to 7 weeks, followed by 1 week rest. Toxicity The dose limiting toxicity is ▶myelosuppression, with 25 and 5% of patients experiencing severe (G3-4) ▶neutropenia and ▶thrombocytopenia, respectively. At the standard regimen hematological toxicity is of short duration, and mild gastrointestinal toxicities (nausea, vomiting), alteration in liver and renal functions, diarrhea and stomatitis are also observed.

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Serious renal or liver toxicities are rare but not reversible. Other toxicities include edema, cutaneous toxicity associated to pruritus, fever, neurotoxicity and dyspnea (uncommon but severe). Indications Gemcitabine is approved for the treatment of several solid tumors such as pancreatic cancer, advanced ▶nonsmall cell lung carcinoma (NSCLC), ▶bladder cancer, ▶ovarian cancer and ▶breast cancer. NSCLC. Gemcitabine is approved for use in combination with ▶cisplatin for first-line treatment of patients with inoperable, locally advanced (Stage IIIA or IIIB) or metastatic (Stage IV) NSCLC. Two schedules have been investigated and the optimal schedule has not been determined. The combination with cisplatin was investigated on the basis of preclinical studies showing inhibition of repair of platinum-induced DNA damage, and of the results of Phase II studies with single agent gemcitabine in more than 400 patients producing response rates consistently above 20%. Promising results are reported in a Phase III study combining gemcitabine to carboplatin, a platinum-analog less myelotoxic than cisplatin. The combination produced better results, in terms of response rate and time to progression than gemcitabine alone. Several Phase II trials have investigated the combination with non-platinum derivatives such as ▶docetaxel, ▶paclitaxel or vinorelbine, but no one has proven advantages when compared to the standard platinum-based regimen. Due to the low toxicity profile gemcitabine is under investigation in elderly patients with advanced NSCLC, either as single-agent or in combination with other drugs. Pancreatic Cancer. Gemcitabine is approved for the treatment of patients with locally advanced (nonresectable Stage II or III) or metastatic (Stage IV) ▶adenocarcinoma of pancreas both for first-line treatment and for patients previously treated with a ▶5-fluorouracil-containing regimen. The approval for first-line treatment was based on the results of a randomized study showing improvement in median survival, 1-year survival and clinical benefits over weekly 5-fluorouracil. Several randomized trials have compared gemcitabine with a gemcitabine-based combination, none of which having shown any incremental benefit in terms of survival or quality of life. Improvement in survival might be achieved by prolonged infusion strategy of gemcitabine and is currently under evaluation. Breast Cancer. Gemcitabine is approved for use in combination with ▶paclitaxel for the first-line treatment of patients with metastatic breast cancer after failure of prior ▶anthracycline-containing adjuvant chemotherapy. The combination of gemcitabine and ▶taxanes (docetaxel or paclitaxel) was tested in clinical

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Gemtuzumab Ozogamicin

trials based on the good single-agent activity of each drug. In a Phase III study gemcitabine in combination with paclitaxel significantly improved response rate, time to progression and survival versus paclitaxel in metastatic breast cancer, with no significant increase in toxicity. The best schedule for administration of gemcitabine-taxane combination and the relevance of the combination in ▶Her-2/neu positive patients are currently under investigation. Bladder Cancer. Systemic chemotherapy has reached a plateau in the treatment of ▶transitional cell carcinoma of the bladder. Gemcitabine is approved for such disease in combination with cisplatin. Such regimen has equivalent response rate and survival, but improved toxicity profile, compared to the previous standard regimen including ▶methotrexate, ▶vinblastine, ▶adriamycin and ▶cisplatin. Promising results have been observed with intrabladder infusion of gemcitabine. Ovarian Cancer. Gemcitabine is approved in combination with ▶carboplatin in several European countries for the treatment of recurrent epithelial ovarian cancer. It has been recently approved also in the USA on the basis of a randomized trial comparing the combination against carboplatin alone in 356 women with recurrent ovarian cancer, whose tumors had previously responded to first-line therapy. The median progression-free survival was 8.6 months for patients taking carboplatin and gemcitabine, and 5.8 months for patients taking carboplatin alone. Quality-of-life was comparable in the two groups of patients. Multicenter Phase III trials are ongoing comparing paclitaxel plus carboplatin with gemcitabine plus carboplatin. The results of a single-institution Phase II study in 24 patients, indicate an acceptable toxicity (with bone marrow toxicity as dose-limiting) with high overall response rate (91%) for the combination gemcitabine plus carboplatin.

Clinical Pharmacology Pharmacokinetic studies indicate that the drug is eliminated in the urine. Gemcitabine is rapidly converted to the inactive uracil metabolite (recovered in the urine for more than 90%), which has a long half life. Gemcitabine pharmacokinetic is linear and described by a 2-compartment model. ▶Clearance and ▶volume of distribution vary with duration of infusion, age and gender. Gemcitabine plasma protein binding is negligible.

Future Perspective Due to its low bone marrow toxicity, gemcitabine might be a suitable drug to be combined with ▶immunotherapy, and preclinical studies support such expectation.

Gemcitabine can induce massive tumor-cell apoptosis both in in vitro and in vivo systems. Uptake of dead cells by ▶antigen presenting cells, still alive after gemcitabine treatment, will result in increase of cross presenting tumor antigens to T lymphocytes in tumor draining lymph nodes. Moreover gemcitabine, which is particularly toxic for B lymphocytes, by impairing the antitumor antibody response might favorite the generation of ▶cytotoxic T lymphocytes, which have to be generated for immunotherapy to be effective. Again, chemotherapy may upregulate cellular death receptors, which are used by T cells to kill targets. Gemcitabine followed by CpG-oligodeoxynucleotide, a modulator of the natural ▶immune response, results in a strong therapeutic synergism in a pancreatic tumor model. Thus, in addition to more combination studies with established or novel antitumor drugs, future clinical studies should address the role of gemcitabine in combination with various immunotherapies for the treatment of cancer.

References 1. Galmarini CM, Mackey JR, Dumontet C (2001) Nucleoside analogues: mechanisms of drug resistance and reversal strategies. Leukemia 15:875–890 2. Lake RA, Robinson BWS (2005) Immunotherapy and chemotherapy – a practical partnership. Nat Rev 5:397–405 3. Peters GJ, Jansen G (2002) Antimetabolites. In: Souhami RL, Tannock I, Hohenberger P, Hoiot J-C (eds) Oxford textbook of oncology, 2nd edn. Oxford University Press, New York pp 663–713 4. Smith IE (2006) Overview of gemcitabine activity in advanced breast cancer. Semin Oncol 33:19–23 5. Toschi L, Finocchiaro G, Bartolini S et al. (2005) Multistep Role of gemcitabine in cancer therapy. Future Oncol 1:7–17

Gemtuzumab Ozogamicin Definition Is a humanized IgG4κ anti-▶CD33 antibody linked to the anti-tumor antibiotic calicheamicin. Gemtuzumab ozogamicin is approved for treatment of relapsed ▶acute myeloic leukemia in patients older than 60 years. ▶Monoclonal Antibody Therapy

Gene Knockout

GEMZAR ▶Gemcitabine

Gene Amplification Definition Amplification of the numbers of copies of one or more genes; synthesis of additional copies of an original gene.

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Gene Expression Profile Definition Messenger-RNA from relevant cells is copied to labeled (with fluorescence dye or radioactive nucleotide) complementary cDNA probes. The probes are incubated on a DNA array containing spots of DNA from known genes being investigated. Hybridization of a given probe signifies the presence of the mRNA in question in the cell or tissue studied, i.e., the gene is active. The method allows estimation of activity genes in one assay. Firstarray many was developed in early 1980 as “Oncogip” for profilin of ▶oncogene expression. ▶Microarray (cDNA) Technology

▶Amplification ▶Neuroblastoma

Gene Expression Profiling Gene Battery

Definition

Definition

A microarray-based methodology to study gene expression (RNA) patterns. Parallel analysis of the expressed transcriptome of a given cell line or tissue sample, first developed in early 1980ies for ▶oncogene profiling.

Refers to a group of genes regulated by a particular transcription factor.

▶Microarray (cDNA) Technology

▶Dioxin

Gene Gun Gene Directed Enzyme-Prodrug Therapy ▶HSV-TK/Ganciclovir Mediated Toxicity

Definition Is a way for in vivo transformation of cells or organisms used for gene therapy and genetic immunization. This gun uses particle bombardment where DNA- (or RNA-) coated gold particles are loaded into the gun. A low pressure helium pulse delivers the coated gold particles into virtually any target cell or tissue. ▶DNA Vaccination

Gene Expression Cassette Definition Stretch of recombinant DNA typically comprising a ▶promoter, a gene and a polyadenylation signal; often used in ▶gene therapy.

Gene Knockout Definition Is a genetically engineered organism that carries one or more genes in its chromosomes that have been made

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Gene Profile

inoperative (have been “knocked out” of the organism). This is done for research purposes. Also known as knockout organisms (▶knock-out mice) or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals.

Gene Profile Definition A extensive survey of genes leading to RNA encompassing known coding sequences in the genome. ▶Gene Expression Profile

Gene Therapy VALERIE B OSCH Forschungsschwerpunkt Infektion und Krebs, F020, German Cancer Research Center (DKFZ), Heidelberg, Germany

Definition Directed introduction and expression of new genetic information in cells of an organism for therapeutic purposes. Only somatic gene therapy is presently permitted, and the introduction of genetic material into germ-line cells is not allowed.

for the development of gene therapeutic intervention strategies. Transfer of Genes Very many different methods have been developed to transfer therapeutic genes to patient cells. A consideration which is central to the choice of gene transfer vehicle (▶vector) is whether stable or only transient gene expression is required. Stable gene expression is necessary for the treatment of hereditary genetic defects whereas transient gene expression is sufficient, and may even be desirable, when employing genes encoding toxic gene products e.g. in the treatment of cancer. Many gene transfer vehicles employ components of viruses (viral vector-mediated gene transfer) since viruses have evolved to efficiently transfer their own genes to cells and to express the respective gene products at high levels. Since it may not be possible to completely eliminate potential safety problems with viral vectors, non-viral gene transfer vehicles have been developed in parallel. These transfer vehicles are based on lipids/▶liposomes (lipoplexes), on polycations (polyplexes), on branched polymer structures (dendriplexes) or consist simply of naked DNA (▶Nonviral vectors for cancer therapy). In general, the transfer efficiencies and gene expression levels achieved with non-viral vectors are poorer than with viral vectors. The commonly used gene transfer vectors, their advantages and their disadvantages are summarized in Table 2. In many situations, in addition to high gene transfer efficiency, it is necessary that the transfer vector is selective i.e. mediates expression of the therapeutic gene exclusively in targeted diseased cells. Achieving selectivity is a major hurdle which is being approached in several ways. Gene vectors are being manipulated (on the surface of the gene vector particle) such that targeted cells are exclusively accessed. Selectivity can be achieved by ensuring that, even in the situation in which many cells have been accessed, expression of the therapeutic gene can only occur in specific targeted cells (e.g. by employing tissue-specific promoter/enhancer elements to control therapeutic gene expression).

Characteristics The science of gene therapy has its beginnings in the early 1980s, subsequent to the identification of several disease-related genes and the development of technologies for gene isolation, purification and transfer to cells in culture. Many different human diseases represent theoretical targets for gene therapeutic intervention and the strategies developed depend on the nature of the disease to be treated. Table 1 illustrates several classes of human disease and the respective treatment strategies currently being followed. It goes without saying that detailed knowledge of the molecular mechanisms of disease development and progression is a prerequisite

Clinical Relevance Therapeutic genes can be introduced into patient cells either ex vivo or in vivo (see Fig. 1). In the ex vivo application, a patient biopsy is genetically manipulated outside the body and subsequently reinjected or transplanted. This procedure has the advantage that only the cells in the biopsy come in contact with the transfer vector. Furthermore, the circumstances in cell culture generally allow more efficient gene transfer than on in vivo application. In addition, in some cases it is possible to expand the cells in the biopsy and create a pool of genetically modified cells which can be

Gene Therapy Gene Therapy. Table 1

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Examples of gene therapeutic strategies for different disease groups

Disease group

Genetic basis of disease

Strategy

Hereditary monogenic disease e.g. haemophilia, cystic fibrosis Multifactorial disease e.g. cardiac disease, diabetes

Defective gene which results in a single gene product being missing or non-functional Many genes and their gene products directly or indirectly involved

Cancer

Somatic mutation of cellular genes (several)

Introduction of wild-type gene (e.g. into liver or muscle for the production of a protein in the blood circulation, into lung in the case of cystic fibrosis) At present too complex. However, restenosis. (i.e. tissue proliferation after surgical dilation of a blood vessel) can be treated by locally expressing genes which inhibit cell proliferation 1. Induction of cell death employing genes encoding toxic gene products 2. Stimulation of the immune system to recognise and destroy cancer cells. (▶Cancer vaccines ▶DNA vaccination) 3. Inhibition of “cancer genes.” (▶Cell-cycle targets for cancer therapy) 4. Prevention of tumor ▶angiogenesis

Infectious diseases e.g. AIDS

Gene Therapy. Table 2

New genetic material from the infectious agent (e.g. virus) induces pathogenic processes

1. Inhibition of expression of genes encoded by the infectious agent employing e.g. antisense, ribozyme or small-interfering (si) RNA approaches. (▶Antisense DNA therapy) 2. Vaccination against viral gene products expressed from transferred heterologous viral vectors or from naked DNA i.e. infection prevented or eliminated by the patient’s immune system

Gene transfer vehicles, their advantages and disadvantages

Gene vehicle Retrovirus vectors including those based on lentiviruses

▶Adenovirus vectors

Advantages

Disadvantages

1. Integration of vector into host genome leading to stable gene expression 2. No cellular toxicity 3. Lentiviral vectors also infect (transduce) differentiated non-dividing cells 1. Very high titers (1013/ml) 2. Very high, but transient, gene expression

1. Titer not as high as e.g. adenoviral vectors 2. Vector integration into host genome is potentially mutagenic

Adeno-associated virus (▶AAV) 1. AAV is not pathogenic in humans vectors 2. Non-toxic

Lipoplexes, polyplexes and dendriplexes Naked DNA

3. Infects non-dividing cells No viral genes, no toxic effects No viral genes, no toxic effects

reapplied to the patient as required. The main disadvantage of the ex vivo procedure is that it is technically cumbersome, time-consuming and very expensive. The most straightforward and desirable situation would be to

1.Only transient gene expression 2. Immune response to vector and transgene 1. lower coding capacity than with retroviral and adenoviral vectors 2. Gene expression in proliferating tissue only transient Mostly lower and transient gene expression Mostly lower and transient gene expression

apply the gene transfer vehicle in vivo either directly into specific tissues or organs (e.g. directly into tumor tissue) or by injection into the blood circulation. In vivo application requires that the transfer vector efficiently

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Gene Transfection

Gene Transfection Definition Synonym Gene Transduction, Gene Transfer; Introduction of genes into cells. ▶Transfection

Gene Therapy. Figure 1 Application routes for gene therapy.

reaches the appropriate diseased cells and at present, this is very often not the case. In fact, major problems hampering gene therapeutic approaches today concern the efficiency and the selectivity of the transfer vectors when applied in vivo. Further problems are related to the induction of host immune reactions towards the (viral) vector and the transgene. Permission for the first clinical gene therapy study was granted in 1989 and since then numerous ▶clinical trials, for the most part with only relatively small numbers of patients, have been carried out. Lack of sufficient selectivity and efficiency of transfer as well as lack of stability of transgene expression, the results of most of these trials did not establish any statistically verified positive effects on disease progression or mortality. Recently, however, more favorable results have been obtained. Thus, as a result of a gene therapeutic intervention with a retroviral vector, several individuals were cured of severe X-linked combined immunodeficiency. Unfortunately, however, within this same trial, severe adverse side-effects were observed in three treated patients. In the meantime, the reasons for these adverse effects have been largely elucidated and subsequent protocols will be adjusted accordingly. At present, basic research is focussed on improving gene vector properties and on gaining a better understanding of the interactions between the gene vector, the transgene and the patient’s immune system. It is to be anticipated that the combined knowledge gained from these efforts will allow gene therapeutic protocols to be developed which will represent valid treatments for diseases which have been difficult or impossible to therapy up until now.

Gene-Environment Interaction Definition The combined effect of genetic susceptibility and exposure to non-genetic factors, broadly defined as “environmental.” ▶Cancer Epidemiology

Genetic Association Definition

Studies of genetic association test whether ▶allele or ▶genotype frequencies are different between groups of individuals (for example, subjects with and without cancer). ▶Linkage Disequilibrium

Genetic Association Study ▶Case Control Association Study

Genetic Epidemiology

Reference

Definition

1. Thomas CE, Ehrhardt A, Kay MA (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4:346–358

The branch of epidemiology which aims at elucidating and quantifying the genetic causes of cancer. Genetic epidemiology investigates the aggregation of cancers in

Genetic Instability

families (linkage studies), and the presence of high-risk genetic variants in sporadic cases (association studies). ▶Cancer Epidemiology ▶Epidemiology of Cancer

Genetic Haplotype Definition

Is a term used in the context of ▶case–control association study where instead of analyzing the association between trait and each genetic marker singly, multiple markers are analyzed simultaneously for a combined effect. These multiple markers when combined together are called a haplotype. There are two rationales for examining haplotypes. First, using haplotypes allows multiple potentially causal markers to be tested simultaneously for association. However, for haplotypes to be superior to individual markers, multiple functional markers must have a strong interaction when combined and yet have no detectable effect when considered individually. Second, haplotypes can be tested for association because they may be a proxy for untyped causal markers. Some have pointed out that the additional association tests entailed in haplotype-based analyses can actually result in a loss of power, and hence lower efficiency, once corrections for multiple testing are taken into account. ▶Case–Control Association Study ▶Haplotype

Genetic Immunization ▶DNA Vaccination

Genetic Instability Definition Synonym genomic instability, seems to be the hallmark of cancer cells, in contrast to the normal cells that, with few exceptions such as e.g. telomeres (▶telomerase) or ▶V(D)J-recombination, are genetically stable. The

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principal concept here is that when genes involved in the maintenance of genetic stability of the cellular genome undergo mutation, the repair of ▶DNA damage often will be incomplete, due to impaired DNA repair (▶repair of DNA) systems. As the consequence, during tumor ▶multistep development, tumor cells assume a ▶mutator phenotype and will accumulate mutations leading to the evolution, by Darwinian selection, of tumor cells that escape cellular growth signals and become more and more malignant. This process is not necessarily related, as often postulated, to very rapid tumor cell proliferation. Cells of many types of tumors actually multiply rather slowly and, in contrast, normal cells in various sites, like the epithelium of the intestine, multiply very rapidly but still remain normal. The ensuing genetic instability drives tumor ▶progression by generating mutations in ▶oncogenes and ▶tumorsuppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumor. Genetic instability can be at the level of the chromosome (▶chromosomal instability; CIN) or can be expressed as ▶microsatellite instability (MIN; also referred to as MSI). Chromosomal instability (CIN) is a defining characteristic of most human cancers. Mutation of CIN genes increases the probability that whole chromosomes or large fractions of chromosomes are gained or lost during cell division. The consequence of CIN is an imbalance in the number of chromosomes per cell (▶aneuploidy) and an enhanced rate of ▶loss of heterozygosity. Microsatellite instability is a condition manifested by damaged DNA due to defects in the normal DNA repair process. MSI is a key factor in several cancers including ▶colon cancer, ▶endometrial cancer, ▶ovarian cancer and ▶gastric cancer. For colon cancer, a prominent form is hereditary non-polyposis colorectal cancer (HNPCC) or ▶Lynch Syndrome, where an inherited mutation in a ▶mismatch repair gene causes ▶microsatellite instability. The replication error results in a ▶frameshift mutation that inactivates or alters ▶tumor suppressor genes. Genetic changes in genetically unstable cells will occurs at random possibly affecting, in humans, any of the estimated 30,000 genes present in the genome. Because of this randomness, genomic instability eventually will lead to tumor cell populations that are genetically different, both within a given tumor in the individual patient and in the same tumor type of different patients. Because the biological behavior of tumor cells is dictated by the profile of genetic changes, the heterogenous populations of cells will respond differently to therapeutic treatments, such as ▶chemotherapy or any other form of therapy. Very often, large populations of tumor cells, due to the pattern of genetic changes, may disappear by therapy-induced ▶apoptosis. Still, minor populations, again because of their

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particular genetic pattern, may be inherently ▶drug resistant and may develop into a new tumor that now is fully resistant against therapeutic drugs. For the same reason, tumors in different patients may respond differently.

Definition

▶Chromosomal Instability

An inherited increase in the risk of developing a specific disease.

Genetic Susceptibility

▶Mutagen Sensitivity

Genetic Knock-out Definition A gene knockout is a genetically engineered organism that carries one or more genes in its chromosomes that have been made inoperative (have been “knocked out” of the organism). Also known as knockout organisms or simply knockouts (▶knock-out mice), they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Researchers draw inferences about gene function from the difference between the knockout organism and normal individuals.

Genetic Toxicology J OSEPH R. L ANDOLPH J R . Cancer Research Laboratory, USC/Norris Comprehensive Cancer Center, Keck School of Medicine/School of Pharmacy, University of Southern California, Los Angeles, CA, USA

Synonyms Chemical mutagenesis; Clastogenesis; Chemical carcinogenesis

▶Orphan Nuclear Receptors

Definition

Genetic Polymorphism Definition Genetic polymorphism is the presence of multiple inheritable forms of a gene within the population; a genetic trait where the least common ▶allele is found in approximately at least 1% of the population ▶Detoxification ▶Modifier Loci ▶Pharmacogenomics in Multidrug Resistance

The study of the processes and mechanisms by which chemicals or radiations cause damage, including ▶mutations, DNA single-strand breaks and doublestrand breaks, ▶gene rearrangements and gene ▶amplifications, and ▶chromosome damage in prokaryotes or eukaryotic cells. This includes aspects of mutagenesis, i.e., the process by which specific chemicals induce changes in the sequence of DNA bases in genomes. It includes also ▶chemical carcinogenesis, i.e., the molecular mechanisms by which chemicals carcinogens induce mutation in ▶tumor suppressor genes, inactivating them, and induce mutations, rearrangements, or amplifications of ▶oncogenes, deregulating them, causing fifteen such changes. These five to eight changes, consisting of inactivation of tumor suppressor genes and activation of oncogenes, leads to further derangements in the expression of ▶numerous genes, which results in the cell acquiring a tumorigenic phenotype and the eventual development of cancer.

Genetic Recombination Characteristics Definition Is the process whereby DNA from one chromosomal location is exchanged for, or replaces, another region of the genome.

Genetic Toxicology Genetic Toxicology, or the science of the study of chemicals and radiations (▶Radiation-induced cancer) that can cause damage to genes and the genome, is now a very advanced science. There are now many

Genetic Toxicology

assay systems, both in bacteria, yeast, Drosophila, mice, and in cultured murine and human cells, which can be used to detect ▶DNA damage. Such damage includes mutations induced by chemicals or radiation, gene rearrangements, gene amplification, chromosome damage (gaps, breaks, fragments, dicentrics, and satellites), single-strand breaks in DNA, and doublestrand breaks in DNA. Important assay detection systems include the ▶Ames assay that detect reversion of His − Salmonella back to His + Salmonella, and which also utilizes addition of exogenous ▶cytochrome P450 to metabolically activate premutagens, such as ▶polycyclic aromatic hydrocarbons to epoxides and diol epoxides. Mutation and recombination can also be studied in yeast. In addition, there are assays to study mutation in mice, using the “spot test.” Plasmids can also be added to mice and retrieved from mice treated with suspect chemical mutagens, and then analyzed to detect mutations in these plasmids. There is now an extensive body of data indicating that specific chemicals and radiations can be assayed in mammalian cells, both rodent cells (murine, rat, and hamster cells) and cultured human cells, for their ability to cause mutation. Examples of these assays include the assay for mutation to 6-thioguanine (or 8-agazguanine) resistance, which detects base substitution, frameshift, and deletion mutations caused by chemicals and radiations. There is an assay for mutation to ouabain resistance, which detects base substitution mutations in the (Na, K) adenosine triphosphatase (ATPase) in rodent and human cells. There is also an assay in L5178Y mouse lymphoma cells that detects large chromosomal mutations and also point mutations, in which the cells are assayed that are resistant to bromodeoxyuridine. These cells have mutations in or deletions of, the thymidine kinase gene. There is also a simple assay in which cells are exposed to chemical mutagens or radiations, and then the cells are lysed, and DNA damage is assayed in a gel electrophoretic assay (“▶COMET assay”). From results of these assays, we now know that we can easily detect chemicals that cause genetic toxicity, in the form of DNA damage, mutations, gene amplification, or breaks in the single- or double-strands of DNA, or damage to chromosomes (gaps, breaks, fragments, dicentrics, satellites) (▶Clastogenesis). In terms of correlations of genetic toxicity with cancercausing ability, it has also been established that 45% of all chemical carcinogens are mutagens. Hence, these assays detect mutagenesis/DNA damage, and will therefore detect mutagenic chemical carcinogens and radiations. Many chemical carcinogens are also clastogenic agents. The other classes of carcinogens involve those that cause ▶methylation of tumor suppressor genes, rendering them transcriptionally quiescent, or demethylation of oncogenes rendering them transcriptionally active at inappropriate times. Another group of

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carcinogens are certain hormones (▶Hormonal carcinogenesis), such as ▶diethylstilbestrol, estrogen, and ▶testosterone, which act by inducing cell division. Inappropriately high numbers of cell division can lead to spontaneous mutations in cells, or can drive cell division to replicate the DNA of cells that have been adducted by chemical mutagens or damaged by radiations, leading to fixation of chemical mutations. Chemical, Viral, and Radiation Carcinogens Broadly defined, a ▶carcinogen is any material, whether radiation, chemical, or virus, which can induce tumors in lower animals or in humans. ▶Tobacco contains 4,500 different chemicals, and over 20 different known strong human carcinogens, as well as ▶tumor promoters and ▶cocarcinogens. Exposure to tobacco and tobacco products is thought to account for 30% of all human cancers. These include ▶lung cancer, ▶bladder cancer, ▶oral cancer, ▶pharyngeal cancer, ▶laryngeal cancer, ▶nasopharyngeal cancer, ▶esophageal cancer, ▶kidney cancer, ▶liver cancer, and ▶pancreatic cancer, according to the latest US Surgeon General’s report. ▶Second-hand tobacco smoke, or environmental tobacco smoke, also causes cancer in exposed individuals. A second grouping of human cancers is thought to be due to exposure to excess levels of normal hormones, including ▶estrogens and ▶testosterone. Excess estrogen is believed to lead to ▶breast cancer by causing an excessive stimulation of cell division, leading to spontaneous mutations in the oncogenes and tumor suppressor genes of breast epithelial cells. An excessive level of cell division in the epithelial cells of the prostate, driven by testosterone and its metabolites, is thought to contribute to the induction of prostate cancer. Human ▶endometrial cancer is thought to be caused by estrogens without progestogens. ▶Ovarian cancer can be induced by ovulation and the accompanying hormonal changes. Exposure to excess levels of hormones is estimated to induce 30% of new human cancer cases. Dietary influences are very important in human cancer induction. Dietary influences are estimated to contribute to 15% of human cancers. Cancers influenced by diet include ▶stomach cancer, ▶colorectal cancer, and nasopharyngeal cancer. Consumption of an excess of animal fat and excess caloric intake predispose to cancer induction. Conversely, high consumption of green leafy and yellow vegetables and fresh fruits and a diet high in fiber inversely correlate with cancer induction. There are also a number of dietary mutagens formed by pyrolysis of foods. These include the tryptophan metabolites, TRP P1 and TRY P2, and other metabolites such as PhiP and MeIQX. ▶Colon cancer in particular is thought to be induced by an excess of animal fat and deficiency of dietary fiber. Caloric excess in the diet correlates with

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endometrial cancer induction. Excessive ▶alcohol consumption correlates with increased risk of induction of pharyngeal cancer, laryngeal cancer, liver cancer esophageal cancer, oral cancer, colon cancer, and breast cancer (▶Alcohal-rediated Cancer). Another specific example of a known human chemical carcinogen occurring during food consumption is ▶aflatoxin B1, a biocidal metabolite of the mold, Aspergillus flavus. A. flavus biosynthesizes and then utilizes aflatoxin B1 as a biocide against other microorganisms in order to establish its own ecological niche. Aflatoxin B1 is metabolized by cytochrome P450 in mammals to a mutagenic metabolite, aflatoxin B1 2,3-epoxide, which binds covalently to DNA and induces mutations in the ▶p53 tumor suppressor gene and in other genes. This process leads liver cancer in humans. Aflatoxin B1induced liver cancer is common in The People’s Republic of China, Taiwan, Africa, and Mozambique, where hot, wet climates favor the growth of A. flavus, and this fungus contaminates fruits and grains, which are ingested by humans, leading to liver cancer. Exposure to oncogenic viruses is estimated to induce 10% of all human cancers. Notable and well-studied viruses that are oncogenic include the ▶human papillomaviruses, including HPV16 and 18, which induce human ▶cervical cancer. ▶Hepatitis B virus and ▶hepatitis C virus induce human liver cancer in Taiwan, in The People’s Republic of China, and in Mozambique. The ▶human T cell leukemia virus (HTLV) induces human T cell leukemias in Japan and the Caribbean. Infection of humans with the human immunodeficiency virus (HIV) is known to lead to ▶lymphomas. Exposure to ▶Epstein-Barr Virus can lead to benign conditions such as mononucleosis, and also at lower incidences, to Burkitt Lymphoma in Africa and to ▶nasopharyngeal carcinoma in The People’s Republic of China. A fifth category of agents that induce human cancer are drugs, ▶x-rays, and ▶UV radiation. This combined category of carcinogens is estimated to induce 10% of all human cancers. Certain medically approved drugs, such as a fraction of cancer chemotherapeutic agents, including ▶alkylating agents, ▶adriamycin, and ▶tamifoxen, can induce secondary malignancies (▶secondary tumor). Certain analgesics, such as phenacetin, can induce renal pelvic cancer. Prominent examples of radiation carcinogens include ▶ultraviolet light associated with ▶skin cancer and ionizing radiations – gamma rays, x-rays, beta particles, alpha particles, and neutrons. Studies of the atomic bomb blasts in Hiroshima and Nagasaki during World War II have provided the best estimates of dose–response curves for radiation-induced carcinogenesis. UV light from the sun and in tanning salons can induce skin cancer. The sixth broad category of carcinogens that can induce human cancer occurs in the occupational

setting. Occupationally induced cancer is estimated to contribute 5% of all human cancers. In this context, exposure to ▶benzene has been linked in epidemiological studies to the induction of ▶acute myelogenous leukemia, ▶non-Hodgkin lymphoma, ▶chronic myelogenous leukemia, and many other types of leukemia in rubber workers and in workers in shoe factories in the past. Exposure of nickel refinery workers to mixtures of soluble and insoluble ▶nickel compounds as aerosols has been correlated with increased incidences of nasal sinus and respiratory cancers. Similarly, exposure of workers to hexavalent ▶chromium compounds in the chromate manufacturing industry and in the chrome plating industry correlates with induction of lung cancer. Vinyl chloride exposure has led to human lung, liver, and brain cancer, and to the immune-mediated disease, vinyl chloride disease, which is similar to scleroderma. Other examples of occupational carcinogens include bis-chloromethyl ether (lung cancer), ▶asbestos (lung cancer and ▶mesothelioma), and trichloroethylene (cancer at multiple sites) and perchloroethylene. ▶Dioxin (TCDD, or 2,3,7,8-tetrachloro-p-dibenzo-dioxin), a by-product of paper manufacturing, is a potent carcinogen and tumor promoter. ▶Arsenic, a by-product of copper smelting, is also found as an environmental carcinogen in drinking water contaminated by arsenate leached from iron sulfide-bearing rocks. Environmental carcinogenesis is thought to be important also. However, at the present time, no solid estimates of the amount of human cancer that environmental pollution with carcinogens causes can be made with confidence due to our lack of knowledge in this area. Chemical Carcinogenesis Chemical carcinogenesis is the process by which chemical carcinogens (and radiations) can induce tumors in lower animals or in humans. Broadly viewed, chemical carcinogenesis begins as the process in which the very complicated series of ▶signal transduction pathways mediating cellular growth and proliferation and their negative regulators are disrupted and deregulated. Chemical carcinogenesis is clearly a multistep process. Chemical carcinogenesis has been divided conceptually and experimentally in the l930s by Isaac Berenblum, of the Weizmann Institute in Israel, into two stages, initiation and promotion (▶Tumor promotion), when studying carcinogenesis on the backs of shaved mice. Berenblum used small doses of the ▶polycyclic aromatic hydrocarbon 7,12-dimethylbenza(a)anthracene (▶DMBA) as the initiator and the mixture croton oil, a biocide isolated from the plant, Euphorbia lathyris, as a promoter. Initiation is thought to be a mutation, likely in an oncogene such as the ▶RAS cellular oncogene. Stimulation of the initiated

Genetic Toxicology

cells bearing mutations in the RAS or other cellular oncogenes with croton oil stimulates the cells to grow. With repeated croton oil stimulation, the initiated cells form a benign tumor called a ▶papilloma. With further croton oil stimulation, the cells convert into a malignant tumor, called a ▶carcinoma. Further damage to the cells of this carcinoma can result in a metastatic carcinoma. For many decades, investigators have used tetradecanoyl-phorbol acetate (▶TPA), a chemical purified form croton oil, as the tumor promoter. TPA is one of the most potent tumor promoters known, and it binds to ▶protein kinase C to stimulate cell division of initiated cells. Studies of how tumor cells interact with fibroblasts and immune effector cells to enable them to continuously grow and break free of the homeostatic mechanisms of the host that mediate tissue integrity are subjects of intense current interest. Chemically Induced Morphological and Neoplastic Transformation and the Molecular Biology of Carcinogenesis/Cell Transformation and Human Cancer At the cellular level, chemical carcinogenesis first involves the conversion of a normal cell into a tumor cell. There are a number of in vitro cell culture systems in which this process can be studied in a systematic way. They involve studying the loss of contact inhibition of cell division, the loss of anchorage dependence of cell division, escape from calcium ion-induced terminal differentiation, and the eventual acquisition of the property of tumorigenicity. These include C3H/10T1/ 2 Cl 8 mouse embryo fibroblastic cells, Balb/c 3T3 cells, Syrian hamster cells, mouse epidermal keratinocytes, and rat tracheal hamster epithelial cells among the rodent cell systems. Additional human cell systems involve diploid human fibroblasts and human epidermal keratinocytes. This process of malignant cell transformation proceeds first through a series of mutations, rearrangements, or amplifications of oncogenes, or in epigenetic changes involving methylation status of oncogenes, leading to inappropriate expression of the normal oncogene product, or to expression of a mutated oncogene product. Such expression can lead to inappropriately high expression of various proteins involved in signal transduction pathways downstream of the oncogene product in the signal transduction pathway. A number of oncogenes contribute to cellular progression chemical carcinogenesis. Oncogenes are a set of genes controlling cellular growth and proliferation, and consist of a large number of gene families. Prominent members of these families include the ▶RAS gene family, the ▶MYC gene family, ▶ABL, ▶FOS, ▶JUN, ▶ERBB2, FMS, ▶KIT, RAF, SIS, ERBA, ETS, REL, ▶HER2/neu, ▶SRC, ▶BCL-2, BCL-3, BCL-6, HOX1, RHOM-1, RHOM-2, TAL-1, TAL-2, TAN-1, and many others.

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Secondly, this process involves the inactivation of a number of ▶tumor suppressor genes. Tumor suppressor genes are negative regulators of cell growth and proliferation. Prominent members of this large group of genes include the genes ▶RB1, ▶FHIT, ▶VHL, ▶APC, ▶WT1, ▶NF1, ▶NF2, ▶TP53, ▶MEN1, ▶PTEN, and many others. Inactivation of the RB1 tumor suppressor gene leads to release of the transcription factor EF-2 from the Rb–EF2 complex, which can cause cell cycle progression. Mutational inactivation or deletion of TP53 can lead to failure to stop cell cycle progression and allow DNA repair to proceed, leading to an accumulation of mutations in cells. Additionally, inactivation of TP53 can lead to a failure of cells bearing many mutations to undergo ▶apoptosis. This allows accumulation of cells bearing mutations, and allows these cells to progress toward malignancy. The accumulation of mutations, gene amplifications, gene rearrangements, or deletions is thought to lead to conversion of a normal cell into a malignant cell. These include events that activate a number of cellular oncogenes, and that inactivate tumor suppressor genes. Hence, a number of cellular oncogenes would be mutated and activated, or amplified, or rearranged and placed under the control of strong promoters. This would lead to expression of mutant oncogene product, or to higher steady-state levels of normal oncogene product. These protein products then would impact upon signal transduction pathways to stimulate these pathways. In the same manner, mutational inactivation of tumor suppressor genes, methylation of the promoters of these genes to transcriptionally inactivate them, and breakage and loss of part of the chromosome bearing these genes, or loss of the entire chromosome bearing these genes, can also contribute to carcinogenesis. It is thought that fifteen events, including activation of oncogenes and inactivation of tumor suppressor genes, lead to carcinogenesis. What has only recently become known is that fifteen such events can have far-reaching effects on global gene expression, leading to aberrant expression of 100–300 genes in cells, which results in the malignant phenotype. This is because mutation or overexpression of each oncogene can lead to increased expression of 10 additional genes in signal transduction pathways in which this gene participates. Similarly, each tumor suppressor gene may control expression of approximately an additional 10 genes. When this suppressor gene is inactivated, stimulation of its expression of these additional 10 genes is also lost. Hence, physiologically, tumor cells suffer global derangement of gene expression. Our knowledge of this complexity paradoxically provides many opportunities for therapeutic intervention to kill tumor cells, cause them to apoptose, or to cause them to differentiate into nontumorigenic cells.

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References 1. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70 2. Klaasen CD (ed) (2001) Casarett and Doull’s toxicology: the basic science of poisons, 6th edn. Mc-Graw-Hill, New York 3. Verma R, Ramnath J, Clemens F et al. (2004) Molecular biology of nickel carcinogenesis: identification of differentially expressed genes in morphologically transformed C3H/10T1/2 Cl8 mouse embry fibroblast cell lines induced by specific insoluble nickel compounds. Mol Cell Biochem 255:2013–2216 4. Warshawsky D, Landolph JR Jr (eds) (2006) Molecular carcinogenesis and the molecular biology of human cancer. CRC/Taylor and Francis Group, Boca Raton, Florida, New York and London, UK 5. Weinberg RA (2007) The biology of cancer. Garland Science, The Taylor and Francis Group, LLC, Boca Raton, Florida, New York

Genistein. Figure 1 The chemical structure of Genistein.

Definition

Genistein is an ▶isoflavone, derived principally from soybeans, that has cancer preventive and therapeutic effects (see Fig. 1).

Characteristics

Genetically Engineered Mice ▶Mouse Models

Genetically Engineered Model Definition Cancer models created by genetically altering an animal (usually a mouse) to make it more susceptible to cancer. The genetic alteration can cause the animal to overexpress a gene that promotes tumor development or inactivate a gene that suppresses tumor development. ▶Ultrasound Micro-Imaging ▶Gene Knockout ▶Transgenic ▶Knock-out mice

Genistein RUIWEN Z HANG University of Alabama at Birmingham, Birmingham, AL, USA

Synonyms 4′,5,7-Trihydroxyisoflavone; Soy phytoestrogen

Soy and Cancer Numerous epidemiological studies have indicated that a diet high in soy products can decrease the risk of developing cancers, including ▶breast cancer, ▶prostate cancer, ▶colon cancer, ▶lung cancer and ▶skin cancer. In countries where soy products are frequently consumed, there is a lower incidence of these and other cancers. A study of more than 600 women in Singapore (420 healthy controls and 200 women with confirmed breast cancer) demonstrated that ingestion of soy products correlated with a decrease in cancer risk. Moreover, a similar study with men showed that soy consumption decreased prostate cancer mortality. Two isoflavones, genistein and daidzein, appear to be the major mediators of the cancer preventive activity of soybeans. For genistein, which is probably the primary active component, a body of work related to its cancer preventive and therapeutic effects, as well as to its mechanisms of action, has emerged. Cancer Prevention by Genistein Studies of ▶chemical carcinogenesis and cancer development in transgenic animals have demonstrated that administration of genistein can decrease the incidence of cancer and decrease the multiplicity of tumors that develop. For example, treatment of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with 100–500 mg genistein/kg diet reduced the incidence of advanced-stage prostate tumors in a dose-dependent manner. A high-isoflavone diet also inhibited methylnitrosourea-induced prostate tumors in Lobund-Wistar rats. Treatment of mouse mammary tumor virus (MMTV)-neu mice with diets containing 250 mg/kg of genistein or daidzein increased the latency for spontaneous breast tumors but did not affect tumor size or multiplicity. Topically applied to ▶DMBA-initiated and ▶TPA-promoted Sencar mice,

Genomic Imbalance

genistein reduced the incidence and multiplicity of skin tumors by 20% and 50%, respectively. In addition to its chemopreventive effects, genistein can be used therapeutically. It increases the survival of animals bearing ▶xenograft, chemically induced and spontaneous (transgenic) tumors and improves the response to conventional therapies. Genistein can act additively or synergistically with chemotherapeutic agents, hormone therapy, ▶immunotherapy or ▶radiation therapy. Epidemiological studies with cancer patients suggest that increased soy consumption may increase survival. Moreover, while randomized trials are still ongoing (or have yet to be performed for many types of cancers), genistein appears to have little or no toxicity. Mechanisms of Action of Genistein Although the mechanisms of action for the anticancer effects of genistein have not been established, there are a number of candidate pathways. Reported in vitro and/or in vivo activities of genistein include estrogen agonism/antagonism; inhibition of protein tyrosine phosphorylation; ▶topoisomerase inhibition; suppression of ▶angiogenesis; scavenging of free radicals; and inhibition of ▶matrix metalloproteinases, ▶NF-κB and oncogenes, such as ▶MDM2. Any or all of these effects can decrease carcinogenesis and tumor progression, lead to the induction of apoptosis and cell differentiation, and suppress ▶metastasis.

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Other Indications Because of its estrogenic effects, genistein has been used to ameliorate the effects of menopause. Studies so far have been inconclusive, although some suggest that genistein can improve the symptoms. Future studies, especially those involving use of randomized placebo controls, will be needed to determine its efficacy. Based on its estrogenic, anti-oxidant and other properties, genistein has also been suggested to prevent osteoporosis, improve cognitive function, decrease cholesterol, and improve cardiovascular health.

References 1. Dixon RA, Ferreira D (2002) Genistein. Phytochemistry 60:205–211 2. Sarkar FH, Li Y (2004) The role of isoflavones in cancer chemoprevention. Front Biosci 9:2714–2724 3. Lambert JD, Hong J, Yang GY et al. (2005) Inhibition of carcinogenesis by polyphenols: evidence from laboratory investigations. Am J Clin Nutr 81:284S–291S 4. Ravindranath MH, Muthugounder S, Presser N et al. (2004) Anticancer therapeutic potential of soy isoflavone, genistein. Adv Exp Med Biol 546:121–165 5. Li M, Zhang Z, Hill DL et al. (2005) Genistein, a dietary isoflavone, down-regulates the MDM2 oncogene at both transcriptional and posttranslational levels. Cancer Res 65:8200–8208

Genomic Imbalance Bioavailability and Safety The ▶bioavailability of genistein appears to be greater than that of many other natural compounds. Plasma concentrations of genistein in tumor-bearing ▶nude mice fed a diet containing 1 mg/g genistein were 3.4 μmol/L; in humans, a single oral dose of 460 mg resulted in peak plasma concentrations of 20–25 μmol/L. In most commercially available products, isoflavones are present as glycosides, which may require hydrolysis by intestinal beta-glucosidases. After absorption, ▶phytoestrogens are primarily converted to glucuronic acid derivatives. Extensive metabolism, accomplished by bacteria in the gut, leads to the formation of equol and lignans. Although genistein is generally regarded as safe, and toxic effects of genistein do not occur when cells are exposed to physiologically relevant levels of the compound, there are some undesirable effects, most of which have been observed in animal models and are due to its hormone-like properties. Because there is evidence from in vitro and animal studies that it can stimulate the growth of hormone-dependent cancers (especially ▶estrogen receptor alpha positive breast cancers), use of genistein to treat these cancers is controversial.

R OBERTA VANNI Department of Biomedical Science & Technology, University of Cagliari, Monserrato (CA), Italy

Definition Refers to a genome showing any loss or gain of DNA sequences compared with the reference DNA whole sequence of the genome of interest. The term usually indicates extensive disequilibrium in the number of chromosomes or chromosome segments per cell.

Characteristics Genomic Imbalance Concept The relative dosage of genes governing cell physiology, is the product of evolution. Human somatic cells have evolved their “genome balance” as ▶diploid, and variation in the number of chromosome copies as compared to the euploidy which has evolved, leads to genomic imbalance. This affects gene dosage and, consequently, promotes imbalances in cellular pathways. Most cancer cells acquire genomic imbalance as a consequence of ▶aneuploidy, i.e. an abnormal copy number

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Genomic Imbalance

of genomic elements, identified as gain and/or loss of whole chromosome(s) (aneuploidy) or chromosome segment(s) (partial aneuploidy). Genomic imbalance reflects the ▶karyotype complexity of the cancer cells. Cancer as a Genetic Disease Cancer as a genetic diseasse involving genomic imbalance is a concept that has been explored since the end of the nineteenth century. In 1890 Paul von Hansemann (1858–1920), noticed that chromosomes in carcinomas segregate in unbalanced fashions, and at the beginning of the twentieth century, in 1914, Theodor Boveri (1862–1915) proposed his “chromosome theory of cancer,” according to which cancer results from abnormal chromosomal composition within cells. Nowadays, this concept is widely accepted, based on molecular evidence, according to which cancer is a biological process characterized not only by accumulation of chromosome changes, but also by gene mutations, all contributing to the cell transformation process. The loss of critical cellular checks and balances throws proliferation processes into disorder by means of sequential steps, broadly including ▶initiation, ▶progression, ▶invasion and ▶metastasis. Cause of Genomic Imbalance in Cancer Genomic imbalance is the product of a phenomenon called ▶chromosome instability (CIN), resulting in the increased probability that during cell division, whole chromosomes or chromosome segments will be acquired or lost. CIN is actually an ongoing process, responsible for variation in the rate of chromosome changes according to Darwinian selection, and may end with the production of stable aneuploid cells having proliferative advantages. A certain degree of cytogenetic heterogeneity within a tumor is maintained by CIN, and cytogenetically related or unrelated clones often persist alongside the favored one, contributing to intratumoral genomic imbalance. Many aggressive cancers, such those of the bladder, brain, breast, bone, cervix, colon, gallbladder, head and neck, liver, lung, ovary, pancreas, prostate, and testis show an accumulation of complex rearranged chromosome patterns, which may include, in addition to aneuploidy, ▶double-minutes and the formation of ▶homogeneously-staining regions. CIN, and the consequent genomic imbalance, calls for the interaction of various outcomes, including, abnormal mitotic segregation, abnormal shortening of ▶telomeres and ▶mitotic checkpoint deficiency. . Abnormal mitotic segregation. Chromosome segregational defects may be due to different mechanisms, such as ▶multipolar spindles formation, loss of ▶sister chromatid cohesion, ▶kinetochore defects during spindle attachment.

Multipolar spindles may develop in the presence of supernumerary ▶centrosomes. Centrosome amplification is frequently seen in almost all types of solid tumors, and to a lesser extent in leukemia and lymphoma. Its occurrence is strongly associated with a high degree of aneuploidy. Amplification of STK15/ aurora kinaseA gene, inactivation of tumor suppressor genes, such as TP53 (Tumor Protein 53), RB1 (Retinoblastoma), and BRCA1 (breast cancer1) as well as overexpression of cyclins D and E, play a prominent role in this process. Loss of sister chromatid cohesion is crucial in the metaphase-to-anaphase transition and the correct separation of genetic material in the cell. Cohesion is maintained by proteins called ▶cohesins, which have to be degraded to lead to sister chromatid separation. Cohesins are released through a number of catabolic steps driven by the anaphase-promoting complex (APC), a ▶ubiquitin ligase, targeting proteins for degradation. In yeast, APC acts on ▶securin proteins, which in turn prevent ▶separin (a cysteine protease, also called separase) from promoting sister chromatid separation. Human homologs of yeast separin and securin are respectively ESP1 (Extra Spindle Poles-like1) and ▶PTTG1 (Pituitary Tumor-Transforming Gene1) proteins, and their inactivation interferes with the correct distribution of genetic material at ▶anaphase. ▶Kinetochore defects during spindle attachment may also have a major impact on chromosome malsegregation. Kinetochore is a highly-complex proteinaceous structure located in centromeric DNA and providing dynamic attachments to spindle microtubules, mediating the chromosome bi-orientation necessary to facilitate accurate segregation. Disturbances in this process result in aneuploid cells. . Abnormal shortening of telomeres. This phenomenon is observed in both benign and malignant tumors. Shortening of ▶telomeres during normal cell development and aging is the rule; however, in somatic cells that have lost proliferation control, it gives rise to genome instability and abnormal cell proliferation. Such abnormal shortening causes telomeres to lose their ability to protect chromosome ends from fusion. The products of chromosome fusion contribute to CIN by the formation of anaphase bridges, leading to structural and numerical chromosome changes or, through cell division inhibition, to poliploydization and subsequent multipolar spindle formation. . Mutations at mitotic checkpoint. The regular mitotic phase of the ▶cell cycle involves an orderly set of events, subdivided into phases: the prophase, prometaphase, metaphase, anaphase and telophase. In a normal cell, anaphase can start only when all chromosomes have congressed to the metaphase plate and achieved bipolar attachment to the mitotic

Genomic Imprinting

spindle. This scenario requires a powerful ▶mitotic checkpoint, based on a dynamic balance of ▶ubiquitination (by APC) and deubiquitination (possibly by ▶USP44, Ubiquitin-Specific Protease44) of target proteins. Breakdown of mitotic checkpoint signaling brings on cell death, whereas its weakening, i.e. minor changes in checkpoint protein levels, does not compromise cell viability but promotes aneuploidy, since the mitotic checkpoint does not recognize a single or a few chromosomes lacking spindle attachment. Although the role of genomic imbalance in tumorigenesis is recognized, and for some tumors diverse genomic imbalances correlate with prognosis, whether or not it may actually initiate tumorigenesis is still being debated.

References 1. Von Hansemann D (1890) Ueber asymmetrische Zellteilung in Epithelkrebsen und deren biologische Bedeutung. Virschows Arch Pathol Anat 119:299–326 2. Boveri T (1914) Zur Frage der Entstehung Maligner Tumoren. Gustav Fischer Verlag, Jena 3. Stegmeier F, Rape M, Draviam VM et al. (2007) Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446:876–881 4. Duesberg P, Li R, Helmann R (2006) Aneuploidy and cancer: from correlation to causation. In: Dittmar T, Zaenker KS, Schmidt A (eds) Infection and inflammation: impacts on oncogenesis (Contributions to Microbiology), vol 13 Basel, Karger, pp 16–44 5. Michor F, Iwasa Y, Vogelstein B et al. (2006) Can chromosomal instability initiate tumorigenesis? Semin Cancer Biol 15:43–49

Genomic Imprinting A NDREW P. F EINBERG Department of Medicine and Center for Epigenetics, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Definition

Is an ▶epigenetic alteration (i.e., not involving a change in base sequence) of a specific parental ▶allele of a gene, or the chromosome on which it resides, in the gamete or zygote, leading to differential expression of the two alleles of the gene in somatic cells of the offspring. Genomic imprinting challenges two

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assumptions of conventional Mendelian genetics applied to human disease: that the maternal and paternal alleles of a gene are equivalent, and that two functional copies of a gene always are associated with health (Table 1).

Characteristics Imprinted genes probably account for many examples of developmental malformations in humans, as uniparental disomy (UPD) of several chromosomes is associated with a variety of recognized defects, but UPD affecting most or all of a chromosome is rarely seen. Imprinting may have arisen in mammals as a result of an evolutionary conflict between maternal and paternal genomes. There is a strong and sometimes surprising relationship between imprinted genes and growth, including both prenatal growth and postnatal growth related to nurturing ability. Imprinting is also thought to underlie some quantitative trait loci for growth, with considerable potential commercial application in animal husbandry, and imprinting may be a potential barrier to stem cell transplantation. For all these reasons, genomic imprinting has generated intense interest. Imprinted Genes and their Regulation Despite this interest, the comprehensive identity of all imprinted genes remains unknown. At present over 80 imprinted genes are suspected in one or more species (see http://www.geneimprint.com). Imprinted genes appear to be organized within genomic domains. Evidence for this idea comes from studies of the region of 15q11-13 involved in Prader-Willi and Angelman syndromes, which cause mental retardation and neurological problems. This region harbors at least six imprinted genes and the region of the IGF2R gene contains at least three. Similarly, band 11p15 contains a domain of imprinted genes distributed over *1Mb. These genes play diverse roles including hormonemediated growth stimulation (insulin-like growth factor II, or IGF2), control of the cell cycle (p57KIP2) and ion trafficking (KVLQT1). Both cis-acting and trans-acting factors appear to be important in the regulation of genomic imprinting. It is believed that cis-acting sequences can regulate imprinting over large distances. Evidence for this idea comes from the identification of patients with microdeletions involving upstream exons of the small nuclear ribonucleoprotein N gene (SNRPN), in the Prader-Willi region of chromosome 15. These deletions lead to disrupted imprinting extending over several megabases. This deletion site has therefore been termed an “imprinting center” for this chromosomal region. However, the molecular basis for the function of the imprinting center is as yet unknown.

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Genomic Imprinting. Table 1

Key ideas of genomic imprinting and cancer

Genomic imprinting

An epigenetic alteration in the gamete leading to differential allele expression in the offspring

Examples of imprinting in disease

UPD with birth defects Specific disorders (PWS, AS) Human cancer Loss of imprinting (LOI) in childhood and adult tumors Both gene silencing and gene activation Link to cancer risk DNA methylation Conserved regulatory elements and genomic domains Modifier genes

Imprinting in cancer

Mechanisms

Imprinting and DNA Methylation Cytosine DNA ▶methylation also appears to be important in the regulation of genomic imprinting, as loss of the cytosine DNA methyltransferase I gene (Dnmt1) disrupts normal genomic imprinting in mice. DNA methylation is a covalent modification of DNA in which a methyl group is transferred from S-adenosyl methionine to the C-5 position of cytosine. DNA methylation occurs almost exclusively at CpG dinucleotides. CpG islands, which are sequences unusually rich in ▶CpG dinucleotides, are usually found in the vicinity of imprinted genes. Recently, one mediator of imprinting was discovered. It is the CCCTC-binding factor (▶CTCF), a transcription factor that binds to an unmethylated, GC-rich sequence *2kb upstream of the H19 gene. The latter has previously been shown to be necessary for normal imprinting of H19 and ▶IGF2. The same CTCF does not bind to methylated DNA. CTCF is known to be an insulator binding protein, and its binding prevents access by IGF2 to a shared enhancer 3′ to H19 (and about 200 kb telomeric to IGF2 itself). Loss of Imprinting (LOI) in Cancer In 1993, it was discovered that IGF2 can undergo loss of imprinting (LOI) in cancer, with abnormal expression of the normally silent maternal allele. LOI of IGF2 has been found in a wide variety of tumors, including embryonal tumors and adult malignancies. In the case of ▶colon cancer, LOI was found not only in the tumors but in the normal tissues of patients with colon cancer. Furthermore, multiple well-controlled studies have now shown an association of LOI with a positive family history and personal history of colorectal tumors, both benign and malignant. Studies of mouse models with LOI and a gene mutation causing intestinal tumors show that LOI increases tumor initiation, and acts by increasing and altering the tissue stem cell compartment. Thus, tests for LOI may eventually prove useful

for identifying patients at risk of cancer, thereby reducing overall cancer mortality (Fig. 1). In the case of embryonal tumors, this activation of the maternal allele of IGF2 is coupled to silencing of the maternal H19 allele and methylation of a normally unmethylated maternal H19 ▶CpG island. However, LOI of IGF2 occurs independently of H19 in adult tumors, such as ▶cervical cancer and ▶brain cancer. LOI of IGF2 is now recognized as one of the most common genetic alterations in human cancer. LOI of IGF2 is also found in about 15% of patients with ▶Beckwith-Wiedemann syndrome (BWS). BWS is a disorder of prenatal overgrowth, birth defects and cancer, which is transmitted as an ▶autosomal dominant trait, although most cases arise sporadically. In addition, LOI of the LIT1 gene, also on 11p15, is found in about 40% of BWS patients. This gene is particularly interesting, as it is an antisense transcript normally expressed from the paternal allele, that lies entirely within the maternally expressed KVLQT1 gene. A link between LOI and DNA methylation is also suggested by studies using the drug 5-aza-2′deoxycytidine, which inhibits DNA methylation. This drug can restore a normal pattern of imprinting to tumor cells with LOI, suggesting that this or other pharmacological agents might eventually be used to treat cancers specifically with LOI or as chemopreventive agents in patients with LOI in their normal tissues. Frequent loss of heterozygosity (▶LOH) of 11p15 has also been observed generally in embryonal tumors, including ▶Wilms tumor, ▶rhabdomyosarcoma and ▶hepatoblastoma. LOH of 11p15 is one of the most common genetic changes in cancer and is found in many adult malignancies, including those of the stomach bladder, ovary, breast and lung. Further strong support for the existence of an embryonal ▶tumor suppressor gene on 11p15 derives from genetic complementation experiments. ▶Microcell-mediated chromosome transfer of a human chromosome 11 suppresses the growth of rhabdomyosarcoma cells

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sequences is by comparative genomics, in which the mouse sequence is obtained and compared to human sequence, in order to identify species conserved orthologous elements. These elements can then be tested in functional assays and be analyzed for mutations or deletions in patients with cancer or ▶BWS. Proteins that interact with such sequences could include as yet unidentified modifiers of DNA methylation and/or genomic imprinting. Thus, one of the most important implications of the study of imprinting is that the lessons learned may be applicable to understanding the regulation of genomic domains generally, and their dysregulation may provide novel insights into the mechanism of cancer.

References

Genomic Imprinting. Figure 1 Upper panel shows a normally imprinted IGF2 and H19 gene, with IGF2 expressed (drawn large) from the paternal allele, and H19 from the maternal allele. A shared enhancer (green) cannot cross an unmethylated CpG island (small open circles) upstream of the maternal H19 allele, presumably because of binding of CTCF to this island. The lower panel shows loss of imprinting (LOI) in cancer, with a switch of the paternal chromosome to a maternal epigenotype. Here the H19 CpG island is also methylated on the maternal chromosome, allowing the enhancer to interact with IGF2, with activation of the maternal IGF2 allele and silencing of the maternal H19 allele.

in vitro. Furthermore, subchromosomal transferable fragments limited to 11p15 do suppress tumor cell growth. However, in the investigation of an 11p15 tumor suppressor gene it is puzzling that in virtually all cases of LOH of 11p15 in embryonal tumors it is the maternal allele that is lost, an observation made before the discovery of human imprinted genes or such genes on 11p15 in particular. To address this problem, Sapienza argued that a gene that is not normally imprinted might become so aberrantly. According to Sapienza, a specific parental allele (in this case the paternal) would become silenced in some individuals. One or more of the maternally expressed genes on 11p15 might fulfill this role. One of the most exciting frontiers in the study of genomic imprinting and its role in cancer is the identification of sequences that lie between genes, which might serve a regulatory role in the maintenance of normal imprinting of large genomic domains, such as on 11p15. One recent approach to identifying such

1. Moore T, Haig D (1991) Genomic imprinting in mammalian development: a parental tug-of war. Trends Genet 7:45–49 2. Nicholls RD, Saitoh S, Horsthemke B (1998) Imprinting in Prader-Willi and Angelman syndromes. Trends Genet 14:194–200 3. Feinberg AP (2001) Genomic imprinting and cancer. In: Scriver CR et al. (eds) The metabolic and molecular bases of inherited disease, 8th edn. McGraw-Hill, New York, pp 525–537 4. Cui H, Horon IL, Ohlsson R et al. (1998) Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability. Nat Med 4:1276–1280 5. Sakatani T, Kaneda A, Iacobuzio-Donahue CA et al. (2005) Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice. Science 307:1976–1978

Genomic Instability Definition

▶Genetic Instability

Genomics Definition Denotes the complete study of the hereditary material of living beings: both the coding and noncoding portions. Structural genomics involves creating “maps” of genomes, carrying out DNA sequencing, and determining the localization and the regulatory regions of genes on ▶chromosomes. Functional genomics is the branch of genomics that studies the biological function of the

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Genotoxic

genes and their products, the coded proteins, their expression and regulation, as well as the interactions between different genes.

Genotype Definition

Genotoxic Definition Causes damage to DNA. Examples of genotoxic agents are chemicals or radiations that cause mutation to cells, chromosome breakage in cells, gene ▶amplification in cells, or single-stranded or ▶DNA-double strand breaks. ▶Chemically Induced Cell Transformation ▶Chemical Carcinogenesis ▶DNA Damage

Catalog of individual’s two alleles at a particular DNA location. Refers to the complete genetic composition of a single cell or organism. It also can refer to the specific allelic composition of a particular gene or group of genes. ▶Arylamine N-Acetyltransferases (NAT) ▶Biomonitoring ▶Linkage Disequilibrium

Geranylgeranyl Definition

Genotoxic Carcinogen Definition

C20 isoprenoid lipid, an intermediate in the HMG-CoA reductase mevalonate biosynthetic pathway, used in the biosynthesis of geranylgeranylated proteins. ▶Rho Family Proteins

A carcinogen capable of causing a change to the structure of the genome. ▶Toxicological Carcinogenesis

Genotoxicity Tests Definition

Geranylgeranylation Definition Post-translational modification of proteins by the attachment of an isoprenoid to C-terminal cysteine residues.

These represent a range of standardized assays designed to test chemicals for interaction with DNA, i.e. genotoxicity or mutagenicity. These comprise a number of different tests both in vitro and in vivo. The most usual study conducted early in the development of a new drug is the ▶Ames Assay that utilizes mutants of Salmonella typhimurium. Various mammalian cell culture systems are also employed such as Chinese hamster ovary (CHO) cells. Clastogenic activity is assessed by exposing cells to chemicals and examining cells microscopically for chromosome damage. An in vivo study is also often performed examination of the bone marrow in treated rodents.

▶Statins

▶Preclinical Testing ▶Clastogen

▶Cowden Syndrome ▶Germ Cell Tumors

Germ Cell Layers Definition Collection of cells formed during embryogenesis into the mesoderm, endoderm and ectoderm that go on to form specific organs of the body.

Germ Cell Tumors

Germ Cell Tumors C RAIG KOVITZ 1 , P HILLIPPE E. S PIESS 2 , N IZAR M. TANNIR 3 1

Department of Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA 2 Department of Urologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA 3 Department of Genitourinary Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Synonyms Testicular tumors; Gonadal neoplasms; Dysgerminomas

Definition

▶Testicular cancer represents a group of histologically heterogeneous neoplasms typically arising in gonadal tissue and, uncommonly, arising in extragonadal sites such as the retroperitoneum or mediastinum. A disease of young men which, when metastatic was previously uniformly fatal, testicular cancer is now usually cured.

Characteristics Incidence Germ cell tumors represent the most common cancer in young men between the ages of 20 years and 40 years. These tumors have a bimodal age distribution being most common in men ages 15–25 with a second, smaller peak at about age 60. It is estimated that 8,000 new cases of testicular cancer were diagnosed in the United States in 2005. During the past century, the worldwide incidence of testicular neoplasms has nearly doubled, with the highest increases reported in the United States, Great Britain and Northern Europe. Risk Factors Numerous case-control and cohort studies have established cryptorchidism as the major identifiable risk factor, although only about 10% of cases are associated with this phenomenon. This increased risk is true for the contralateral testicle even if it is normally descended. Additional risk factors include a personal history of testicular cancer as well as the presence of a first degree relative with the disease. Scrotal trauma and toxic exposures have not clearly been associated with the occurrence of germ cell tumors. Histological Classification The main histological categories of germ cell tumors are seminomas (▶Seminomatous Germ Cell Tumor) and non-seminomas (▶Non-seminomatous Gene Cell

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Tumor). Non-seminomas are further subcategorized as embryonal carcinomas, endodermal sinus tumors (also known as yolk sac tumors), choriocarcinoma and ▶teratoma. Tumors that contain more than one histological subtype are termed mixed germ cell tumors. For classification and treatment purposes, any tumor not histologically a pure seminoma is classified as a non-seminoma. Clinical Presentation and Diagnosis The majority of patients with testicular cancer presents with a painless testicular swelling or a palpable mass. In many cases, testicular swelling can be accompanied by pain secondary to bleeding or infarction within the tumor. Systemic symptoms at presentation such as abdominal pain, decreased appetite with or without associated weight loss, night sweats, chest pain, shortness of breath or hemoptysis usually indicate either an advanced stage of disease or an extragonadal primary tumor. If the diagnosis of a germ cell tumor is suspected, a bilateral high-resolution testicular ultrasound should be performed. In addition, the tumor markers human chorionic ▶gonadotropin (hCG), ▶alpha-fetoprotein (AFP) and lactate dehydrogenase (LDH) have unique diagnostic and prognostic significance in germ cell tumors and levels should be obtained on all patients with suspected germ cell tumors. Though not specific for germ cell tumors, AFP is produced by tumors with endodermal sinus or embryonal components as well as by immature teratomas. HCG is a hormonal product of syncitiotrophoblasts and can be expressed by choriocarcinomas, mixed germ cell tumors and, sometimes, by seminomas. LDH is a cellular protein expressed in numerous tissues and can be produced by non-seminomas. These markers are important for ▶staging of tumors, monitoring therapy, deciding when to apply surgical consolidation as well as to sensitively detect residual or recurrent disease. Additional preoperative workup typically includes a chest radiograph and discussion of sperm banking with the patient. When a testicular mass is found on ultrasonography, a radical inguinal ▶orchiectomy should be performed, unless the patient is ill, and initiation of systemic therapy is deemed urgent. In this instance, it would be appropriate to proceed with ▶chemotherapy without tissue diagnosis, if the markers are elevated and the clinical picture is compatible with the diagnosis of germ cell tumor. Trans-scrotal biopsies should be avoided as they can disrupt regional lymphatics, potentially altering the typically predictable lymphatic spread of these tumors. Postoperatively (or preoperatively if this will not delay surgery) an abdominal and pelvic computed tomography (CT) scan should be obtained and, if clinically indicated, brain magnetic resonance imaging (MRI) and a bone scan. CT scans can reveal clinically significant adenopathy

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that will be important in clinical decision-making. The preferred sites of initial spread for right-sided tumors are typically the infra-renal paracaval, inter-aortocaval and possibly paraortic nodes. In contrast, left-sided tumors preferentially spread to the infra-renal paraortic nodes initially. Staging and Risk Stratification As with all staging systems, the purpose is to classify patients with respect to prognosis and to allow one to offer treatments having a toxicity profile commensurate with the burden of disease and risk of recurrence. Given the importance of tumor markers in the management of germ cell tumors, the staging for germ cell tumors takes these into account along with histology, site of origin and anatomic extent of disease. The standard risk stratification used for these tumors is that developed by the International Germ Cell Cancer Consensus Group (IGCCCG). Through a retrospective multivariate analysis of nearly 6,000 patients with germ cell tumors, this group identified the clinical features strongly associated with prognosis: primary site of disease, the presence of non-pulmonary visceral metastases and marker levels at the initiation of therapy. Using these features, they divided non-seminomatous germ cell tumors into good-, intermediate- and poor-risk categories and seminomas into good- and intermediate-risk categories (Table 1). This risk stratification system provided the basis for the

Germ Cell Tumors. Table 1

Good risk

Intermediate risk

Poor risk

most recent American Joint Commission on Cancer (AJCC) TNM staging system for germ cell tumors. In general, Stage I disease is confined to the testis, stage II disease is disease that does not spread beyond the retroperitoneum and stage III disease involves the nodal regions beyond the retroperitoneum or non-nodal metastatic disease. Elevated serum tumor markers can help to define higher stages of disease. Management As the biology and management of seminomas and non-seminomas are quite different, we will consider the management of these distinct histologies separately. Seminomas By IGCCCG risk stratification, all patients with seminomas without non-pulmonary visceral metastates are categorized as having good-risk disease. Amongst these patients are those with Stages I and II disease and some with Stage III disease. The majority of patients with Stage I disease will be cured with radical inguinal orchiectomy alone, however, ~15–20% of patients with Stage I seminoma will have recurrent disease. As a result, there exists a need to identify those features of Stage I disease that would indicate a higher risk of relapse so that such patients could be offered adjuvant therapy. It has been reported that there is a subset of patients with Stage I seminoma with a primary tumor

International germ cell cancer consensus group classification prognostic risk stratification Seminoma

Nonseminoma

Any primary site No non-pulmonary visceral metastases Normal AFP, any hCG, any LDH

Testis/retroperitoneal primary and No non-pulmonary visceral metastases and AFP < 1,000 ng/mL and hCG < 5,000 mIu/mL and LDH < 1.5 × upper limits of normal range (ULN) 86% 5-year PFS; 90% 5-year OS Testis/retroperitoneal primary and No non-pulmonary visceral metastases and AFP 1,000–10,000 ng/mL and hCG 5,000–50,000 mIu/mL and LDH 1.5–10 × ULN 75% 5-year PFS; 80% 5-year OS Mediastinal primary or Non-pulmonary visceral metastases or AFP > 10,000 ng/mL or HCG > 50,000 mIu/mL or LDH > 10 × ULN 41% 5-year PFS; 48% 5-year OS

82% 5-year PFS; 86% 5-year OS Any primary site Non-pulmonary visceral metastases Normal AFP, any hCG, any LDH

67% 5-year PFS; 72% 5-year OS –

Germ Cell Tumors

less than 4 cm in size and without rete testis involvement who have a relapse free survival of 88%, and thus may constitute a group of patients most appropriate for surveillance. At present, surveillance is considered a reasonable option for highly motivated patients with Stage I seminoma after orchiectomy, though given the fact that nearly one third of patients with recurrent disease will require systemic chemotherapy, this has not been the most commonly used approach. At present, the majority of patients with Stage I seminoma are treated with infra-diaphragmatic radiotherapy (20 Gy) to the para-aortics. Such treatment results in 5-year survival rates of 98–99%. Data exists to support the use of a single dose of ▶carboplatin as ▶adjuvant therapy in Stage I seminoma, but this practice has not been widely adopted at present. Recently published retrospective data from the Princess Margaret Hospital in Toronto, Canada, showed comparable long-term results for patients with stage I seminoma treated with radiation prophylactically versus those with stage I who elected to be followed with active surveillance with initiation of therapy at the time of recurrence. Patients with Stage II disease are divided into those with non-bulky disease (those with nodal metastases no larger than 5 cm) and those with bulky disease (those with nodal metastases greater than 5 cm). For patients with non-bulky disease (Stage IIA-B), infradiaphragmatic radiation therapy (20 Gy) to include the para-aortics and ipsilateral iliac nodes with a boost (6–10 Gy) to the involved site is the standard therapy. Residual abnormalities are sometimes encountered following radiation therapy, but observation is generally recommended. For patients with advanced seminoma, defined as having Stage IIC or Stage III disease, chemotherapy is the treatment modality of choice. For those patients with good-risk disease, chemotherapy with four cycles of ▶etoposide and ▶cisplatin (EP) is generally offered. ▶Bleomycin is generally excluded as the risk of pulmonary toxicity outweighs the small incremental benefit afforded by its use. In the same light, ▶carboplatin has been demonstrated to be inferior to cisplatin and is thus not substituted in this scenario. For those patients with intermediate-risk disease, chemotherapy is usually administered with four cycles of ▶bleomycin, ▶etoposide and ▶cisplatin (BEP). After completion of chemotherapy, patients with advanced seminoma are restaged with chest, abdominal and pelvic CT scans as well as serum tumor markers. If no residual mass is found, or the residual mass measures 3 cm in size, a ▶positron emission tomography (PET) scan is performed, if available, to assess for the presence of viable tumor. In the presence

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of a positive PET scan, salvage radiation therapy is offered at our institution. For those patients unable to undergo PET scan, the size of the residual mass guides post-chemotherapy management, with masses greater than 3 cm having a higher risk of relapse. In this case, a surgical biopsy or consolidative radiation therapy are the interventions of choice. Patients who are found to have progressive disease after initial therapy are generally treated with salvage chemotherapy. Non-Seminomatous Germ Cell Tumors (NSGCT) NSGCT are risk stratified according to the IGCCCG classification schema based on the location of the primary tumor, the presence of non-pulmonary visceral metastases and the level of tumor marker elevation. Treatment options vary by stage and can include observation, chemotherapy and/or retroperitoneal lymph node dissection (RPLND). Patients with Stage IA NSGCT (tumor limited to the testis and epididymis without lympho-vascular invasion and normal post-orchiectomy tumor markers) are generally managed with either surveillance (in reliable patients) or RPLND. The reason for surveillance as an option is that the majority of patients with Stage I NSGCT will not have recurrence and of those approximately 30% who do have recurrent disease, there is effective chemotherapy which can result in long-term survival for most patients. RPLND is often used because it usually leads to accurate staging and can be curative in the majority of patients. The presence of N1 or N2 disease found at RPLND can then be managed either with surveillance or two cycles of EP or BEP chemotherapy. Patients with N3 disease found at RPLND are typically managed as good-risk advanced stage patients and are treated with EP for four cycles or BEP for three cycles. Patients with Stage IB NSGCT are generally managed with RPLND, although either active surveillance (for T2 disease only) or chemotherapy with two cycles of BEP is also appropriate. PostRPLND management is as stated above for Stage IA disease. Patients with Stage IS disease (persistent marker elevation after orchiectomy) are managed with chemotherapy (either four cycles of EP or three cycles of BEP). Stage IIA NSGCT in the presence of negative postorchiectomy tumor markers are generally approached with either RPLND or primary chemotherapy with EP for four cycles or BEP for three cycles. Those patients with Stage IIA disease who have persistent tumor marker elevations are managed with chemotherapy alone. Patients with Stage IIB disease and negative markers may undergo RPLND as long as lymph node metastases are within lymphatic drainage sites. PostRPLND management is the same as for those patients who have RPLND for Stage I disease. If the patient

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Germinal Center

has multi-focal lymph node metastases, adjuvant chemotherapy is considered the management option of choice. For patients who have received chemotherapy and have a residual retroperitoneal mass posttreatment, RPLND is generally performed to look for viable tumor, or teratoma. Patients with advanced disease (Stage IIC and III) disease are risk stratified by the IGCCCG into three categories (good, intermediate and poor). Those with good-risk disease (testis or retroperitoneal primary; no non-pulmonary visceral metastases as well as AFP >f

Lung

1st–8th Dec.; peak 2nd/3rd Dec. Median 40y

Solitary, rarely multifocal 50% Multifocal

f>m

>50% Multifocal

Liver

2nd–9th Dec.

f>m

>50% Multifocal

Mortality (%)

35

The majority of primary epithelioid hemangioendothelioma arises from soft tissue, bone, lung, and liver. The worst outcome is seen in the lung and hepatic tumors. There is a difference in age and gender distribution of EH in various sites [1].

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Hepatic Epithelioid Hemangioendothelioma

Hepatic Epithelioid Hemangioendothelioma. Figure 1 Hepatic epithelioid ▶hemangioendothelioma. (a) Macroscopy: Multifocal growth pattern of nodules and coalescence of them to form confluent masses specifically in the periphery with extension to and retraction of liver capsule. (b) Hematoxylin–eosin stain: The tumor cells form intracytoplasmic lumina containing erythrocytes. (c) Tissue stain for factor VIII-related antigen: In addition to the intracytoplasmic lumens, the pleomorphic cells of tumor are immunoreactive for factor VIII related Ag in this tissue section.

imaging, ▶low uptake is the major finding, which can be useful for the follow-up of patients under therapy. Angiographic findings are nonspecific ranging from hypo- to hyperperfusion. It is noteworthy that imaging studies can only lead to a strong suspicion regarding the presence of HEH and its pattern. Histopathology. Definitive diagnosis requires a histopathological examination. Often, a laparoscopic wedge or core biopsy is sufficient to depict architectural features of the HEH such as the intravascular characteristics. The diagnosis is mostly confirmed with ▶immunohistochemical (Immunohistochemistry) evidence of endothelial differentiation (Fig. 1b and c). Differential Diagnosis. More than two thirds of HEH cases may be initially misdiagnosed. The most common misdiagnoses are ▶cholangiocarcinoma, angiosarcoma, ▶hepatocellular carcinoma (HCC), ▶metastatic carcinoma (▶Metastasis), and sclerosing hemangioma.

Hepatic Epithelioid Hemangioendothelioma. Figure 2 MRI sections of HEH T2-weighted hyperintense lesions and central necrosis showing a target appearance.

Hepatic Ethanol Metabolism

Some important features for differential diagnosis include the infiltrative growth pattern with preservation of the hepatic acinar (▶Hepatic acinus) landmarks such as portal areas, the characteristic vascular invasion with tufting of portal vein branches and terminal hepatic venules, the identification of epithelioid tumor cells especially with intracytoplasmic lumina, and the delay of staining for epithelial differentiation markers. Therapy There is no generally accepted strategy for the treatment of HEH due to its heterogeneous dignity and variable clinical outcome. Theoretically, ▶liver resection is the first choice for curative treatment of HEH, but in the majority of the cases an oncological resection is impossible due to multicentricity of the lesions or anatomical difficulties. ▶Liver transplantation is generally the most common treatment modality. According to the Pychlmayr classification of hepatic malignancies, HEH is placed among the favorable indications for liver transplantation. Life expectancy of the patients with HEH is potentially good. Long-term disease-free survival after liver transplantation is reported in patients with disseminated disease at the time of diagnosis; conversely some patients with disease confined to the liver developed rapid recurrence and metastases following liver transplantation. The experience with other therapeutic modalities such as systemic or regional chemotherapy, ▶transarterial chemoembolization (▶Chemotherapy), and ▶radiotherapy (▶Chemoradiotherapy) is limited and variable, therefore, generally these treatments are of limited value especially as the first line therapy. Considering only follow-up without any treatment as a management option is controversial. There are some cases with long-term survival or even complete spontaneous tumor regression without receiving any treatment but until now, it is impossible to reliably identify HEH patients with a nonaggressive tumor and to consider them for a “wait and see” strategy. The mode of hepatic involvement and presence or absence of extrahepatic involvement are the main factors in the decision of the treatment modality. When less than one lobe is involved and there is no extrahepatic involvement, liver resection could be done as the first choice in treatment of HEH. The therapeutic strategy in the presence of extrahepatic involvement is especially controversial. When extrahepatic involvement exists, independent of performing liver resection or not, ▶adjuvant chemotherapy (▶Adjuvant chemoendocrine therapy) may be considered. In the case of massive involvement of the liver, liver transplantation is the best therapeutic choice. Extrahepatic involvement by itself does not exclude liver transplantation. Although chemotherapy in this situation is questionable, it may control the growth of the extrahepatic tumor. It is noteworthy that the clinical course of HEH is variable, ranging from a

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favorable disease with prolonged survival, even without therapy, to a rapidly progressive disease with a grave outcome. Therefore, the decision of the treatment strategy has to be tailored for each case, and the individual rate of progression, severity of signs and symptoms, and response to other treatment modalities may be important determinants for decision making. Clinical Outcome Three main causes for tumor recurrence and treatment failure after liver transplantation are error in the pretransplant evaluation, enhanced tumor growth under immunosuppressive therapy, and lack of effective anticancer therapy following surgery. The 5-year survival in different reports varies from 45 to 70%. Generally, the surgical therapies such as liver resection or transplantation have the best survival rates and chemoradiotherapy or no treatment lead to the worst clinical outcome. The presence of tumor necrosis may be associated with poor outcome, while typical indicators of biologic aggression such as ▶nuclear atypia, capsule penetration, and number of ▶mitosis are found to be unrelated to clinical outcome. The unpredictable natural course and prognosis of HEH makes it difficult to give a correlation between the morphological grading or clinical staging and outcome.

References 1. Mehrabi A, Kashfi A, Fonouni H et al. (2006) Primary malignant hepatic epithelioid hemangioendothelioma: a comprehensive review of the literature with emphasis on the surgical therapy. Cancer 107(9):2108–2121 2. Ishak KG, Sesterhenn IA, Goodman ZD et al. (1984) Epithelioid hemangioendothelioma of the liver: a clinicopathologic and follow-up study of 32 cases. Hum Pathol 15:839–852 3. Makhlouf HR, Ishak KG, Goodman ZD (1999) Epithelioid hemangioendothelioma of the liver: a clinicopathologic study of 137 cases. Cancer 85:562–582 4. Lauffer JM, Zimmermann A, Krahenbuhl L et al. (1996) Epithelioid hemangioendothelioma of the liver. A rare hepatic tumor. Cancer 78:2318–2327

Hepatic Ethanol Metabolism I AIN H. M C K ILLOP, L AURA W. S CHRUM Department of Biology, The University of North Carolina at Charlotte, Charlotte, NC, USA

Definition

The majority of ▶ethanol metabolism occurs in the ▶liver following the ingestion of alcoholic beverages.

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The enzymatic reactions involved in ethanol metabolism can in turn lead to a variety of deleterious effects on cells both within the liver and at the systemic level which has led to chronic ethanol consumption being identified as a major risk factor in the development of ▶liver cancer and a significant risk factor for the development of non-hepatic tumors.

Characteristics

The liver demonstrates a “dual circulation” vasculature in which oxygenated blood is delivered via the hepatic artery and deoxygenated blood, containing substances that have been absorbed from the gastro-intestinal tract, via the hepatic portal vein. Following ingestion ethanol is rapidly absorbed and enters the hepatic portal vein where it is delivered to the functional subunits of the liver termed the hepatic lobules. As blood flows through the hepatic vasculature (sinusoids) specialized epithelial cells, termed ▶hepatocytes, that line the vasculature process materials absorbed in the GI tract to maintain normal physiological homeostasis. In addition to performing essential physiological functions such as protein, lipid and carbohydrate metabolism, bile production and regulation of vitamin A storage the liver also plays a central role in drug and hormone metabolism. Following ethanol consumption metabolism occurs in the hepatocyte via three main enzymatic pathways; ▶alcohol dehydrogenase (ADH), ▶cytochrome p450 2 E1 (CYP2E1) and catalase. The metabolism of ethanol is a two step process, the first involves the conversion of ethanol to ▶acetaldehyde (via ADH, CYP2E1 or catalase) and the second requires the

conversion of acetaldehyde to acetate by the ▶aldehyde dehydrogenase (ALDH) enzyme (Fig. 1). In both instances, these reactions lead to the production of the reduced form of nicotinamide dinucleotide (▶NADH). These metabolic pathways for ethanol directly contribute to many of the deleterious effects of ethanol intake on normal hepatic and systemic physiology. While the metabolism of acetaldehyde by ALDH is an efficient metabolic pathway, acetaldehyde is a highly reactive species that, if allowed to accumulate, can cause significant protein damage and formation of ▶adducts to DNA and ▶DNA damage. In addition to the damaging effects of acetaldehyde/NADH, once formed, must be recycled to the oxidized NAD+ form by the electron transport chain in the mitochondria of hepatocytes. This process requires increased oxygen demand and can in turn lead to the production of reactive oxygen species (▶ROS) and increase cellular ▶oxidative stress that can cause ▶oxidative DNA damage and protein/lipid peroxidation. In the instance of moderate ethanol consumption the ADH–ALDH system is sufficient to metabolize ethanol. However, following chronic, excessive ethanol intake the CYP2E1 enzyme becomes induced leading to increased acetaldehyde/NADH production and further elevating ROS synthesis and hepatic oxidative stress (Fig. 1). Although ethanol metabolism is localized primarily in the hepatocytes, the nonparenchymal cells of the liver including ▶hepatic stellate cells (HSCs) and ▶Kupffer cells (KCs) have also been shown to express ethanol metabolizing genes. Human HSCs express both ADH and ALDH, and CYP2E1 expression has been observed

Hepatic Ethanol Metabolism. Figure 1 Hepatic ethanol metabolism as it pertains to potential mechanisms of liver damage. Ethanol is metabolized by a two-step process to acetaldehyde that, in turn, is metabolized by acetaldehyde dehydrogenase (ALDH) to acetate.


in both HSCs and KCs, therefore further contributing to the detrimental effects of ethanol metabolism in the liver. Depending on the level and period of ethanol consumption normal hepatic function becomes increasingly compromised with complications ranging from moderate steatosis (fatty liver) to acute alcoholic hepatitis and eventually the development of fibrosis and cirrhosis. In addition, epidemiological data indicates that chronic ethanol consumption acts synergistically to accelerate the progression of ▶HCC in patients exposed to other common risk factors including exposure to ▶aflatoxins and ▶hepatitis virus associated HCC. The damage caused to the liver through acetaldehyde adduct formation and/or ROS generation/oxidative stress is thought to be a major risk factor for ethanol related liver cancer development. Greater than 80% of all primary tumors diagnosed in the liver arise as a result of hepatocyte cell transformation (▶hepatocellular carcinoma; HCC). Unlike other common malignancies HCC occurs most commonly in the absence of familial patterns and largely within the realms of known risk factors. Chronic ethanol consumption, metabolism and the cellular and genetic damage associated with these events thus represent a significant risk factor for hepatic damage, cell transformation and the development of HCC. In addition to the direct effects of ethanol metabolism on the hepatocyte integrity, such as impaired microtubule polymerization, several other direct and indirect factors can combine to augment the effects of ethanol consumption in the liver and the body as a whole. Glutathione (▶GSH) is an antioxidant important in reducing the oxidative stress caused by the synthesis and release of ROS. In the mitochondria, GSH is the only source of hydrogen peroxide metabolism and becomes significantly depleted following chronic ethanol ingestion due, at least in part, to decreased GSH transport into the mitochondria. While these detrimental effects on hepatocyte function directly increase the probability of cell transformation, increasing evidence suggests that the sequential nature of tumor formation and/or other systemic effects may play an increasing role in the effects of ethanol during the development and progression of HCC. Ethanol abuse accounts for the majority of liver fibrosis and cirrhosis cases in the western world; however, even though the clinical progression is well-described, the ▶molecular pathology is less well understood. Current models propose that alcoholic liver disease is initiated by an inflammatory response due to the activation of KCs as well as the infiltration of leukocytes including macrophages, neutrophils and lymphocytes. The activation of these inflammatory cells has been shown to be due to elevated gut-derived endotoxin plasma levels. Ethanol alters gut permeability to macromolecules, decreases gut motility and increases growth of Gram-negative bacteria in the intestinal microflora which ultimately leads to the introduction of endotoxin

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into the portal microcirculation. The activation and recruitment of inflammatory cells lead to the production of several profibrogenic cytokines. These cytokines likely induce and perpetuate the activation of HSCs leading to increased deposition of extracellular matrix (ECM) like type I collagen. The HSCs themselves can also exacerbate the inflammatory response through the secretion of chemoattractants and adhesion molecules necessary for leukocyte adhesion and infiltration. The presence and activation of KCs, other infiltrating inflammatory cells and damaged hepatocytes, lead to increased ROS resulting in the activation of quiescent HSCs. The quiescent HSCs reside in the perisinusoidal space of Disse, and one of their primary functions in the healthy liver is the storage and homeostasis of vitamin A, namely retinol, retinal and retinoic acid. Upon a fibrogenic stimulus, including ethanol, the HSC transdifferentiates from a quiescent, vitamin A storing cell to that of an activated myofibroblast-like cell which proliferates, migrates to the site of injury and is responsible for the excessive accumulation of ECM. This continued deposition of ECM leads to scarring of the liver and ultimately liver dysfunction. Reduced levels of serum and hepatic vitamin A have been reported in persons with alcoholic liver disease (▶ALD). Ethanol exposure to HSCs inhibits retinoic acid production and intracellular retinol levels. Possible mechanisms which interfere with retinoid metabolism in the cell may include reduced vitamin A uptake and enhanced degradation of vitamin A. In addition to oxidative damage incurred directly by the hepatocyte, liver damage also affects cell membrane integrity due to ethanol metabolism via a nonoxidative pathway leading to the generation of fatty acid ethyl esters (FAEE). Fatty acid ethyl esters accumulate in the cell plasma membrane as well as in the mitochondrial membrane leading to organelle dysfunction. Accumulation of FAEE, particularly linolenic acid ethyl ester (LAEE) has been reported to activate signaling pathways important in regulating collagen expression which may further contribute to ethanol-induced fibrosis and subsequently HCC progression. In addition to the effects of ethanol and ethanol metabolism on hepatocytes and other cell populations other factors must also be considered as to how ethanol can affect tumorigenesis. For example, the high incidence of cigarette smoking in ethanol dependent patients, regional differences in dietary intake, the type of beverage consumed and the socio-economic status and relative balance of diet in ethanol dependent patients can all affect the incidence and rate of hepatic tumor development and progression. Similarly, the induction of CYP2E1 (following chronic ethanol intake) has also been demonstrated to play a significant role in pro-carcinogen and ▶carcinogen metabolism, many of which are present in cigarette smoke and alcoholic beverages.

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Since oxidative stress has been linked to the development of ALD, the use of antioxidants as possible therapeutic strategies has been explored. The addition of antioxidants such as vitamin E, superoxide dismutase, GSH precursors such as S-adenosyl-L-methionine (SAMe) and the green tea extract, (–)-epigallocatechin3-gallate (EGCG), have been shown to prevent or ameliorate ethanol-induced liver injury in a variety of animal models of ALD and HCC. However, the use of antioxidants should be approached with caution due to the possible toxic properties of antioxidants under certain conditions. Similarly, the use of “over the counter” antioxidants raises the possibility of drug–drug interactions and altered endogenous and exogenous agent metabolism caused by antioxidant intake, many of which are currently poorly studied and reported.

References 1. McKillop IH, Moran DM, Jin X et al. (2006) Molecular pathogenesis of hepatocellular carcinoma. J Surg Res 136:125–135 2. McKillop IH, Schrum LW (2005) Alcohol and liver cancer. Alcohol 35:195–203

Hepatic (Liver) Fixed Macrophages ▶Kupffer Cells

Hepatic Stellate Cell Definition HSC; Pericytes located in the perisinusoidal space of the liver responsible for vitamin A storage. In the damaged liver activation of HSCs leads to depleted Vitamin A storage and synthesis of extracellular matrix proteins (scarring). ▶Hepatic Ethanol Metabolism

Hepatitis Definition

Hepatic Excretion Definition Is the excretion of a drug by the liver by way of the gall bladder emptying into the intestine and ultimately into the feces. ▶ADMET Screen

Is liver inflammation and necrosis of varying severity and etiologies, such as toxic (drugs, alcohol), metabolic, autoimmune or viral. ▶Hepatitis C Virus ▶Hepatic Ethanol Metabolism

Hepatitis B Virus Definition

Is a basic leucine zipper protein defined by a PAR domain that plays a critical role in hematopoietic specific expression of the LMO2 gene. A role for HLF in HSC self-renewal is supported by studies showing that ectopic expression of HLF enhanced HSC engraftment and inhibited apotosis.

HBV; is a member of the Hepadnaviridae family (hepatotropic DNA viruses). The HBV genome is a partially relaxed double-stranded circular DNA of 3,200 base pairs, and encodes four partly overlapping open reading frames (ORFs). Thirty to fifty percent of persons who acquire the infection before the age of 5 years develop chronic HBV infection. HBV is a high-risk factor for ▶hepatocellular carcinoma. ▶Hepatocellular Carcinoma – Clinical Oncology; Hepatocellular carcinoma – etiology, risk factors and prevention; ▶liver cancer – molecular biology.

▶NUP98-HOXA9 Fusion ▶E2A-PBX1

▶Hepatocellular Carcinoma ▶Hepatitis Virus Associated Heptocellular Carcinoma

Hepatic Leukemia Factor (HLF) Definition

Hepatitis B Virus x Antigen Associated Hepatocellular Carcinoma

Hepatitis B Virus x Antigen Associated Hepatocellular Carcinoma M ARK A. F EITELSON Department of Biology, Temple University, Philadelphia, PA

Synonyms Liver cancer; Hepatocellular carcinoma; HCC

Definition The term hepatitis B x antigen, or HBxAg, refers to the gene product of hepatitis B virus (HBV) that contributes importantly to virus replication, the pathogenesis of chronic liver disease, and to the development of HCC.

Characteristics There are an estimated 350 million people worldwide who are chronically infected with HBV and often replicate virus for many years or decades. These people are at high risk for the development of chronic hepatitis, which may progress onto cirrhosis (end stage liver disease) and then HCC. Although the pathogenesis of infection is immune mediated, this is characterized most often by responses that trigger hepatocellular damage and destruction but do not clear virus. An important characteristic of ▶chronic liver disease (CLD) is that ▶liver cell regeneration provides opportunities for integration of virus DNA into the replication forks of cellular DNA. It turns out that the region encoding HBxAg is the most frequently integrated region of HBV DNA into the host genome. This region often encodes HBxAg, which is a ▶transactivating protein that alters the expression of cellular genes in many chromosomes throughout the host genome. This is quite an accomplishment for a small protein of only 17 kDa, and is achieved by constitutively activating a number of signal transduction pathways in the cytoplasm (such as JAK/STAT [▶signal transducers and activators of transcription in oncogenesis], ▶NF-κB, ▶AP-1, AP-2, ▶ras, src, PI3K/Akt [▶PI3K signaling; ▶Akt signal transduction pathways in oncogenesis] and β-catenin [▶Wnt signaling], among others) and by binding to transcription factors in the nucleus (such as Oct-1, CREB, ATF-2, b-zip, TBP, and other basal transcription factors) or sequestering them in the cytoplasm (e.g., p53). HBxAg also alters gene expression at the post-transcriptional level by blocking the activity of the ▶proteasome, which normally degrades proteins, and by altering the integrity of translation. Some of these pathways have been shown to be operative in the mechanism(s) whereby HBxAg

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promotes cell growth in culture dishes and anchorage independent growth in soft agar (a property often associated with cancer cells). The findings that HBxAg is capable of conferring tumorigenicity upon nonmalignant liver cells, and that the sustained over-expression of HBxAg in transgenic mice gives rise to HCC, further underscores the centrality of HBxAg expression to the development of HCC. It is likely that inadequate immune responses permit the persistence of infected hepatocytes during chronic infection. However, there is increasing evidence that HBxAg inhibits a number of ▶apoptotic pathways (▶apoptosis signaling) (e.g., Fas and tumor necrosis factor alpha (TNFα), both of which trigger programmed cell death) during chronic infection, thereby promoting the persistence of infected cells during CLD. HBxAg is also promotes ▶fibrogenesis, in that it promotes the expression of extracellular matrix proteins such as ▶fibronectin, promotes the cross-linking of collagen, and potentiates ▶TGFβ1 signaling, in part, by inhibiting expression of the TGFβ1 binding partner, α-2macroglobulin. In this context, it is not surprising to find a strong direct correlation between intrahepatic HBxAg expression and CLD. This relationship would protect virus infected cells from immune destruction, but these same features also promote carcinogenesis, since tumor cells are also resistant to apoptosis and demonstrate constitutively active signaling pathways that had been previously altered in preneoplastic tissue by HBxAg. There are few natural effectors of HBxAg that are known to be responsible for these profound biological changes that ultimately result in cancer. HBxAg appears to stimulate ▶insulin-like growth factor 2 (IGF2), which promotes hepatocellular growth, activates wild type β-catenin (and its effector, c-myc), trans-activates several unique “oncogenes,” and promotes ▶angiogenesis. HBxAg also binds to and inactivates the ▶tumor suppressors, ▶p53 and ▶PTEN, down-regulates expression of the natural cell cycle inhibitor, ▶p21WAF1/SDI1/CIP1, and promotes phosphorylation (inactivation) of the Rb tumor suppressor protein (▶retinoblastoma protein), suggesting the molecular basis upon which HBxAg stimulates unchecked cell growth. HBxAg also binds to and inactivates a novel ▶senescence factor, p55sen, suggesting that HBxAg contributes to carcinogenesis, in part, by overcoming cellular senescence. The observation that HBxAg also down-regulates ▶E-cadherin expression, the latter of which is involved in cell adhesion, provides at least part of the explanation as to how HBxAg stimulates cell motility (metastasis) in carcinogenesis. ▶Oxidative stress is a common feature of CLD, resulting in the activation of HBxAg in CLD and HCC. Over-expression of HBV envelope associated polypeptides, cytokine signaling, and cell mediated immunity against HBV infected cells, all promote the development

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of oxidative stress. This was associated with decreased intrahepatic levels of superoxide dismutase (Cu/Zn), which normally protects cells from ▶reactive oxygen species. These conditions stimulate HBxAg, which compromises transcription coupled DNA repair and ▶nucleotide excision repair, resulting in increased chromosomal alterations (genetic instability) and micronuclei formation (where metaphase plates separate prior to complete DNA replication). In addition, HBxAg is associated with the outer mitochondrial membrane, where it binds to the voltage dependent anion channel (VDAC3), resulting in decreased mitochrondrial membrane potential and the further development of oxidative stress. In this way, oxidative stress augments HBxAg function in propagating chronic infection and in stepwise hepatocarcinogenesis.

References 1. Breuhahn K, Longerich T, Schirmacher P (2006) Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 25:3787–3800 2. Arbuthnot P, Capovilla A, Kew M (2000) Putative role of hepatitis B virus X protein in hepatocarcinogenesis: effects on apoptosis, DNA repair, mitogen-activated protein kinase and JAK/STAT pathways. J Gastroenterol Hepatol 15:357–368 3. Waris G, Siddiqui A (2003) Regulatory mechanisms of viral hepatitis B and C. J Biosci 28:311–321 4. Jin YM, Yun C, Park C et al. (2001) Expression of hepatitis B virus X protein is closely correlated with the high periportal inflammatory activity of liver diseases. J Viral Hepatitis 8:322–330 5. Feitelson MA (2004) Molecular and genetic determinants of primary liver malignancy. In: Khatri VP, Schneider PD (eds) Surgical clinics of North America. Liver surgery: Modern concepts and techniques, vol. 84. WB Saunders, Philadelphia, pp. 339–354

Hepatitis C Virus WOLFGANG H. C ASELMANN Medizinische Klinik und Poliklinik I, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany

Synonyms HCV

Definition

▶Hepatitis C virus (HCV) belongs to the flaviviridae family (Hepaciviruses genus) and is a pathogenic human RNA virus, which causes chronic liver disease and

hepatocellular carcinoma (▶liver cancer, molecular biology) (HCC).

Characteristics

The size of this enveloped virus is 60 nm. Its nucleocapsid contains a single-stranded RNA genome of 9.6 kb genome (Fig. 1) of plus(+) strand polarity that carries a single open reading frame. At least six major genotypes (1–6) with up to three subtypes (a to c) exist, which differ not only in their nucleic acid sequence but also in their pathophysiological properties. All structural and non-structural viral proteins are processed from a polyprotein precursor of 3,010–3,033 amino acids in the cytoplasm or endoplasmic reticulum of the infected cell. Untranslated Regions The 5′- and 3′-untranslated regions (UTR) are highly conserved within the different genotypes. The 341–344 nucleotides 5′-UTR (Fig. 1) forms extensive stem-loop structures, which are important in translation initiation. It also harbors an internal ribosomal entry site. The 3′-UTR is composed of a poorly conserved variable region (28–42 nucleotides), a variable polypyrimidine stretch and conserved 98 nucleotides at the 3′ end (x region). Both UTRs seem to be crucial for regulation of HCV translation and possibly also for controlling HCV replication, and are therefore candidate targets for experimental antiviral strategies. Structural Proteins The core protein C (21 kD) polymerizes to an icosaedric capsid and binds RNA to form the nucleocapsid. The envelope proteins E1 (31 kD) and E2 (70 kD) form heterodimers whose formation is mediated by the chaperon calnexin. These dimers are embedded in a host-derived lipid bilayer. Within the E2 sequence there are two hot spots of mutations (hypervariable regions, HVR1 and HVR2). Mutations occurring during HCV infections are due to a selection driven by the host immune system and account for the regular existence of a variety of ▶quasispecies. The E2 protein is produced as two precursors, E2/NS2 and E2/p7 that differ in their C-terminus. The hydrophobic p7 is probably important for membrane anchoring of E2. The function of p7 or E2/p7 during the HCV life cycle is not known. All structural protein processing is performed by cellular signal peptidases. Non-structural Proteins The non-structural protein NS2 (23 kD) is released from the polyprotein precursor both by host signal peptidases at the C-terminus of p7 and by a NS2/3 proteinase at the NS2-NS3 junction. NS3 (68 kD) has

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Hepatitis C Virus. Figure 1 Genomic organization and designation of HCV RNA, and amino acid positions and function of HCV proteins.

three different functions: It is part of the NS2/3 proteinase, its N-terminal third contains a serine proteinase and at the C-terminus a RNA-dependent NTPase/ helicase was discovered. Together with NS4A (6 kD) as a stable complexed cofactor, the N-terminal NS3 serine proteinase catalyses the cleavage between NS3NS4A, followed by cleavage between NS5A-NS5B, NS4A-NS4B and NS4B-NS5A. The NS3 helicase can unwind double-stranded (ds)RNA, dsDNA and RNA/ DNA heteroduplex molecules. For this purpose any NTP or dNTP is used as source of energy. NS4B is a 26 kD membrane associated protein of unknown function. NS5A (56–58 kD) represents two cytoplasmatic proteins, p56 and p58, which are both phosporylated at serine residues. This process may be essential for the viral replication cycle, but the biological functions of these proteins are not understood. There is some evidence that NS5A mediates interferon-alpha resistance of HCV. The NS5B (65 kD) protein is responsible for HCV RNA replication; it represents an RNA-dependent RNA-polymerase that is not found in humans and may therefore be another attractive target for antiviral strategies. The RNA minus(−) strand is transcribed in the host cytoplasm into a plus(+) strand RNA. This serves as a template to produce new minus(−) strands for packaging into the envelope. Both steps are accomplished by the NS5B protein.

Cellular and Molecular Regulation Although the HCV genome does not harbor acutely transforming oncogenes, the HCV core as well as the NS3 gene are candidate genes whose products may mediate malignant hepatocyte transformation. Oncogene complementation assays using HCV core and H-▶ras or c-▶myc oncogenes showed that HCV core cooperates with these oncogenes and transforms primary rat embryo fibroblasts to the tumorgenic phenotype. Focus formation, soft agar growth and tumor development was also shown in nude mice using Rat-1 fibroblasts. Also in this model a loss of contact inhibition, morphological changes and anchorage- and serumindependent growth occurred when HCV core and v-H-ras were cooperatively expressed. The most striking evidence of an oncogenic potential of HCV comes from an HCV core-transgenic mouse model, in which hepatic tumors arose in up to 30% of animals of two different strains. Interestingly, all tumors occurred in male animals. However, other mice transgenic for HCV core or C-terminally truncated (amino acid 384–715) HCV core failed to develop histological or biochemical signs of liver disease. The postulated underlying molecular mechanisms of HCV core-induced hepatocyte transformation are manifold. They comprise sequestration of LZIP in the cytoplasm leading to a loss of CRE-dependent

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transcription and regulation of cell proliferation and subsequently to morphological transformation of NIH 3T3 cells. Other mechanisms may be interference with ▶apoptosis or ▶transactivation of cellular c-fos, c-myc, p53 or β-interferon promoters. Stable expression of the 5′-portion of the NS3 gene was able to induce focus formation and soft agar growth in NIH 3T3 cells. Only recently it was demonstrated that internal cleavage of the NS3 protein occurs at cleavage sites FCH(1395)//S(1396)KK and IPT(1428)// S(1429) GD within the HCV RNA helicase domain in presence of NS4A. These findings were confirmed in two different isolates of HCV of genotype 1b. The 5′-portion of NS3 was more oncogenic than the fulllength NS3 protein. Clinical Relevance An estimated 2% of the world’s population is chronically infected with HCV. It accounts for 20% of acute and 70% of chronic hepatitis cases. HCV is mainly transmitted by blood. Since screening and treatment of blood products for HCV has been routinely performed, post-transfusion hepatitis has become extremely rare. Intravenous drug addiction is today a major way of HCV transmission. Acute hepatitis is often clinically inapparent, and in chronic hepatitis symptoms occur mainly in later stages of disease. A high chronicity rate of up to 75% of acute infection and its silent course account for the pathogenic potential of this virus, which includes extrahepatic manifestations. Twenty percent of chronically infected patients develop ▶liver cirrhosis and 2–5% per year will progress to hepatocellular carcinoma (HCC). Therapy for HCV infection comprises the combined use of 3–6 MU of α-interferon administered subcutaneously three times a week and 800–1200 mg of the nucleoside analog ▶ribavirin p.o daily for 6–12 months dependent on the genotype and viral load. Roughly 40–45% of previously untreated or relapse patients benefit from the treatment, i.e. eliminate HCV and normalize liver enzymes. In addition, this treatment is capable of improving histological signs of liver damage. Its effect on HCC prevention can not clearly be judged at present. Recent therapeutic innovations comprise ▶pegylated interferons that allow single weekly dosing due to slow effector release from subcutaneous depots and a decrease of systemic side effects. Sustained response rates up to 70 % can be reached. A synthetic consensus interferon-alpha showed promising effects in monotherapeutic use in a preliminary trial and is presently being evaluated in combination with ribavirin. Major future achievements are expected from the development of inhibitors of HCV protease, helicase or RNA-dependent RNA polymerase, which may improve effectivity of antiviral treatment similarly to

the situation in HIV infection, but are not yet available for clinical use. Therapeutic nucleic acids such as antisense oligodeoxynucleotides, RNAs or ribozymes may be another experimental concept for the treatment of HCV infection in the future.

References 1. Moriya K, Fujie H, Fukuda K et al. (1998) The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 4:1065–1067 2. Wedemeyer H, Caselmann WH, Manns MP (1998) Combination therapy of chronic hepatitis C – an important step but not the final goal. J Hepatol 29:1010–1014

Hepatoblastoma T ORSTEN P IETSCH 1 , D IETRICH 1

VON

S CHWEINITZ 2

Institut für Neuropathologie, Universitätskliniken Bonn, Bonn, Germany Kinderchirurgie 2 Universitäts-Kinderspital beider Basel (UKBB), Basel, Switzerland

Definition Heptoblastoma is a childhood malignant embryonal liver tumor consisting of immature epithelial cells with or without additional mesenchymal component.

Characteristics Hepatoblastomas are the most frequent malignant liver tumors of childhood. Their annual incidence is 0.5–1 cases per million children under 15 years of age in Western countries. The affected children are most frequently between 6 months and 3 years old. Hepatoblastoma has also been detected in utero by prenatal ultrasound examination. Since liver tumors lack early clinical symptoms, hepatoblastoma patients often present with locally extended tumors at diagnosis. However, distant metatases usually occur very late in the disease progression. The patients frequently have highly elevated ▶α-fetoprotein levels. In these cases this ▶oncofetal antigen can be useful as a sensitive diagnostic marker and also as a marker for the monitoring of treatment response. Pathological Classification Hepatoblastomas always consist of immature epithelial liver cells resembling fetal or embryonal liver cells. Approximately one third of the cases contain additional mesenchymal components and are then termed “mixed” hepatoblastomas. According to Weinberg and Finegold (1983) the epithelial component is further classified into

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Hepatoblastoma. Figure 1 Histopathology of an epithelial hepatoblastoma showing foci of hematopoietic cells.

well differentiated “fetal,” less differentiated “embryonal” and undifferentiated “small cell anaplastic” categories. The latter is rare, as are “macrotrabecular” or “teratoid” variants. Small cell anaplastic as well as macrotrabecular variants indicate a bad prognosis. A histopathological hallmark of hepatoblastomas, in particular of the fetal differentiated cases, is the occurrence of hematopoietic foci mainly consisting of erythropoietic or thrombopoietic progenitor cells mimicking fetal hematopoiesis in the liver (Fig. 1).

with prolonged preoperative chemotherapy (SIOP, USA) or with mega-therapy (Germany).

Staging In the last 30 years the treatment modalities and outcome of hepatoblastoma patients has significantly improved. It turned out that hepatoblastomas are responsive to chemotherapy. Today, most hepatoblastoma patients are enrolled in multicenter studies and receive a stage and risk adapted multimodal therapy. Different staging systems are used including the American and German fourgraded postoperative staging system, the Japanese TNM classification and the European SIOP PreTreatment Extent of Disease (PRETEXT) grouping system. The patients are stratified into standard and high risk patients, the latter often presenting with extended, multifocal or metastatic disease.

Predictive Factors Several histological, clinical as well as serological factors have been evaluated for their predictive value. In particular, the extension of the tumor in the liver, multifocality, vascular invasion and the presence of metastases have been of predictive importance in most studies. The decline of alpha-fetoprotein levels under chemotherapy predicts the clinical response. In contrast, the impact of distinct histological features such as mixed versus epithelial, or fetal versus embryonal differentiation and chromosome ▶ploidy of the tumors is still under discussion and the data is controversial. The etiology of hepatoblastomas is unknown. Environmental factors do not seem to play a major role in the pathogenesis of hepatoblastomas. Preterm infants may have an elevated risk for the development of hepatoblastomas. Although most hepatoblastomas occur sporadically, familial cases have been described. The incidence is highly elevated in families with ▶adenomatous polyposis coli (FAP [▶APC Gene in Familial Adenomatous Polyposis]) and ▶Beckwith-Wiedemann Syndrome.

Therapy and Outsome In current protocols most patients receive a ▶neoadjuvant chemotherapy with ▶cisplatin and ▶doxorubicin, in some studies combined with ifosphamid, 5-fluorouracil or carboplatin. The combination carboplatin and etoposide has also been effective. The overall survival after chemotherapy and resection is 70–80%. Unfortunately, approximately one quarter of the patients still die from the disease, so that predictive factors are important to early recognize these high risk patients and to offer them an intensified therapy. High risk patients have been treated

Genetic and Molecular Characteristics Cytogenetic analyses have revealed several recurrent numerical aberrations as well as structural alterations. Most frequently trisomies of chromosomes 1, 2, 8 and 20 have been described as well as a recurrent translocation t(1;4)(q12;q34) in 15% of hepatoblastomas. Approximately 50% of cases show gain of material of chromosome 2q. A few cases have an ▶amplification of material from chromosomal band 2q24, indicating the location of a hepatoblastoma related oncogene. ▶Microsatellite analyses have uncovered

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frequent allelic losses of chromosomal regions of chromosome arms 1p and 11p. The losses of chromosomal region 11p15.5 are always of maternal origin indicating the existence of one or more imprinted hepatoblastoma associated gene(s) expressed from the maternal ▶allele. Studies on the mutational status of the ▶TP53 gene resulted in conflicting data. Whereas TP53 mutations were described in a small Japanese tumor collection, these were absent in other studies. Similarly, somatic missense mutations were described in one study but not confirmed by others. The activation of the ▶WNT signaling pathway by activating mutations in the ▶β-catenin gene is a frequent event in hepatoblastomas. Approximately 50% of the hepatoblastomas save point mutations or deletions of exon 3 encoding the protein degradation targeting site of the β-catenin protein, resulting in a destruction of N-terminal phosphorylation sites that are necessary for protein degradation. This leads to the accumulation of a mutated protein, which transfers oncogenetic signals to the nucleus and increases transcription of specific target genes of the ▶TCF/LEF family of transcriptions factors such as ▶cyclin D1 and ▶c-myc. Similar mutations have been described in other tumor entities including colorectal cancer. However, hepatoblastoma represents the malignant tumor with the highest incidence for β-catenin mutations. Cellular Characteristics Hepatoblastoma cells resemble liver progenitor cells during embryonic and fetal development suggesting that hepatoblastomas are derived from such progenitors. Hepatoblastoma cells are still dependent on growth factors and use specific growth factor signaling systems. The important fetal mitogen, insulin-like growth factor-II, has been demonstrated to be highly over-expressed in hepatoblastomas. The encoding ▶IGF2 gene maps to chromosome 11p15.5, a region frequently altered in hepatoblastomas and related embryonal tumors. They also produce several hematopoietic ▶cytokines such as ▶interleukin-1, ▶stem cell factor, ▶erythropoietin and ▶thrombopoietin that induce hematopoietic foci in hepatoblastomas. Hepatoblastoma cells over-express the receptor ▶Met for hepatocyte growth factor (HGF; ▶scatter factor) and proliferate in response to HGF in vitro. Concentrations needed for this effect are usually found in the serum of patients after liver surgery. This may explain why hepatoblastomas often show rapid regrowth after partial resection. Therefore it is now common sense that the tumors should undergo primary resection, only when they can be removed without residual tumor cells.

References 1. Weinberg AG, Finegold MJ (1983) Primary hepatic tumors of childhood. Hum Pathol 14:512–537

2. Brown J, Perilongo G, Shafford E et al. (2000) Pretreatment prognostic factors for children with hepatoblastoma – results from the International Society of Paediatric Oncology (SIOP) study SIOPEL 1. Eur J Cancer 36: 1418–1425 3. Mann JR, Kasthuri N, Raafat F et al. (1990) Malignant hepatic tumours in children: incidence, clinical features and aetiology. Paediatr Perinat Epidemiol 4:276–289 4. Von Schweinitz D, Hecker H, Schmidt-von-Arndt G et al. (1997) Prognostic factors and staging systems in childhood hepatoblastoma. Int J Cancer 74:593–599 5. Von Schweinitz D, Byrd DJ, Hecker H et al. (1997) Efficiency and toxicity of ifosfamide, cisplatin and doxorubicin in the treatment of childhood hepatoblastoma. Study Committee of the Cooperative Paediatric Liver Tumour Study HB89 of the German Society for Paediatric Oncology and Haematology. Eur J Cancer 33:1243–1249

Hepatocellular Carcinoma T OSHIHIKO M IZUTA Department of Internal Medicine, Saga Medical School, Saga, Japan

Synonyms Liver cancer; Hepatoma; Hepatic carcinoma; Hepatitis B Virus x Antigen Associated Hepatocellular Carcinoma; Primary liver cancer; Primary hepatic carcinoma; Liver cell carcinoma

Definition Hepatocellular carcinoma (HCC) is a primary cancer that arises from hepatocytes, the major cell type of the liver. Most cases of HCC are secondary to either hepatitis virus (usually type B or C) infection or cirrhosis.

Characteristics Epidemiology HCC is the fifth most common cancer in men and the eighth most common cancer in women worldwide. An estimated more than half a million new cases are diagnosed annually, while there are major geographical differences in incidence. The annual incidence rates in Eastern Asia and sub-Saharan Africa exceed 15/10,000 inhabitants, while the figures are intermediate (between 5/10,000 and 15/10,000) in the Mediterranean Basin and Southern Europe, and very low (50% chance of surviving 3 years, even without treatment. In contrast, a patient with multiple tumors involving both lobes of the liver with decompensated cirrhosis is unlikely to survive more than 6 months, even with treatment. AFP levels have been shown to be prognostically important, with the median survival of AFP-negative patients significantly longer than that of AFP-positive patients. Other prognostic variables include performance status (measure of general wellbeing), liver functions, and the presence or absence of cirrhosis and its severity in relation to the ▶Child-Pugh classification. Prognostic Staging System A clinical staging system provides guidance for patient assessment and appropriate therapy. It is useful for the decision to treat certain patients aggressively while avoiding the overtreatment of other patients who would not tolerate therapy or whose life expectancy rules out any chance of success. Clinical staging is also an essential tool for comparison between groups in therapeutic trials and between different studies. The current classifications most commonly used for HCC are the ▶Okuda staging system, the ▶Child-Pugh staging system, tumor node metastasis (TNM) staging, and Cancer of the Liver Italian Program (CLIP) score (Table 1). Among these, the CLIP score is currently the most commonly used integrated staging score, for both tumor and liver disease stages. The Japan Integrated Staging (JIS) system, a new system that is based on a combination of the Child-Pugh system and the Liver Cancer Study Group of Japan (LCSGJ) system – the LCSGJ system is also concordant with TNM classification for HCC by the International Hepato-Pancreato-Biliary Association and the International Union Against Cancer (UICC) – has recently been proposed in Japan (Table 2). The stratification ability of the JIS scoring system is much better than that of the CLIP scoring system. The JIS scoring system also performed better than the CLIP scoring system in selecting the best prognostic patient group. Treatment There is no worldwide agreement on a common treatment strategy for patients with HCC, and several proposals have been published (Fig. 1).

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Hepatocellular Carcinoma. Table 1

Definitions of the CLIP score Score

Variables Child-Turcotte-Pugh stage Tumor morphology AFP (ng/ml) Portal vein thrombosis

0

1

2

A

B

C

Unilobular and extension ≤50% 50% − −

Hepatocellular Carcinoma. Table 2

Japan Integrated staging (JIS) scoring system Score

Variables

0

1

2

3

Child-Pugh grade TNM stage by LCSGJ

A I

B II

C III

IV

Definition of TNM stage by the Liver Cancer Study Group of Japan (LCSGJ)

T factor T1 T2 T3 T4 Stage I Stage II Stage III

I. Single

II. 20%, and chromosome arms 6q, 9p, 13q, 16p, and 17p LOH have been linked to inactivation of the tumorsuppressor insulin-like growth factor 2 receptor (IGF2R), p16, retinoblastoma (RB1), axin 1, and p53. The genetic changes involved in hepatocarcinogenesis can be divided into at least five pathways: the p53 pathway, which is involved in the response to DNA damage or genomic instability; the p16/p27/RB1 pathway, which is involved in cell-cycle control; the transforming growth factor-β (TGF-β) pathway, which is involved in growth inhibition and apoptosis of hepatocytes; the Wnt/β-catenin/APC pathway, which is involved in intercellular interactions; and the E-cadherin/ integrin and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways, which are involved in cancer cell migration and metastasis.

Hepatocellular Carcinoma – Etiology, Risk Factors and Prevention

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H Hepatocellular Carcinoma – Etiology, Risk Factors and Prevention. Figure 1 Possible mechanisms of hepatocarcinogenesis. Cell-cycle regulators involved in hepatocarcinogenesis.

Among the different signaling pathways involved in hepatocarcinogenesis, deregulation of the cell-cycle machinery is thought to be closely involved in the progression of HCC. Cell-cycle progression in mammalian cells is mainly governed by cyclin A, cyclin D, and cyclin E, along with cyclin-dependent kinase 2 (CDK2) and CDK4. The kinase activities of these proteins are negatively regulated by the CDK inhibitors (CDKIs) p16 and p27. RB1, which is phosphorylated by CDKs and activates the E2F family of transcription factors, is mutated in 15% of HCCs. The cyclin D1 gene, which is involved in the G1 progression and G1/S transition phases of the cell cycle, has been shown to be amplified in 10–20% of HCCs. By contrast, both p16 and p27 are frequently inactivated in HCCs. P16 is inactivated in 50% of HCCs, mainly due to the de novo methylation of the DNA-promoter region. Somatic mutation of the p16 gene has also been described in some cases of HCC. Recently, p16 DNA methylation was identified in preneoplastic liver tissues of individuals with chronic hepatitis virus infections. P27 is a member of the KIP family of CDKIs, and its expression is reduced in 40– 60% of HCCs. Decreased p27 expression is closely associated with poor prognoses of individuals with HCCs, indicating that it is an adverse prognostic factor. In some cases of HCC, increased cell proliferation has been noted despite relatively high levels of p27 expression, suggesting the inactivation of p27 by sequestration into cyclin D1–CDK4-containing complexes. Although many genes appear to be altered in HCC, the frequencies of individual mutations are low, and the patterns of genetic alteration differ among patients. Connections between different pathways might thus be plausible mechanisms underlying hepatocarcinogenesis.

Diagnosis Serum alpha-fetoprotein (AFP) and des-γ-carboxy prothrombin, which is also known as protein induced by vitamin K antagonist (PIVKA II), are useful serological markers of HCC. Although AFP exists in normal human tissues (both fetal and adult), serum levels are sensitive to the presence of HCC. The overall diagnostic accuracy of AFP is 80%. By contrast, PIVKA II is only present in HCC tissues, so its specificity is relatively high, and its overall diagnostic accuracy is 55%. The simultaneous measurement of AFP and PIVKA II could therefore be useful for the detection of HCC. However, as elevated AFP levels can be the result of liver cirrhosis, it is important to determine whether they are caused by active liver cirrhosis or by HCC that has arisen from liver cirrhosis. The elevation of serum AFP reflected as regeneration of hepatocytes. The levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are also often elevated in the serum of patients with HCC. However, the specificities of these markers are lower than those of AFP and PIVKAII. Total imaging is important for detecting small HCCs. The majority of HCCs arise from chronic hepatitis and liver cirrhosis; periodical checking using ultrasound scanning and computed tomography (CT) is therefore crucial to detect the early stages of HCCs. Recent advances in the use of imaging tools, such as CT scanning with contrast medium and magnetic resonance imaging (MRI), have enabled the precise detection of HCCs. Accuracy levels have reached >90% using enhanced CT and MRI techniques, and the current limit of detection for HCCs is 10 mm diameter.

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HCCS can develop from small nodules into large nodules when given access to an arterial blood supply rather than a portal blood supply. Small HCCs with poor arterial blood supplies are difficult to distinguish from adenomatous hyperplasia, large regenerative nodules, or angiomyolipoma. Direct tumor biopsy guided by ultrasound with a fine needle is therefore useful for making a final diagnosis in such cases. Treatment There are several treatment modalities for HCC. Surgical treatment is divided into two categories: hepatic resection and liver transplantation. The former is based upon the hepatic functional reserve, tumor location, and tumor numbers, and includes many procedures ranging from partial resection to extended right or left hepatic lobectomy. The latter is based on the tumor size and tumor numbers alone. The Milan criteria (that is, the presence of one nodule 10% during the previous six months, documented fever, and night sweats. There is a slight male

Hodgkin and Reed/Sternberg Cell

predominance and a bimodal age distribution in Western populations, with peaks of incidence in young adulthood and old age. Among economically disadvantaged populations, the first peak of incidence occurs earlier in childhood, particularly for males. There is a relatively low incidence of Hodgkin disease among Asian populations. Hodgkin disease usually starts at a single site and progresses in an orderly manner through the lymphatic system to local lymph nodes before disseminating hematogenously to distant sites, particularly the bone marrow, spleen, or liver. Hodgkin disease is typically staged from I to IV. Stage I represents involvement of a single lymph node structure, stage II is involvement of two or more lymph node regions on the same side of the diaphragm, stage III is involvement of lymph node regions or structures on both sides of the diaphragm, and stage IV is involvement of extranodal sites. Prognosis is directly related to stage. Hodgkin disease is typically treated by multidrug chemotherapy and/or radiotherapy, with an overall 5-year survival of greater than 80%. Patients who relapse usually do so within the first three years after treatment, and have a significantly worse prognosis. High-dose chemotherapy with ▶autologous bone marrow transplantation or peripheral blood stem-cell transplantation has become a standard therapy for patients who fail conventional chemotherapy regimens. Novel immunotherapies have been proposed, including treatment with interleukin-2, bi-specific antibodies, immunotoxins, and radioimmunoconjugates. Patients with the nodular lymphocyte predominance subtype may behave differently than the classical forms of Hodgkin disease, with a better overall survival, but a greater number of recurrences, which are independent of time after treatment.

References 1. Hansmann M-L, Kuppers R (2005) The Hodgkin and Reed/Sternberg cell. Int J Biochem Cell Biol 37:511–517 2. Hoppe RT, Advani RH, Bierman PJ et al. (2006) National Comprehensive Cancer Network. Hodgkin disease/lymphoma. Clinical practice guidlelines in oncology. J Natl Compr Canc Netw 4:210–230 3. Hoppe RT, Mauch PM, Armitage JO et al. (2007) Hodgkin Disease Lippincott Williams & Wilkins, Baltimore

Hodgkin Lymphoma Definition

▶Hodgkin disease ▶Hodgkin Disease, Clinical Oncology

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Hodgkin and Non-Hodgkin Lymphomas ▶B-cell Tumors

Hodgkin and Reed/Sternberg Cell R ALF K U¨ PPERS Institute for Cell Biology (Tumor Research), University of Duisburg-Essen, Medical School, Essen, Germany

Definition Hodgkin and Reed/Sternberg (HRS) cells are large cells with a peculiar morphology and immunophenotype that does not resemble any other normal cell in the body. The cells are called Hodgkin cells when they are mononucleated and Reed/Sternberg cells when they are multinucleated. HRS cells in nearly all instances derive from B lymphocytes, but in rare cases they originate from T cells. HRS cells are the hallmark cells of Hodgkin lymphoma, in which they represent the tumor cell clone.

Characteristics Associated Pathologies The first cases of Hodgkin lymphoma (HL) were described by Thomas Hodgkin in 1832. A peculiar type of cells that is a hallmark of HL was first characterized in detail about 100 years ago by Dorothy Reed and Carl Sternberg. These cells are called Hodgkin cells when they are mononucleated and Reed/Sternberg cells when they are bi- or multinucleated. HRS cells are large cells ten times or more of the size of small lymphocytes with prominent nucleoli. Reed/Sternberg cells most likely derive from Hodgkin cells by endomitosis, i.e., nuclear division without cell division. In HL, the HRS cells usually represent less than 1% of cells in the tumor tissue (Fig. 1). They are surrounded by a mixture of various types of cells, including T cells, B cells, plasma cells, macrophages, eosinophils, and others. In about 40% of cases of HL in the Western world, the HRS cells are latently infected by the ▶Epstein–Barr virus (EBV). HL is one of the most frequent malignant lymphomas in the Western world, with an incidence of about 2.5–3 new cases per 100,000 people per year. Nowadays, about 90% of the patients can be cured by chemo- and/or radiation therapy. Based on differences in the histological picture and the cellular

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morphology and to some extend the immunophenotype of HRS cells. As HRS cells have most extensively been analyzed in HL, the following discussion of HRS cells refers to HRS cells in HL.

Hodgkin and Reed/Sternberg Cell. Figure 1 HRS cells in Hodgkin lymphoma tissue. CD30 immunostaining (red) of a HL lymph node tissue section with CD30-positive HRS cells. Nuclei of the cells are stained in blue with haemalaun.

composition of the lymphoma, HL is subdivided in four types of classical HL (nodular sclerosis, mixed cellularity, lymphocyte depletion, and lymphocyte-rich classical) and lymphocyte-predominant HL. This latter type represents about 5% of cases. In lymphocytepredominant HL, the tumor cells have a distinct morphology and immunophenotype and are normally not multinucleated. These cells are not called HRS cells but lymphocytic and histiocytic ▶(L&H) cells. It is very difficult to grow HRS cells in culture, and only a few cell lines could be established from HL biopsies. Nevertheless, these cell lines are valuable models for the functional analysis of HRS cells. The cell lines still available and considered as HRS cellderived include the B cell lineage lines L428, L1236, KMH2, L591 (the latter is the only EBV-positive line) and the T cell lineage HL lines HDLM2 and L540. Although HRS cells are the pathognomonic cells in HL, HRS or HRS-like cells are also occasionally observed in other lymphomas. These include some ▶diffuse large B cell lymphomas and ▶B cell chronic lymphocytic leukemia (B-CLL). In B-CLL, the HRSlike cells can either be clonally related to the B-CLL lymphoma clone, or they can represent an independent cell clone. HRS cells are also regularly found in an infectious disease, i.e., ▶infectious mononucleosis. This disease is caused by EBV and can occur if the primary infection of an individual by EBV does not occur in young children (when it is usually asymptomatic) but is delayed into adolescence or adulthood. In infectious mononucleosis many B cells are infected by EBV and can expand to large clones. Some of the EBV-infected B cells for unknown reasons acquire the

Cellular Origin As the HRS cells usually represent only a small fraction of the cells in the HL tissue and as they have an unusual immunophenotype that does not resemble any normal hematopoietic cell type (see later) the cellular origin of these cells was unclear for a long time. However, about 10 years ago, the molecular analysis of isolated HRS cells revealed that these cells derive from B lymphocytes in nearly all cases. HRS cells carry rearranged immunoglobulin (Ig) genes which is specific for B cells. These rearranged Ig genes carry with few exceptions a high load of somatic mutations. The acquisition of such mutations through the process of ▶somatic hypermutation takes place in antigenactivated B cells in the course of T cell-dependent immune responses in specific histological structures of lymph nodes and other lymphoid organs – the ▶germinal centres. Therefore, it is very likely that HRS cells derive from such mature germinal centre B cells. Curiously, HRS cells frequently carry destructive somatic mutations that prevent expression of a functional B cell antigen receptor. As normal germinal centre B cells acquiring such mutations undergo ▶apoptosis, HRS cells may represent transformed preapoptotic germinal centre B cells. The detection of rearranged T cell receptor genes in a few cases of HL indicates that in rare instances (less than 2% of cases), HRS cells may also derive from T cells. Immunophenotype HRS cells show an unusual immunophenotype with coexpression of markers of distinct hematopoietic cell lineages. B cell antigens (e.g., CD19, CD20) or T cell antigens (e.g., CD3, CD4) are detectable on variable proportions of HRS cells in about 10–20% of cases. Several markers of dendritic cells are regularly expressed (e.g., thymus and activation regulated chemokine (TARC), fascin, and restin), but also markers of myeloid cells are found (CD15). As HRS cells are in nearly all cases derived from B cells, it is evident that many of these markers are aberrantly expressed by HRS cells. It also became clear that HRS cells lost expression of most typical B cell markers. For the diagnosis of HL, it is important that HRS cells in all cases of classical HL express CD30, an activation marker and member of the tumor necrosis factor receptor family. Cell Differentiation and Function HRS cells can be considered as hyperactivated cells, as a multitude of signaling pathways is chronically

Hoechst-33342 Dye

activated in these cells. HRS cells show constitutive activity of the transcription factor NFκB, which regulates the expression of many genes, including important antiapoptotic factors. Indeed, inhibition of NFκB in HRS cell lines induced apoptosis of the cells. Also the PI3 kinase/AKT signaling pathway and the activator protein 1 (AP-1) are active in HRS cells and contribute to cell survival and/or proliferation. Remarkable is Notch-1 activity in HRS cells, as this transcription factor is normally active only in T cells and suppresses expression of B cell-specific genes. Thus, the deregulated activity of Notch-1 in HRS cells may contribute to the downregulation of B cell genes, as discussed earlier. In HL tissues, many cytokines are produced by HRS cells and surrounding cells. On this basis, it is perhaps not surprising that HRS cells have an active Jak/STAT pathway, as many cytokines signal through this pathway. There appears to be even an autocrine interleukin stimulation in HRS cells, as they express interleukin 13 and the interleukin 13 receptor. Three of the STAT transcription factors have been identified as constitutively active in HRS cells, STAT3, 5 and 6. Another important family of signaling molecules is receptor tyrosine kinases. In several cancers, specific members of this family show deregulated activity that contributes to pathogenesis. In HL, multiple receptor tyrosine kinases are expressed and active, which is in its extent unique among human tumors. Transforming Mechanisms of HRS Cells As mentioned earlier, in about 40% of cases of HL in the Western world, HRS cells are infected by EBV. In some instances (e.g., in childhood cases in Central America) the association can be even up to 90%. In EBV-positive cases, three latent viral proteins are expressed, the EBV nuclear antigen EBNA1, and the latent membrane proteins LMP1 and LMP2A. EBNA1 is essential for the replication of the viral genome in proliferating cells. Interestingly, LMP1 and LMP2A functionally mimic two main survival signals for germinal centre B cells, i.e., CD40 signaling and B cell receptor signaling. In this way, EBV can rescue the HRS cell precursors from apoptosis and contributes to the malignant transformation. HRS cells usually show multiple numerical and structural chromosomal abnormalities. Some of these are shared by all HRS cells, while others are found only in fractions of cells of a tumor clone, indicating a significant genomic instability. Due to the multitude of genomic aberrations, it has been difficult to identify events causally involved in the transformation of HRS cells. However, some genomic imbalances occur recurrent and are therefore candidates for pathogenetic events. These include, for example, amplification of the genomic region including the c-Rel gene, a member

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of the NFκB family. HRS cells frequently also harbor translocations involving the immunoglobulin loci, but the partner genes involved in these translocations have not yet been identified. Isolated HRS cells were also analyzed for mutations in proto-oncogenes and tumor suppressor genes. Mutations in the tumor suppressor genes p53 and CD95 were found in less than 10% of cases investigated. Inactivating mutations in a main inhibitor of the NFκB transcription factor, IκBα, are present in about 20% of cases. Rare cases may also carry mutations in IκBε, another inhibitor of NFκB. Mutations in these NFκB inhibitors contribute to the constitutive activity of this factor, which has an important role as an antiapoptotic factor in HRS cells (see earlier). Mutations were recently also found in the SOCS1 gene, which is an inhibitor of STAT transcription factors. Thus, these mutations likely contribute to the constitutive activity of STATs in HRS cells. In summary, the transforming events involved in the generation of the malignant HRS cells in classical HL have only partly been revealed, and it appears so far that multiple different combinations of genetic aberrations can cause the pathogenesis of HL.

References 1. Pileri SA, Ascani S, Leoncini L et al. (2001) Hodgkin’s lymphoma: the pathologist’s viewpoint. J Clin Pathol 55:162–176 2. Re D, Thomas RK, Behringer K et al. (2005) From Hodgkin disease to Hodgkin lymphoma: biologic insights and therapeutic potential. Blood 105:4553–4560 3. Küppers R, Re D (2007) Nature of Reed-Sternberg and L&H cells, and their molecular biology in Hodgkin lymphoma. In: Hoppe RT, Armitage JO, Diehl V, Mauch PM, Weiss LM (eds) Hodgkin lymphoma. Lippincott Williams & Wilkins, Philadelphia 4. Küppers R (2002) Molecular biology of Hodgkin's lymphoma. Adv Cancer Res 84:277–312

Hoechst-33342 Dye Definition Is a UV-excitable nucleic acid stain that is readily taken up by all cells, except those cells that express the transporterABCG2. Hoechst-dim cells can be detected by flow cytometry or by fluorescence microscopy. ▶Stem Cell Markers ▶FISH

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Hollow Fiber Assay

Hollow Fiber Assay I RENE V. B IJNSDORP, G ODEFRIDUS J. P ETERS Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands

Definition The hollow fiber assay (▶HFA) is a fast in vivo assay to determine the cytotoxic effect of drugs, as well as their pharmacodynamic (▶Pharmacodynamics) effects on human tumor cell lines grown in hollow fibers that are implanted subcutaneously or intraperitoneally in mice or rats.

Characteristics Various in vivo models exist to asses the efficacy of potential anticancer agents, including subcutaneously implanted ▶xenografts, ▶orthotopic models, and the HFA. The HFA has been optimized at the National Cancer Institute (▶NCI) and is a unique in vivo model for ▶drug discovery. It permits the simultaneous evaluation of the efficacy of anticancer agents against multiple tumor cell lines in two physiological compartments of one animal. In this assay, semipermeable biocompatible fibers are used, which can be filled with cancer cells. These cancer cells can be derived from different tissue origins and can have different cellular characteristics. The fibers are made of polyvinylidene fluoride (PVDF) and have an internal diameter of 0.5–1 mm and are 2 cm long. The fiber has a molecular weight exclusion of 500,000 Da. Therefore soluble bioactive agents (e.g., proteins, such as growth factors produced by tumor cells or host) can bypass the fiber, but tumor cell to host cell contact is not possible. The fibers are mostly implanted in mice, but it is also possible to implant the fibers in rats and other animals for ▶preclinical testing of anticancer agents. One mouse can support the growth of up to six cancer cell lines, and therefore enables to test more than one cell line simultaneously in one animal. The fibers can be transplanted in both immunocompromized and in immunocompetent mouse strains, since the immune cells of the host can not infiltrate into the hollow fiber and therefore can not activate an immune response or impede the growth of the tumor cells. The fiber can be implanted either intraperitoneally (▶i.p.) or subcutaneously (▶s.c.), therefore differences of intravenous (▶i.v.), oral, i.p., and s.c. administration of drugs can aid in assessing the effects of hepatic pass through, thus providing information for dose estimations and administration routes for more extensive in vivo testing of both cytotoxic and antiangiogenic ▶chemotherapy. After treatment, active cell proliferation and cellular

characteristics, including ▶cell cycle distribution, ▶DNA damage induction, cell death induction (▶Apoptosis), protein expression levels and cell morphology can easily be studied (Fig. 1). Besides this, in vivo pharmacokinetic (▶Pharmacokinetics) parameters, including drug transport, pH and pO2 can be analyzed. The NCI currently uses a panel of 12 tumor cell lines for routine hollow fiber screening of anticancer drug activities. It is used as the initial in vivo assessment to determine potential activity of agents that have reproducible activity in the in vitro anticancer drug screen. The HFA prioritizes compounds for secondary ▶xenograft screening and helps to reduce the large number of active compounds generated by the in vitro ▶NCI 60 cell line screen that were forming a bottleneck for entry into secondary xenograft testing. The NCI HFA requires 24 mice for testing of one compound against a panel of 12 cancer cell lines in contrast to the NCI xenograft model, where three xenograft models are used to test one agent with each xenograft model, which requires about 50 mice. The use of the HFA in early ▶preclinical drug screens fits excellently with the philosophy of replacement, refinement and reduction (3Rs) of animals in research, controlling the use of laboratory animals.

Hollow Fiber Assay. Figure 1 Schematic overview of the HFA procedure for preclinical testing the anticancer activity of a compound using cancer cell lines transplanted subcutaneously (s.c.) or intraperitoneally (i.p.) in mice. Method is described according to NCI guidelines, http:// dtp.nci.nih.gov/branches/btb/hfa.html.

Hollow Fiber Assay

Since the development of the HFA for ▶drug screening, other groups have used this assay to independently demonstrate drug activity. Compound efficacy has been demonstrated using the HFA, using both primary human tumor cells and characterized tumor cell lines. Although the NCI uses the HFA successfully to quantitatively define anticancer activity, the role of this in vivo assay could be extended. The HFA has already been adapted for evaluation of antiviral agents and it may also be adapted to use it in other medical fields. The in vivo HFA has demonstrated a good predictivity of xenograft activity. Anticancer activity in xenograft models is defined by a reduction in relative tumor volume. In the HFA the activity is defined as a reduction of the number of viable cells, measured with an ▶MTT assay. Any compound found to be active in xenograft models is often also active in the HFA. A xenograft model does not easily permit elucidation of the mechanism of action of a compound in a tumor. Retrieval of tumor cells from xenograft models for pharmacodynamic analysis is complicated by host cell contamination. Cells grown in a hollow fiber can easily be harvested for mechanistic studies as a single cell suspension without contamination of host cells. When the HFA is used for preclinical pharmacodynamic investigations, compounds may progress to the clinic much faster. On the other hand, more information is required on tumor cell–stroma interaction. The development of such a model using the HFA is a new challenge in its application. The HFA has several advantages over standard animal efficacy models. First, the assay has demonstrated the ability to provide quantitative indices of drug efficacy with minimum expenditures of time (procedure takes less than 2 weeks) and materials (e.g., test compounds). It is not limited by the high costs that are associated with large-scale animal testing using xenograft tumor models. Second, conducting studies in animal models requires substantial amounts of time and resources for the testing procedure. Even when it is possible to conduct such studies, it is possible that the experimental agents will exhibit only minimal antitumor activity. Thus, the HFA will prevent to test inactive compounds in a xenograft model. Third, cancer treatments that appear promising in tissue culture are often less effective in solid tumors. This can be partially because of the proliferative and microenvironmental heterogeneity that develops in these tumors during their growth in a ▶three-dimensional structure; cells in a hollow fiber also grow in a three-dimensional structure. Fourth, this procedure of testing takes the pharmacokinetic behavior of the drug into account, as well as potential biotransformation. Fifth, the HFA is excellently suited to perform initial in vivo experiments to study combinations, because the compounds will have a

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dynamic in vivo interaction. Sixth, the HFA prevents that the mouse suffers from the tumor induced cachexia. The HFA was not developed to replace the classic xenograft model. Complex interactions which occur when the transplanted tumor cells are growing in and interacting with the host’s tissue can not be analyzed. Cells can not metastasize to secondary organs of the animal, since the fiber prevents cellular ▶migration. Although ▶angiogenesis in the tumor will not be induced when these fibers are used, neovascularization occurs to supply the fiber with nutrients. This is possibly induced by growth factors that are released by the tumor cells in the fiber. The hollow fiber can be used perfectly as a preliminary tool to asses the capacity of a compound to reach tumor cells growing in two distinct physiologic compartments (s.c. or i.p.) and to asses whether the drug can reach pharmacologically active concentrations in the tumor cells. Using modern ▶bioluminescent techniques, it will be possible to perform dynamic growth of the cells in the fibers.

Methodology The HFA procedure is as follows (Fig. 1): tumor cells are harvested at the desired cell density from a log phase growing in vitro culture. The cell suspension is flushed into the hollow fibers, which are subsequently heatsealed so that the cells can not escape from the fiber. The density is dependent on the cell line and may vary from 1 to 100 × 105 cells/fiber. Prior to transplantation into the host, fibers are placed into tissue culture medium and incubated at 37°C in 5% CO2 atmosphere for 24–48 h. On the day of implantation, samples of each tumor cell line preparation can be quantified for viable cell mass by a stable endpoint MTT assay. Mice can then be treated with anticancer agents starting few days after fiber implantation and for a maximum of 2 weeks. Following treatment, the fibers are collected from the host and subjected to the stable endpoint MTT assay. The percent net growth for each cell line is calculated and compared to the percent net growth in the vehicle treated controls. Furthermore, at the treatment endpoint, various other cellular analyses can be performed.

References 1. Hollingshead MG, Alley MC, Camalier RF et al. (1995) In vivo cultivation of tumor cells in hollow fibers. Life Sci 57:131–141 2. Suggitt M, Bibby MC (2005) 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clin Cancer Res 11:971–981 3. Decker S, Hollingshead M, Bonomi CA et al. (2004) The hollow fibre model in cancer drug screening: the NCI experience. Eur J Cancer 40:821–826

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4. Temmink OH, Prins HJ, van Gelderop E et al. (2007) The hollow fibre assay as a model for in vivo pharmacodynamics of fluoropyrimidines in colon cancer cells. Br J Cancer 96:61–66 5. Suggitt M, Cooper PA, Shnyder SD et al. (2006) The hollow fibre model – facilitating anti-cancer pre-clinical pharmacodynamics and improving animal welfare. Int J Oncol 29:1493–1499

Homeobox Definition A short usually highly conserved DNA sequence found within genes that are involved in the regulation of development (morphogenesis) of animals, fungi and plants. Genes that have a homeobox are called homeobox genes and form the homeobox gene family. ▶Nucleoporin

Homeobox Genes S TACEY S TEIN , C ORY A BATE -S HEN Center for Advanced Biotechnology and Medicine, UMDMJ – Robert Wood Johnson Medical School, Piscataway, NJ, USA

Definition Homeobox genes are a class of developmental regulatory genes that encode homeoproteins. Homeoproteins function as transcription factors to regulate downstream targets, turning on (activating) or turning off (repressing) other genes that in turn regulate developmental processes. Aberrant expression of homeobox genes has been implicated as causal factors in leukemia and solid tumors.

Characteristics Homeobox genes are often referred to as master control genes. They are active during embryonic development and regulate important processes such as ▶morphogenesis and cellular ▶differentiation. They were first identified in Drosophila as the homeotic cluster (HOM-C) genes and were later found to have homologs in many species. They are now known to be highly evolutionarily conserved and are present in animals, plants and fungi.

Homeobox genes share a common sequence motif (the homeobox), which is 180 nucleotides in length and encodes a 60 amino acid region (the homeodomain). The homeodomain mediates DNA binding to sites containing a ▶TAAT sequence that are found in the transcriptional regulatory elements of target genes. Upon DNA binding, homeoproteins are thought to act as transcription factors to regulate downstream targets. There are two major subclasses of vertebrate homeobox genes: . The clustered genes, or HOX genes . The non-clustered genes The non-clustered genes are sub-divided into many subfamilies, which are classified on the basis of sequence similarities within their homeobox regions. The HOX family of homeobox genes, as well as most other families of homeobox genes, is thought to control cellular proliferation and differentiation during development. The HOX genes are expressed beginning in ▶gastrulation and are involved in patterning the body axis from the branchial arches to the tail. What is most notable about HOX genes is their positioning on four chromosome clusters. Not only are the sequences of the individual genes highly conserved across species, the order of the genes on the chromosomes is conserved as well. Moreover, the physical order of the genes on the chromosome correlates with their spatial and temporal expression patterns along the anteroposterior axis of the embryo. Many studies have reported a link between deregulated homeobox gene expression and abnormal cellular proliferation, which implicates homeobox genes not only as development regulators, but also as potential protooncogenes and tumor suppressor genes. The downstream targets of homeobox genes are postulated to include extracellular matrix proteins, adhesion molecules and growth factors. Because these target genes are likely to be important for tumorigenesis as well as development, their misexpression may upset the delicate balance of cell proliferation, differentiation and apoptosis, thereby contributing to carcinogenesis. Hox Genes and Leukemia In addition to their functions during development, homeobox genes also play important roles in adult tissues. For instance, the HOX genes are expressed in specific patterns during lineage determination in ▶hematopoiesis. HOX genes have been demonstrated to be important for normal blood cell formation; in addition, abnormal expression of HOX and other homeobox genes contributes to the development of leukemia and lymphoma. A common mechanism by which abnormal gene expression contributes to leukemia is through translocation of two chromosomal regions, which may

Homeostasis

produce fusion proteins with novel properties or impaired activities. For example, in human acute myeloid leukemia (AML) a translocation between chromosomes 7 and 11 fuses the nucleophorine gene NUP98 in frame with HOXA9. It is thought that the resulting chimeric protein is no longer able to interact with HOXA9 target genes. Another example of a homeobox translocation in leukemia is the fusion of PBX1 homeobox gene and the E2A gene. Normally, PBX1 forms transcriptionally active protein complexes with HOX proteins; its fusion with E2A alters such interactions, which is thought to direct homeoproteins to different targets, thereby inducing leukemogenesis. Like PBX, other divergent homeoproteins interact with specific HOX proteins to potentiate or modulate their effects, and their genes are potential targets for deregulation in carcinoma. For example, it now appears that the MEIS1 homeobox gene is co-activated with HOXA9 in human myeloid leukemia. In addition, translocation of the MLL gene is a common event having been found to be fused to more than 25 other genes in leukemia. While MLL is not a homeobox gene, it is homologous to a Drosophila protein that is an important regulator of HOM-C genes. The leukemogenic MLL fusion proteins are thought to disrupt HOX gene expression in hematopoietic progenitor cells, thereby contributing to myeloid or lymphoid acute leukemias. Homeobox Genes and Solid Tumors A common feature of HOX gene expression in certain adult organs, such as kidney, lungs and colon, are the significant differences in expression pattern between normal and cancer tissue. For example, in the kidney HOXC11 is present in tumors but not in normal tissue. Conversely, HOXB5 and HOXB9 are normally expressed in the kidney but expression is often lost in tumors. Other HOX genes, such as HOXD4, are expressed in both normal kidney and in kidney tumors, although they express a different-sized transcript in the tumors versus the normal tissue. In each of these cases the significance of these differential gene expression patterns remains undetermined. Similar observations have been made in the colon. Misexpression of some HOX genes (HOXA9, HOXB7 and HOXD11) is found in primary colon cancer and metastatic lesions originating from colorectal tumors. In addition, HOXB6, HOXB8, HOXC8 and HOXC9 are misexpressed at specific stages of colon cancer progression. And, while HOXB7 and HOXD4 are both expressed in normal and neoplastic colon, the size of the transcripts differ. Again, the significance of these observations for disease initiation or progression is unclear. CDX1 and CDX2 homeobox genes are expressed in normal colon, while their expression is reduced in colon

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tumors. Interestingly, there appears to be an inverse correlation between the levels of protein expression and the severity of the dysplasia. This correlation, in conjunction with other data, suggests that expression of CDX1 and CDX2 is important for maintaining normal colon differentiation and that their loss of expression may promote a cancer phenotype. These observations have been supported by mutant mouse models in which loss of CDX genes results in colon tumors. Misexpression of certain homeobox genes has also been implicated in prostate cancer. For example, the GBX2 homeobox gene is overexpressed in several metastatic prostate cell lines, suggesting a possible role in cancer progression. Conversely, loss of function of the NKX3.1 homeobox genes has been implicated in prostate cancer initiation. Notably, NKX3.1 maps to a hotspot that is frequently deleted in many prostate cancer samples, and mutant mice lacking NKX3.1 display prostatic epithelial dysplasia. Like, the CDX genes, these mutant mouse models provide excellent support for a functional role of these homeobox genes in cancer. At this point much of the data is rather circumstantial and based on altered expression patterns. Nonetheless, the available evidence suggests that homeobox genes may provide an important link in understanding the delicate balance of development, cell-cycle control and cancer. The specific molecular events that occur between the misexpression of homeobox genes and the progression to cancer remains a topic of active investigation.

References 1. Cillo C, Faiella A, Cantile M et al. (1999) Homeobox genes and cancer. Exp Cell Res 248:1–9 2. Cristina M, Largman C, Lawrence J (1997) Effects of HOX homeobox genes in blood cell differentiation. J Cell Physiol 173:168–177 3. Look A (1997) Oncogenic transcription factors in the human acute leukemias. Science 278:1059–1064 4. Mark M, Rijli F, Chambon P (1997) Homeobox genes in embryogenesis and pathogenesis. Pediatr Res 42:421–429

Homeostasis Definition Is one of the fundamental characteristics of living things. It is the maintenance of the internal environment within tolerable limits. Homeostasis is maintained by

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means of multiple dynamic equilibrium adjustments, controlled by interrelated regulatory mechanisms, resulting in a stable, constant condition. ▶Apoptosis-Induction for Cancer Therapy

Homeotic Genes Definition Genes involved in developmental patterns and sequences. ▶Polycomb Group

Homer Wright Rosette Definition Is a circular or spherical grouping of dark tumor cells around a pale, eosinophilic, central area that contains neurofibrils but lacks a lumen; seen in some cases of ▶medulloblastoma, ▶neuroblastoma, and ▶retinoblastoma. A rosette structure is also an important morphologic finding in the diagnosis ▶Ewing sarcoma/ ▶pPNET.

target tissues through the circulation. The resultant peptides bind to endothelial (and possibly pericyte) surface proteins that are selectively expressed in the vasculature of individual tissues (Fig. 1). Homing peptides have been obtained for the vasculature in a large number of individual normal tissues that reveal a previously unsuspected degree of endothelial specialization. Screening for tumor homing has produced a collection of peptides that home to tumor vasculature. Atherosclerotic lesions have also been targeted in this manner. The vessels of some normal organs have been known to express specific marker molecules, but in vivo phage has revealed an unprecedented degree of diversification in endothelia. The tissues for which vascular homing peptides have been described in the literature include brain, kidney, lung, heart, skin, pancreas, retina, intestine, muscle, uterus, prostate, fat tissue, and the adrenal gland. Success with so many tissues indicates that many, perhaps all, organs modify the endothelium of the vasculature, and that it is justified to refer to this heterogeneity as “vascular zip codes.” The vasculature of tumors is also specialized. Tumors depend on ▶angiogenesis to grow and undergo ▶metastasis. The actively growing tumor vessels are biochemically and structurally different from normal resting blood vessels. Integrins αvβ3, αvβ3, and α5β1as well as receptors for various vascular endothelial growth factors, are examples of molecules expressed at elevated levels in tumor blood vessels. Significantly, each of these receptors plays a critical role in angiogenesis. Other markers of tumor vasculature include an alternatively

Homing Peptides and Vascular Zip Codes E RKKI R UOSLAHTI Vascular Mapping Center, Burnham Institute for Medical Research at University of California, Santa Barbara, CA, USA

Definition Vascular Zip Codes refers to tissue- and disease-specific molecular differences in vessels.

Characteristics Peptide libraries displayed on phage can be screened in vivo to derive peptides capable of homing to selected

Homing Peptides and Vascular Zip Codes. Figure 1 Homing peptides direct phage binding to specific sites in the vasculature.

Homogeneously Staining Region

spliced form of fibronectin, endosialin, certain aminopeptidases, an anthrax toxin receptor, and intracellular proteins aberrantly expressed at the cell surface (nucleolin, annexin A1). Melanoma-associated proteoglycan NG2 is a marker of pericytes in tumor vessels. Phage library screening in vivo has resulted in the identification of several peptide motifs that selectively direct phage into tumors. One of these motifs contains the sequence RGD (arginine-glycine-aspartic acid) embedded in a peptide motif previously shown to bind selectively to αv integrins. A number of additional tumor-homing peptides and receptors for some of them have been identified. Examples include peptides that recognize aminopeptidase N, cell surface nucleolin and clotted plasma proteins in tumor vessels. Homing peptides have also been used to show that the blood vessels of pre-malignant lesions differ from those of the corresponding normal tissue and from the vessels of fully developed tumors in that tissue. Further, lymphatic vessels in tumors differ molecularly from the lymphatics of normal tissues. Biological Significance Vascular zip code molecules are functionally important in the vessels that express them. A prime example is various angiogenesis markers, many of which are involved in the angiogenesis process. One known function of zip code molecules in the vessels of normal tissues is to serve cell trafficking. Tumor cells appear to make use of such molecules in homing to preferred sites of metastasis. Clinical Significance Coupling of drugs, ▶apoptosis-inducing peptides, protein therapeutics, or imaging agents to tumor-homing peptides enhances the activity of these compounds in mice and lowers their toxicity. The homing peptides are also useful for the targeting of nanoparticles and potentially also cells to tumors. Delivery strategies similar to the tumor targeting should be applicable to targeting of pathological conditions other than tumors.

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Homing receptor ▶CD44

Homo Sapiens Mitotic Arrest Deficient-like 1 Protein ▶Mitotic Arrest-Deficient Protein?

H Homodimer Definition A type of protein interaction in which two of the same protein bind together to form a complex, often necessary for the normal function of receptors. ▶Prostate-Specific Membrane Antigen

Homo- or Heterodimers Definition A protein complex consisting of two identical (homo) or different (hetero) subunits. ▶Early B-cell Factors

References 1. Ruoslahti E (2002) Specialization of tumour vasculature. Nat Rev Cancer 2:83–90 2. Laakkonen P, Porkka K, Hoffman JA et al. (2002) A tumor-homing peptide with a lymphatic vessel-related targeting specificity. Nat Med 8:743–751 3. Brown D, Ruoslahti E (2004) Metadherin, a cell-surface protein in breast tumors that mediates lung metastasis. Cancer Cell 5:365–374 4. Ruoslahti E, (2004) Vascular zipcodes in angiogenesis and metastasis. Biochem Soc Transact 32:397–402 5. Simberg D, Duza T, Park JH et al. (2007) Biomimetic amplification of nanoparticle homing to tumors. Proc Natl Acad Sci USA 104:932–936

Homogeneously Staining Region Definition HSR; is a region within a chromosome lacking the typical banding pattern after staining with Giemsa, indicative of DNA amplification. In tumor cells indicating oncogene amplification, in some cases also amplification of genes encoding proteins for drug metabolism. ▶Amplification

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Homogenous Assays

Homogenous Assays ▶Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery

Homogenous Time Resolved Fluorescence ▶Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery

Homolog Definition Genes that share a high degree of sequence identity or similarity, indicating their common ancestor or function.

Homologous Recombination Repair J OHN T HACKER Medical Research Council, Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK

Definition Homologous recombination repair is a DNA repair process that includes the invasion of an undamaged DNA molecule by a damaged molecule of identical or very similar sequence. Resynthesis of the damaged region is accomplished using the undamaged molecule as a template.

Characteristics Homologous recombination repair has been found in all organisms examined from bacteria to man. It has an important role in repairing DNA damage with high fidelity by correcting damage with the use of information copied from an homologous undamaged molecule. Sister chromatids (duplicated chromosomes following DNA replication) or the paternal and maternal copies of

chromosomes provide the required homology (sequence identity or near-identity over a few hundred DNA base pairs). In somatic cells, if the homologues have some sequence differences the copying process may alter the sequence of the damaged chromosome to the same as that of the undamaged chromosome, potentially revealing mutations (▶loss of heterozygosity). Additionally, recombination between repeat sequences may lead to high frequencies of sequence variation as seen in certain ▶minisatellite sequences. In germ cells, homologous recombination is vital for the reassortment of chromosomes during meiosis to create genetic diversity in organisms. Cellular Regulation The proteins that mediate homologous recombination repair have functions and often structures that have been conserved in evolution. These proteins have to seek out homologous regions of chromosomes, exchange DNA strands, copy sequence from the undamaged strand and finally resolve the DNA structures arising from exchange. This complex series of events is best understood in bacteria but recently many of the components and functions of homologous recombination repair have been identified in mammalian cells. Evidence suggests that DNA double-strand breaks commonly trigger repair by homologous recombination; these breaks may be caused by the interaction of DNA with chemical radicals, produced as a consequence of cellular metabolism, or by external damaging agents such as ionizing radiations. Meiotic recombination is also driven by DNA double-strand breaks, but these are formed enzymatically at specific sites in DNA. In bacteria, the ▶RecA protein is central to homologous recombination through its ability to search for homologous regions of DNA and promote strand exchange. RecA forms a polymer on DNA to give a nucleoprotein filament, acting as a DNA-dependent ATPase. Filament formation occurs very rapidly on single-stranded DNA; in purified solutions the rate can approach 1,000 RecA monomers assembled per minute. RecA will also polymerize on double-stranded DNA but needs a short single-stranded gap to start the process, potentially targeting the protein to sites needing repair. On binding RecA and in the presence of homologous double-stranded DNA, strand exchange occurs to form junctions between the two DNA molecules. A number of other proteins are involved in the early stages of homologous recombination repair in bacteria; to generate the single-stranded DNA required for RecA-mediated strand exchange different proteins (RecBCD, RecJ, RecQ, or RecE) may be used depending on the initial state of the DNA. For example, during bacterial conjugation the RecBCD protein will unwind DNA from a break and cut the unwound DNA at a specific sequence-recognition site,

Homologous Recombination Repair

forming a long 3′ single-stranded region suitable for invading homologous double-stranded DNA. Other proteins assist in the loading of RecA onto singlestranded DNA and/or strand-invasion activities (RecF, RecO, RecR and single-stranded DNA binding protein). During recombination the junctions formed at sites of exchange migrate along the DNA molecules to complete gap repair, and finally the junctions must be cut to free the participating DNA molecules. Again some of these functions are supplied by different proteins, in this case either RuvABC or RecG, indicating the presence of more than one pathway for homologous recombination. While much of the mechanistic detail of homologous recombination repair has been described in bacteria, it is clear that the principles of this mechanism have been retained by all organisms. RecA-like proteins have been found in eukaryotes; in particular the ▶Rad51 protein from the yeast Saccharomyces cerevisiae (Rad51p) has both structural and functional similarities to RecA. Rad51p leads to the formation of nucleoprotein filaments on DNA and the promotion of homologous pairing and strand exchange reactions in vitro. However, in addition to Rad51p there are three other ▶RecA/Rad51-like proteins in yeast; Rad55p, Rad57p and Dmc1p. Biochemical studies suggest that these RecA/Rad51-like proteins do not have redundant functions but rather have distinct roles to play in the early stages of repair. The Dmc1 protein functions exclusively in meiosis, with loss of its function leading to sterility, while the other RecA-like proteins operate in both mitosis and meiosis. Rad55p and Rad57p exist as a dimer and appear to act as a cofactor for the assembly of Rad51p onto single-stranded DNA. A three-protein complex (▶Rad50p/▶Mre11p/Xrs2p) has been found that promotes the early stages of homologous recombination but is also involved in a number of other repair and DNA maintenance functions; the Mre11 protein in particular has nuclease activities that may process double-strand breaks to form single-stranded regions associated with strand invasion. Other yeast recombination proteins, such as ▶Rad52p and ▶Rad54p, interact with Rad51p and promote Rad51-mediated strand exchange. Rad52p interacts with single-stranded DNA binding protein (Rpa) and facilitates the loading of Rad51p onto single-stranded DNA lacking secondary structure. Rad54p is structurally related to a family of putative DNA helicases but its precise function is unknown. Rad51 also occurs in human cells. Despite millions of years of evolutionary divergence, the human protein is remarkably similar in structure and function to the yeast Rad51 protein. Additionally, six further RecA/ Rad51-like proteins have recently been identified in humans. Two of these, Xrcc2 and Xrcc3, were found through their ability to complement the sensitivity of

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certain mammalian cell mutants to DNA-damaging agents. The remainder, including a protein closely similar to the yeast Dmc1, were found by searching for RecA/Rad51-like proteins in the human protein sequence databases. These proteins are presently the subject of intensive study but there is already evidence that some may function like the yeast Rad55 and Rad57 proteins in forming dimers that help Rad51 to function efficiently. The increase in the numbers of RecA/ Rad51-like proteins in higher organisms also suggests that certain specialized functions may have evolved to deal with specific types of DNA damage or with tissuespecific recombination events. The yeast Rad50 and Mre11 proteins are also conserved in mammalian cells, but a structural homologue of the yeast Xrs2 protein has not been found in humans. Instead the Nbs1 (▶Nijmegen breakage syndrome) protein (also known as p95 or nibrin), defective in the radiosensitive and cancer-prone human disorder Nijmegen breakage syndrome, appears to be a functional analogue of Xrs2. The features of Nbs1-deficient cells are very similar to those of ataxiatelangiectasia cells, and recently the human Mre11 protein has been shown to be mutated in individuals with an ataxia-telangiectasia-like disorder. Proteins similar to the yeast Rad52 and Rad54 have also been found in humans, and in the case of Rad54 it has been shown that the human protein is able to function in place of the yeast protein. The Rad52 protein is not so well conserved in structure between yeast and humans, although it is possible that another Rad52-like protein remains to be discovered in humans. In biochemical experiments, however, the human Rad52 protein has been shown to stimulate Rad51-mediated DNA-strand transfer reactions, probably at an early stage of Rad51 filament formation. Proteins involved in the later stages of recombination repair have yet to be identified in yeast or mammalian cells. The interactions of these proteins in the early stages of repair of a DNA doublestrand break by homologous recombination are illustrated in the Fig. 1. To study the effects of loss of recombination genes in mammals, several of the genes have been disrupted in mice (“knockout mice”). Surprisingly, disruption of Rad51 in mice is lethal for embryonic development and cells could not be cultured from tissue derived from the knockout animal. This finding suggests that the mammalian Rad51 gene has an important role in essential aspects of DNA metabolism or in development. It also suggests that the other Rad51-like genes cannot replace the function of Rad51 itself. Disruption of some of the Rad51-like genes in mice also leads to embryonic lethality, showing that these similarly have important functions in the organism. In contrast, knocking-out other recombination genes in mice is not necessarily lethal. Disruption of the meiosisspecific Dmc1 gene, as well as similar knockout

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Homologous Recombination Repair

Homologous Recombination Repair. Figure 1 A model for the repair of a DNA double-strand break by homologous recombination in human cells. The main steps are noted to the left, and the proteins involved (where known) to the right. The Rad51-like proteins include Xrcc2 and Xrcc3. The broken DNA molecule (black) is processed to give long 3′ single stranded regions; these invade an undamaged homologous molecule (red). The branched-out undamaged strand acts as a template to repair the break. Repair synthesis is accompanied by branch migration. Resolution involves cutting the junctions between the two molecules; here it is shown without crossing-over, potentially leading to gene conversion where the homologous molecules differ in sequence.

experiments with the mouse Rad52 and Rad54 genes, yielded viable progeny. With the possible exception of the Rad52 knockout mouse, each has severe defects in aspects of mitotic and/or meiotic processes, consistent with roles in homologous recombination repair. The potential importance of homologous recombination repair genes in genetic stability and in development has been highlighted by several recent discoveries. Firstly, it has been found that the human Rad51 protein interacts physically with the tumor-suppressor protein p53, which has a central role in the control of the cell cycle and apoptosis. Additionally, Rad51 knockout embryos survived longer in a p53-deficient background, suggesting at least part of the growth problems experienced by Rad51 knockouts arises from unrepaired damage triggering ▶cell-cycle checkpoints leading to growth arrest. Equally important, Rad51 interacts with the breast cancer-susceptibility gene products ▶Brca1 and ▶Brca2. Disruption of the Brca1 or Brca2 genes in

mice gives embryonic lethality and shows a similar elevation of chromosomal aberrations as that found in Rad51-disrupted cells. The link between the Brca proteins and homologous recombination repair has been extended by showing that Brca1 and Brca2-deficient cells have a large reduction in ability to repair doublestrand breaks in homologous substrates integrated into the genome. Brca1 is phosphorylated in response to DNA damage and this event is dependent on the protein kinase mutated in the cancer-prone disorder ataxia-telangiectasia (Atm). Atm may act as a sensor of DNA damage, especially double-strand breaks, and there is evidence supporting a model in which Brca1 phosphorylation triggers the activity of Rad51 and Brca2 to initiate homologous recombination repair. However, Brca1 also associates with the human Rad50 protein and has a role in the ▶transcription-coupled repair pathway, so its action is likely to be broader than this model suggests.

Homophilic and Heterophilic Adhesion

The role of recombination proteins in influencing development in multicellular organisms has been illustrated by recent findings in the fruit fly, Drosophila melanogaster. Mutations in the spindle class of genes lead to patterning defects in the oocyte and embryo, apparently due to mis-localization and/or failure to accumulate normal levels of oocyte signaling molecules. The cloning and sequencing of these genes showed that they are homologues of RAD51 or RAD54. Mutation in these genes leads to the accumulation of DNA damage, which triggers a meiotic recombination checkpoint that down-regulates some of the genes essential for embryonic development. Interestingly, oocytes that are mutant for both spindle-class genes and the Drosophila homologue of ATM (mei-41) can bypass this meiotic arrest and show normal developmental patterning, suggesting that loss of ability to signal damage fails to trigger this checkpoint. The downside of this checkpoint loss is that DNA damage may be carried through into later cell divisions, resulting in genomic instability. The mechanisms of homologous recombination repair are still being investigated, but it is clear that this repair pathway is of considerable significance in all organisms. Other pathways can repair certain types of DNA damage; for example, in mammalian cells, ▶non-homologous end joining is an alternative way to repair DNA double-strand breaks. However, simple end joining of broken DNA is prone to loss of sequence (DNA deletion) at the damage site, while homologous recombination repair can rejoin breaks with high fidelity. In bacteria it is clear that a major role of homologous recombination repair is to sort out problems arising during DNA replication, in particular where a replication fork stalls due to damage in its path. In yeast and mammals there is also evidence that homologous recombination proteins are particularly active during DNA replication. Additionally, certain types of DNA damage such as ▶DNA interstrand cross-links formed by agents such as mitomycin-C may be resolved only by the homologous recombination repair pathway. In support of this idea, cell lines lacking homologous recombination repair genes are very sensitive to mitomycin-C and other DNA cross-linking agents. Clinical Relevance Loss of homologous recombination repair leads to unrepaired damage in the genome, which in turn can give rise to genomic instability. Both Mre11 and Nbs1 have been found to be associated with rare human disorders with complex phenotypes, including radiation sensitivity and cancer-proneness (ataxia-telangiectasia and Nijmegen breakage syndrome, respectively). The association of homologous recombination repair

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proteins with p53 and the Brca proteins also suggests that this repair pathway has potentially important connections with predisposition to cancer. The links are strongest with breast cancer, through the Brca proteins and possibly the Atm protein. Additionally, loss of one of the human Rad51-like proteins (Rad51L1) has been associated with uterine leiomyomas.

References 1. Li X, Heyer W-D (2008) Homologous recombination in DNA repair and DNA damage tolerance. Cell Research 18:99–113 2. Thorslund T, West SC (2007) BRCA2: a universal recombinase regulator. Oncogene 26, 7720–7730 3. Helleday T, Lo J, van Gent DC, Engelward BP (2007) DNA double-strand break repair: from mechanistic understanding to cancer treatment. DNA Repair 6:923–935 4. Thacker J (2005) The RAD51 gene family, genetic instability and cancer. Cancer Letters 219:125–135

Homologue of Synaptopodin ▶Myopodin

Homomeric Complex Definition Complex composed of two or more identical subunits. ▶Hamartin

Homophilic and Heterophilic Adhesion Definition Adhesion between two undefined cell types can be mediated by either identical (homophilic) or different (heterophilic) adhesion molecules. ▶Cell Adhesion Molecules

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Homoplasmy

Homoplasmy

Hormonal Carcinogenesis

Definition

S USHANTA K. B ANERJEE

In normal conditions all copies of mtDNA are identical within coding region.

Cancer Research Unit, Research Division, VA Medical Center, Kansas City, MO Division of Hematology and Oncology, School of Medicine, Kansas University of Medical Center, Kansas City, KS, USA

▶Mitochondrial DNA

Definition

Homotypic and Heterotypic Adhesion Definition Adhesion mediated by undefined adhesion molecules between identical cell types (homotypic) or two different cell types (heterotypic). ▶Cell Adhesion Molecules

Homozygous Deletion Definition In a diploid organism, loss of both alleles or portion of both alleles from the genome of a cell, tumor or organism. Physical loss of both copies of the same gene or of the same chromosomal segment of a pair of homologous chromosomes. ▶Fragile Histidine Triad

Horizontal Gene Transfer (HGT) Definition In this process, an organism transfers its genetic material to another cell that is not its offspring. The horizontal transfer of genetic material was originally detected in prokaryotes, but recent data show that it occurs not only in unicellular, but also in multicellular eukaryotes and might be an important evolutionary mechanism. ▶Circulating Nucleic Acids

The process by which a normal cell is transformed into a cancer cell is called “Carcinogenesis”. When the carcinogenic event is either potentiated or promoted by hormones (i.e., natural or synthetic) (▶diethylstilbestrol, estradiol) is considered as “Hormonal Carcinogenesis” (▶Estrogenic hormone and cancer) (▶Hormones and Cancer).

Characteristics There are three major classes of hormones based upon their chemical structures. These are (i) peptide/protein hormones (e.g., leptin, angiotensin II, interleukins, ACTH, gastrin and others), (ii) amino acid or fatty acidderived hormones (e.g., epinephrine, acetylcholine, prostaglandins and others, and (iii) steroid hormones (e.g., estrogen, progesterone, testosterone and others). Each hormone has its unique features and plays specific role under normal physiological conditions. Normally, it requires very minute amounts to exert its biological function through its receptor or other signaling pathways. However, excess or alter form of a hormone or its receptor sometimes exhibits carcinogenic behavior and distort the regulated state of the cells, which ultimately activate the carcinogenic switch or growth of the tumor or aggressive properties of a tumor or all the above events. The ideal example of carcinogenic hormone is natural or synthetic estrogens such as 17β-estradiol (E2) and diethylstilbestrol (DES). In animal system (i.e., mice, rats and hamsters), these two estrogenic compounds induces tumors in various estrogen-depended organs or tissues and the incidence rate is 100%. Moreover, a strong links between estrogens and the development cancer in human have been evident over 300 years ago that showed a protective effect of pregnancy on the development of breast cancer and also underscored that the higher rates of breast cancer among catholic nuns. This finding was reconfirmed by several epidemiological studies that showed a single full-term pregnancy reduces the risk of development of breast cancer by 50% or more. Subsequent births, especially at an early age, further reduce the risk. Moreover, breast cancer risk in the presence of estrogen can be determined by several factors including early menarche, late menopause and obesity in postmenopausal women.


Several cancers have been found to relate with the status of carcinogenic hormones in the body. For instance, breast or prostate cancer development may depend on the supply of estrogen or androgen, respectively, or their cognate receptors in the tumor cells. Therefore, the removal of the source glands (ovary, testis or pituitary) or targeting the receptors by antagonists (i.e., tamoxifen) of the hormones is used to prevent the growth of these tumors. Mechanisms Cancer is a mixed tissue of abnormal cells, and the etiology of this disease remains uncertain. After decades of studies it has been apparent that cancer begins with multiple genetic and epigenetic insults that included mutations of regulatory genes, activation of oncogenes or inactivation of tumor suppressor genes. These molecular insults facilitate the cells to grow abnormally, gain motile behavior and other features needed to grow the cancer cells and invade to distant parts of the body. These fatal abnormalities are probably built up in the normal cells through endogenous or exogenous carcinogens. Estrogens have been used for last several decades to explore the molecular mechanisms of hormonal carcinogenesis by several investigators. These studies found chronic exposure of estrogens involve interplay and cross-talk between genotoxic and epigenetic

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changes that eventually lead to an uncontrolled cell proliferation and cellular transformation (Fig. 1). The genetic alterations are accomplished by estrogens through the formation of DNA adducts and chromosomal abnormalities including chromosome breaks, aneuploidy and telomeric association, while epigenetic alterations are potentiated through DNA methylation/ demethylation or through the alterations in cell signaling molecules such as WISP-2/CCN5 and others. These epigenetic changes through the molecular crosstalk may precede genetic changes in normal or premalignant cells and foster the accumulation of additional genetic and epigenetic hits. How estrogens induce genetic and epigenetic abuses in some cells? Despite decades of investigations, an absolute answer of this question is elusive. Because estrogeninduced carcinogenesis can be blocked by estrogen antagonists (i.e., Tamoxifen or ICI 182,780) or estrogen withdrawal, the estrogen receptors (nuclear or cytoplasmic) may be involved in estrogen-induced carcinogenic switch (Fig. 2). The normal physiological effects of estrogen are usually mediated through two classical estrogen receptors, estrogen receptor-alpha (ER-α) and estrogen receptor-beta (ER-β). These receptors are encoded by two separate genes though they have some common domains and have similar binding affinity with the ligands. Nevertheless, these two receptors play redundant roles in estrogen signaling. In cells where both

Hormonal Carcinogenesis. Figure 1 Putative Pathways involved in hormonal carcinogenesis. Carcinogenic estrogens-induced neoplastic transformation is mediated through genetic and epigenetic abuses in the target tissues or cells. Presumably genetic and epigenetic changes by carcinogenic hormones are interdependent and cross-talk or side-talk with each other to enhance uncontrolled proliferation and genomic instability which are the hallmarks of carcinogenic switch.

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Hormonal Carcinogenesis. Figure 2 The role of nuclear and cytoplasmic receptors in hormonal carcinogenesis. The endogenous or exogenous estrogen binds with either cytoplasmic estrogen receptor or nuclear receptor and subsequently activates estrogen-response genes associated with cellular proliferation and transformation events.

receptors are expressed, the estrogen responsiveness is determined by the ER-α: ER-β ratio. The studies shown that isoforms of ER-β heterodimerize and inhibit the activity of ER-α, which suggest a modulatory role of ER-β. In hormonal carcinogenesis, participations of these two receptors are considered to be crucial. However, some investigators believe that non-receptor pathways are also involved in hormonal carcinogenesis. Like estrogens, other hormones which are having carcinogenic potential exhibit identical effect on the cells for the induction of transformation. The formation of new blood vessels surrounding the tumors from preexisting blood vessel, which is also known as tumor angiogenesis, is the hallmark of cancers. It is necessary for increased nutrient and oxygen supply to the tumor cells. This process is controlled by several positive and negative angiogenic factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factoralpha (TGF-α), TGD-β, thrombospondin-1 (TSP-1) and statin (▶Antiangiogenesis). Some of these angiogenic factors such as PDGF and TGFs are proteins/peptide hormones. Normally, angiogenesis is a highly controlled

Hormonal Carcinogenesis. Figure 3 Angiogenic switch during hormonal carcinogenesis. Estrogen plays dual roles in tumor angiogenic switch. It activates positive regulator of angiogenesis and simultaneously silenced the expression of negative regulator.

process. Uncontrolled and persistent changes in angiogenic process are occurred at different stages of tumor progression. This phenomenon is occurred by imbalance expression of positive and negative angiogenic

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regulators. Multiple studies have shown that tumor angiogenic switch can be regulated by steroid hormones including estrogen and progesterone. These hormones modulate both positive and negative angiogenic regulators for angiogenic switch (Fig. 3).

Definition

References

HRPC; Synonym androgen-independent prostate cancer (AIPC); Prostate cancer that has become refractory, that is, it no longer responds to hormone therapy.

1. Banerjee SK, Islam A, Banerjee S (2005) The regulatory roles of estrogen in carcinogenesis. In: Bagchi D, Preuss HG (eds) Phytopharmaceuticals in cancer chemoprevention. CRC Press, Washington, DC, pp 105–121 2. Norman AW, Litwack G (1997) Hormones, 2nd edn. Academic Press, UK 3. Cavalieri EL, Stack DE, Devanesan PD et al. (1997) Molecular origin of cancer: Catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc Natl Acad Sci USA 94:10937–10942

Hormonal Therapy Definition

▶Endocrine Therapy

Hormonal Treatments Definition Treatment that adds, blocks, or removes hormones. To slow or stop the growth of certain cancers (such as prostate cancer and breast cancer), synthetic hormones or other drugs may be given to block the body’s natural hormones.

Hormone-Refractory Prostate Cancer

Hormone-related Cancers ▶Endocrine-Related Cancers

Hormone Replacement Therapy Definition HRT; Is a medical treatment for menopausal, perimenopausal, and postmenopausal men and women, based on the assumption that it may prevent discomfort and health problems caused by diminished circulating estrogen and progesterone hormones in the female. The treatment involves a series of drugs designed to artificially boost hormone levels. The main types of hormones involved are estrogens, progesterone or progestins, and sometimes testosterone in the case of men. ▶Estrogenic Hormones ▶Estradiol ▶Progestin

▶Temsirolimus ▶Hormones

Hormone-Responsive Element Hormone-induced Cancers ▶Endocrine-Related Cancers

Definition A specific cis-regulatory DNA sequence for a certain hormone that acts by binding to a receptor. This hormone–receptor complex can act as a transcription factor by binding to the hormone responsive element.

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Hormones

Hormones R EKHA M EHTA , J AYADEV R AJU Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HPFB, Health Canada, Ottawa, ONT, Canada

Definition Hormones are complex chemical messengers that bind with high affinity to specific cellular receptors to activate single or multiple ▶signal transduction pathway(s) leading to growth, ▶differentiation, embryogenesis or other biological processes. More than one hormone may work in concert to fulfill a physiological function. Hormones, through their capacity to influence cell growth and differentiation, may further modify the response of the body to carcinogens, the biological behavior of established tumors, and overall ▶cancer risk. Hormones, therefore, play an important role in both the genesis and treatment of cancer.

Characteristics Hormone-dependent malignancy has been most extensively studied in steroid hormone responsive organs. The steroid hormones, androgens, estrogens (▶Estradiol; estrogenic hormones and cancer) and progestins (▶Progestins and cancer), are implicated as key regulators in the development and the cancer process in the breast (▶Breast cancer), prostate (▶Prostate cancer, clinical oncology), ovary (▶Ovarian cancer) and endometrium (▶Endometrial cancer). More recently, however, hormonal influences in cancer are also becoming apparent in steroid hormone independent organs such as the colon. Likewise, non-steroid hormones participate, directly or indirectly, as modifiers of cancer, not only in endocrine system responsive organs, but also at other sites. Mechanisms The precise mechanisms of hormonal influences on cancer are being elucidated. In the context of the multistage theory of ▶carcinogenesis (▶Multistep development), it has been generally recognized that the principal effects of a given hormone are during the ▶cancer promotion stage, occurring predominantly in tissues where normal cellular growth and function are regulated by the specific hormone. Single or multiple hormones may interact to modify carcinogenesis. The tumor promoting effects of hormone(s) may be a result of indirect interaction of the hormone with the genome, culminating in epigenetic (▶Epigenetics) consequences. Recent evidence, however, implicates hormonal modulation during the ▶cancer initiation stage as well by direct

interaction of certain hormonal metabolites with the genetic material.

The Receptor Concept A receptor-mediated mechanism for hormone action during normal physiological functions is now widely accepted (Fig. 1). Hormones play a role as ligands that interact with specific receptors: the protein type binds to trans-membrane receptors, while the steroidal type binds to intracellular receptors. In the case of a protein hormone, a cascade of receptor-ligand mediated cytoplasmic responses is triggered leading to activation of post-receptor secondary messengers. On the other hand, a steroid hormone on entry into the cell forms cytoplasmic – receptor complexes that bind to DNA as a dimer. In both cases, recruitment of other co-factors ultimately regulate the activities of the cell’s general transcription apparatus assembled at the gene’s promoter. The mRNA produced is eventually translated into proteins which elicit and achieve a complex array of cellular responses such as alterations in growth and differentiation of the target tissue. An interplay of a variety of factors can interfere with the normal development and function of the ▶hormone receptors in target tissues, culminating in pathological conditions such as cancers. Thus, hormone receptors may be activated non-specifically by hormone-like endogenous metabolites or xenobiotic (▶Xenobiotics) ligands found as dietary constituents (e.g. ▶phytoestrogens), environmental pollutants, or food contaminating chemical or veterinary drug residues. Inherited genetic polymorphisms or mutations in hormone receptors and/ or hormone metabolizing enzymes may further affect normal hormonal function. In the case of steroid hormones, estrogens function as growth promoters through the nuclear receptors, estrogen receptor-α (ER-α) (▶Estrogen receptor) or ER-β by signal transduction-mediated increase in cellular proliferation, decrease in ▶apoptosis and regulation of growth factor production. ER-α and ER-β are products of two different genes, and ER-α may predominate in breast tumors. Likewise, androgen action on physiological processes such as cellular differentiation, secretory function, metabolism, morphology, proliferation and survival, is exerted in target tissues via binding to another nuclear receptor transcription factor, the ▶androgen receptor (AR). Testosterone, produced mainly in the testis, is able to directly bind to and activate the AR. However, the androgens produced in the adrenal glands, androstenedione and dehydroepiandrosterone, are converted to testosterone in peripheral tissues prior to their interaction with the AR. Similarly, in the prostate, testosterone is first metabolized to a more potent ligand dihydrotestosterone, before its interaction with the AR. The effects of progestins as

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Hormones. Figure 1 Schematic presentation of two types of receptor mediated hormonal action. Hormone binding to a trans-membrane or an intracellular receptor activates a cascade of events which eventually regulate the cell’s transcription and translation machinery to elicit a complex array of functional responses by the cell.

well are mediated through two isoforms of progesterone receptor in a tissue-specific manner. Prolactin exhibits dual characteristics of a circulating hormone and a ▶cytokine, both of which utilize receptor-mediated mechanisms. In its role as a cytokine, prolactin is secreted and regulated in extrapituitary sites where its binding to cytokine superfamily receptors not only can promote cell proliferation and survival, but also increase cell motility, suppress apoptosis, upregulate BRCA1 (▶Breast cancer genes BRCA1/ BRCA2), induce both ER-positive and ER-negative tumors, increase tumor growth rates (▶Prolactin and cancer), support tumor vascularization and enhance tumor metastases. Insulin and ▶insulin-like growth factors (IGF-I) likewise act through a receptor (▶Insulin receptor) -mediated mechanism to regulate energy metabolism. The bioactivity of IGF-I is increased through its enhanced synthesis and by a decrease in several of its binding proteins (IGFBP; IGFBP-1 and -2). Insulin and IGF-I both stimulate ▶anabolism based on the amount of available energy and the necessary substrates such as amino acids. The anabolic signals by insulin or IGF-I can promote tumor development by inhibiting

apoptosis, and by stimulating cell proliferation. Furthermore, both insulin and IGF-I stimulate the synthesis of sex steroids, and inhibit the synthesis of sex hormone-binding globulin, a binding protein that regulates the bioavailability of circulating sex steroids to tissues. Receptor-mediated mechanisms also intercede the action of thyroid hormone, and the gastrointestinal hormones gastrin, cholecystokinin, neurotensin and ▶gastrin-releasing peptide. Hence, the receptor concept provides one common mechanism whereby a steroid or a non-steroid hormone may potentially mediate or modulate cancer under the conditions that an aberration in its receptor-mediated signal transduction system favors an enhancement or prevention of neoplasia. Endogenous Metabolic Activation of Steroids A second mechanism of action implicated in hormonal modification of carcinogenesis involves endogenous metabolism of the steroid hormones, estrogen and androgens by tissue-specific enzymes (Fig. 2). A similar mechanism for non-steroid hormones has not been described so far. Estrogen is biotransformed by several ▶cytochrome P450 isoforms and various peroxidases

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Hormones

Hormones. Figure 2 Metabolic activation of steroid hormones. Aromatase-mediated metabolism of androgens to estrogens, and further oxidative metabolism of estrogens lead to genotoxic effects that may contribute to cancer induction.

to produce estrogenic metabolites that may be protective through their antioxidant properties or growth and ▶angiogenesis inhibitory activities. On the contrary, the more reactive quinone metabolites of estrogen may directly form ▶adducts with DNA and/or cause oxidative damage to lipids and DNA (▶Oxidative DNA damage) through redox cycling processes that produce ▶reactive oxygen species (ROS). Increased production of ROS may further alter regulation of gene expression through effects on transcription factor function. The androgens testosterone, androstenedione and dehydroepiandrosterone may act as additional cancer modifiers in situ in their respective target organs when they are metabolized first to estrogens by aromatase, another cytochrome P-450 enzyme, followed by their metabolic activation to quinones. Epidemiology and Animal Studies Steroid Hormones Breast cancer risk is associated with prolonged exposure to predominantly estrogens. Thus, increased in utero exposure to estrogens, early onset of ▶menarche, late ▶menopause, hormone replacement therapy and postmenopausal obesity enhance breast cancer incidence. Androgens, through their metabolism to estradiol by the aromatase enzyme expressed in epithelial cells of both normal and neoplastic breast, act synergistically with estrogens to directly stimulate tumor growth and increase breast cancer risk in postmenopausal women.

Androgens are further implicated as a causative factor in prostate cancer induction, though with limited epidemiological evidence (▶Epidemiology of cancer). Human plasma levels of androgens do not always correlate with prostate cancer susceptibility. Animal studies, however, support a strong tumor promoting role for androgens acting via androgen receptor-mediated mechanisms, and following a tumor-initiating stimulus. Exposure of the fetus to higher androgen concentrations is a potential ▶risk factor for prostate cancer in later life. However, as in the breast, malignant changes to the prostate gland depend upon both androgenic and estrogenic responses that are accomplished through aromatase mediated conversion of testosterone to estradiol and via estrogen receptors found in both human and rat prostate. Similar synergistic links between androgens and estrogens have been reported in uterine cancer in experimental animals, and clinically, in ovarian and endometrial cancers. Estrogen receptors are likewise expressed in several ▶gastrointestinal cancers such as those of the esophagus, gallbladder, stomach and colon (▶Colon cancer). Estrogens may further interact with additional growth factors and polyamines to modulate growth and progression (▶Cancer progression) of colorectal tumors. However, the epidemiological evidence for a role for estrogens in colorectal cancers is currently inconsistent. Loss or down-regulation of progesterone receptor observed in ovarian carcinoma, and antiproliferative

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and apoptotic effects of progesterone in the ovary, suggest that progestins protect against ovarian cancer. On the contrary, synthetic progestins, globally used as contraceptive agents, are implicated as tumor promoters or ▶cocarcinogens in rodent liver, pituitary and mammary gland.

specific G-protein-coupled receptors through which the gastrointestinal hormones stimulate cancer cell growth. Gastrointestinal hormone receptors have also been noted in breast, lung and prostate cancers where they may modify neoplastic growth and aggressive behavior of the lesions.

Prolactin The neuroendocrine hormone prolactin may play a role in several types of cancers in reproductive and nonreproductive tissues. Its possible role in breast cancer has been widely considered because of its physiological action in the stimulation of mammary gland development and differentiation during puberty, pregnancy, and lactation. While experimental data support a relationship between increased prolactin levels and breast cancer, epidemiological data is discrepant.

Thyroid Hormones An association between thyroid hormones and cancer has been considered extensively with conflicting results. In experimental models, perturbations in thyroid hormones affect estrogen and prolactin secretion, and cause pathological alterations in the ovary, endometrium and breast that are consistent with precancer. However, clinical evidence for the role of thyroid hormones in cancers in these organs is not definitive due to difficulties in differentiating thyroid hormoneinduced cellular alterations from neoplasia-induced cellular atypia.

Insulin Physiologically, the growth hormone insulin-like growth factor-I (IGF-I)-axis is an important modulator of growth and development, as well as a regulator of a wide range of biological functions. Their potent mitogenic and anti-apoptotic effects, for example, play a critical role in the development of the breast, and in regulation of rapidly renewing epithelial cell populations such as those in the colon. In cancer patients, increased insulin production is commonly observed during cancer development. Some types of tumors produce constituents that stimulate insulin secretion by the pancreas, while other more aggressive tumor types produce insulin ectopically. Global variations in cancer incidence rates provide evidence for diet and associated factors such as nutritional energy balance, macronutrient composition of the diet, physical activity and body size (▶Obesity and cancer risk) as among the most important lifestyle factors that influence cancer risk (▶Nutritional status and cancer). Recent studies further suggest that these dietary and related factors may influence the risk of cancers of the colon, pancreas, endometrium, breast, ovary and prostate by affecting the levels of circulating serum insulin and increasing the bioavailability of IGF-I. IGF-I has been positively associated with the risk of colorectal cancer in human studies. In vitro, IGF-I elicits mitogenic and anti-apoptotic actions on colorectal cancer cells. Gastrointestinal Hormones The gastrointestinal hormones (▶GI hormones), gastrin, cholecystokinin, neurotensin and gastrin-releasing peptide (▶Gut peptides), are localized mainly in the pancreas and in the mucosa of the gastrointestinal tract from the stomach to the rectum. Certain gastric (▶Gastric cancer), pancreatic (▶Pancreas cancer, basic and clinical parameters) and colorectal cancers possess

Hormonal Management of Cancer Recognition of the role of estrogens in the stimulation of breast tumor growth has led to the development of several therapeutic endocrine agents. In premenopausal women ▶gonadotrophin-releasing hormone (GnRH) agonists suppress ovarian oestrogen synthesis and reduce estradiol close to postmenopausal levels. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides another way of treating hormone-dependent breast cancer in older patients. Currently available ▶aromatase inhibitors are effective in the management of hormone-dependent breast cancer in post-menopausal women failing antiestrogen or tamoxifen therapy (▶Tamoxifen) in locally advanced or metastatic disease. GnRH agonists used in combination with an aromatase inhibitor suppress estrogen levels even further. Similarly, hormone therapy for prostate cancer has evolved from the use of estrogens to GnRH agonists. Recently, investigational GnRH antagonists have been discovered that cause the inhibition of luteinizing hormone production, eventually leading to a suppression of testosterone and dihydrotestosterone on which continued growth of prostate cancer cells is dependant. Steroid hormone receptors are another well established therapeutic target in cancer, and drug development has continued to focus on agents that either block steroid hormone production or bind to and modulate their receptors. The implication of insulin and IGF-I as additional factors in cancer modulation may further advance development of newer cancer prevention drugs that restore sensitivity to insulin and reduce hyperinsulinemia. ▶Celecoxib ▶Estrogenic Hormones ▶Hormonal Carcinogenesis

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Hornstein–Knickenberg syndrome

References 1. Argiles JM, Lopez Soriano FJ (2001) Insulin and cancer. Int J Oncol 18:683–687 2. Ben Jonathan N, Liby K, McFarland M et al. (2002) Prolactin as an autocrine/paracrine growth factor in human cancer. Trends Endocrinol Metab 3:245–250 3. Bernstein L (2002) Epidemiology of endocrine-related risk factors for breast cancer. J Mammary Gland Biol Neoplasia 7:3–15 4. Bosland MC (2000) The role of steroid hormones in prostate carcinogenesis. J Natl Cancer Inst Monogr 27:39–66 5. Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast cancer. N Engl J Med 354:270–282

may regulate gene expression, morphogenesis, and differentiation. ▶Methylation of this gene may result in the loss of its expression and, since the encoded protein up-regulates the tumor suppressor p53, this protein may play an important role in tumorigenesis. ▶Pleiotrophin ▶P53 Family

HOXA9 Hornstein–Knickenberg syndrome ▶Birt–Hogg–Dubé Syndrome

Definition Is a member of the abdominal-B subclass of HOX genes and plays important roles in normal hematopoiesis as well as in leukemia development. ▶NUP98-HOXA9 Fusion

HOX Definition The HOX homeodomain (HD) proteins are DNAbinding transcription factors that are key regulators of development and hematopoiesis. In vertebrates, there are 39 HOX genes that are organized into four chromosomal clusters (HOXA, B, C and D), which can be classified into 13 paralogous groups based on their extensive sequence homology within the HD and their relative chromosomal locations within a cluster. HOX genes are expressed at various stages during hematopoietic development. Perturbation of orderly HOX gene activation and inactivation results in hematological abnormalities. Increased expression of HOXA and B genes has been documented in human acute myeloid malignancies. ▶NUP98-HOXA9 Fusion

HOXA5 gene

HpaII Tiny Fragments (HTF) Islands ▶CpG Islands

HPAEC Definition

Human pulmonary artery ▶endothelial cells.

HPD

Definition Is part of the A cluster of HOX genes on chromosome 7 and encodes a DNA-binding transcription factor that

Definition

▶Hematoporphyrin derivative

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HPRT

HSC

Definition

Definition

Hypoxanthine phosphoribosyl transferase.

Hematopoietic stem cell. HSCs are multipotent stem cells found in the bone marrow and give rise to all the cell types of both the myeloid and lymphoid lineages.

▶Microcell-Mediated Chromosome Transfer

▶Adult Stem Cells

hPTTG1 HSD18 ▶Securin ▶Methylation-Controlled J Protein

HPV ▶Human Papillomaviruses

HRPC Definition

HSF ▶Interleukin-6

HSF-III ▶Leukemia Inhibitory Factor

▶Hormone refractory prostate cancer

hSNF5 HS1

▶hSNF5/INI1/SMARCB1 Tumor Suppressor Gene

Definition Hematopoietic Specific protein 1 is a protein found exclusively in leukocytes that is functionally similar to cortactin. It is a component of the ZAP 70 and Syk tyrosine kinase signaling pathways activated during the immune response. ▶Cortactin

HSNF5/INI1 Definition

▶Tumor Suppressor hSNF5/INI1.

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hSNF5/INI1/SMARCB1 Tumor Suppressor Gene

hSNF5/INI1/SMARCB1 Tumor Suppressor Gene F RANCK B OURDEAUT 1 , PAUL F RE´ NEAUX 2 1

Département de pédiatrie, INSERM 830, Biologie et génétique des tumeurs, Institut Curie, Paris, France 2 Département de Pathologie, Institut Curie, Paris, France

Synonyms hSNF5; Human non sucrose fermenting 5; SMARCB1; SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily b, member1; BAF47; BRG- and BRM-associated factor, 47 kDa

Definition hSNF5/INI1 is a member of the highly conserved ▶SWI/SNF complex, involved in the ATP-dependent ▶chromatin remodeling. hSNF5/INI1 acts as a ▶tumor suppressor gene.

Characteristics The Chromatin Remodeling and the SWI/SNF Complex In the nucleus of eukaryotic cells, DNA is wrapped around histones, constituting a dense histone–DNA complex referred to as nucleosomes. The closed conformation of DNA in nucleosomes precludes the binding of transcription factors to their target promoters. By regulating the compaction degree of nucleosomes, cells can allow interactions between promoters and transcription factors. This regulation relies on two main mechanisms: (i) stable covalent modifications of histones, including actelyation, methylation, ubiquitination, etc. (ii) ATP-hydrolysis-dependent chromatin remodeling, capable to affect the spatial organization of the

chromatin and the stability of nucleosomes in a dynamic way. The SWI/SNF complex, first described in Saccharomyces cerevisia, is the prototype of ATP-dependent chromatin remodeling complex. Approximately 5% of the genome is affected by the SWI/SNF-dependent regulation of the transcription, either by induction or by repression (Fig. 1). The SWI/SNF complexes are conserved throughout the evolution. In humans, chromatography has lead to identify two fractions of SWI/SNF complexes, PBAF or SWI/SNF-B, characterized by the presence of polybromo protein, and BAF or SWI/SNF-A, which may be the actual homolog of yeast SWI/SNF. The BAF complexes are variably composed of approximately ten subunits, in a cell-type-specific manner. However, all BAF complexes contain one of the two ySnf2 human homologs, hBRM or hBRG1 (Fig. 2). Either of them brings the ATPase activity of the complex. In vitro, hBRG1 and hBRM are sufficient to induce a chromatin remodeling. Nevertheless, the addition of other members of the core complex increases the efficiency of the remodeling. Furthermore, the integrity of the entire complex seems to be indispensable in vivo. Among the BAF proteins, at least three are ubiquitously expressed, demonstrating a critical role in the function of the complex: BAF155, BAF 170 and BAF47. Altogether, these indispensable subunits constitute the core complex of SWI/SNF. The latter, BAF47, maintains the stability of the whole complex in yeasts but might not play such a role in mammals. Since its interaction with the HIV1 integrase IN was its first role identified, it has been called “INI1.” hSNF5/INI1 Gene and Protein Structures In human, hSNF5/INI1 gene is located in 22q11.2. It is composed of nine exons and spreads over 50 kb (Fig. 3a). An alternative splicing in exon 2 produces two

hSNF5/INI1/SMARCB1 Tumor Suppressor Gene. Figure 1 Chromatin remodeling and regulation of transcription. The chromatin is wrapped around histones. Acetyl or methyl radicals are added or removed by histone acetyltransferases (HAT) or histone deacetylases (HDAC) and histone methyl transferases (HMT) and histone demethylases (HDMT) respectively. ATP hydrolysis (ATP = ADP + Pi) also participates to a direct chromatin remodeling through the ATPase activity of remodeling complexes such as SWI/SNF, regulating the accessibility of the chromatin to transcriptions factors (TF).


different transcripts of 1155 and 1128 nucleotides. hSNF5 protein has strong homologies with other SWI/ SNF proteins in various species (yeast Snf5, Drosophila dSNR1, Caenorhabditis elegans CeSnf5). The homology domain encompasses 193 amino acids and two highly conserved repeated sequences, Rpt1 and Rpt2 (Fig. 3b). This repeated domain brings a nuclear export signal, consistent with the subcellular localization of the protein. Rpt1 directly interacts with several partners, such as HIV IN, c-MYC, hBRM or ALL1. A coiled-coil domain at the C-terminal extremity plays a role in the binding to interaction partners. Biological Roles of hSNF5/INI1 Studies of Snf5 inactivation in cell and mouse models have brought numerous insights on Snf5 biological

hSNF5/INI1/SMARCB1 Tumor Suppressor Gene. Figure 2 The SWI/SNF complex. SWI/SNF complexes are composed of up to 12 proteins. Alternatively hBRM or hBRG1 carries the ATPase activity. The other proteins of the core complex are BAF155, BAF170, and BAF47.

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functions. First, there are some strong evidences of Snf5 being involved in cell survival. Indeed, complete inactivation of Snf5 leads to early embryonic lethality and decreases cell survival in vitro. In MEFs, loss of Snf5 also impairs the cell cycle progression and, consistently, alters the expression of various Rb-E2fresponsive genes. Furthermore, the defect of Snf5 enhances sensitivity to DNA damage and subsequent apoptosis through a p53-dependent manner. These paradoxical roles in survival and apoptosis might be time-, cell-type- and stress-dependent. However, the most relevant observation is that conditional inactivation of Snf5 leads to the development of a tumor in 100% of the mice. Moreover, the short median delay of 11 weeks before tumor development is rarely observed with the inactivation of a single gene, and indicates a tremendously potent tumor suppressor role for SNF5. To which extent the tumor suppressor function of SNF5 depends on SWI/SNF remains unclear. However, reexpression of hSNF5/INI1 in rhabdoid cells has demonstrated its critical role in the control of the ▶G1– S transition. This effect could rely either on a repressive effect upon ▶Cyclin D1 expression, or on a strong induction of ▶p16/INK4a. Interestingly, a functional RB protein is required for INI1-dependent cycle control, whereas INI1 is dispensable for normal RBmediated growth arrest (Fig. 4). Hence, INI1 definitely acts upstream RB. However, the control of the G1–S transition may not fully explain the antioncogenic role of hSNF5/INI. Indeed, missense mutations do not affect the replication checkpoint, but might rather lead to polyploidy by promoting chromosomal instability and compromising the mitotic checkpoint. Furthermore, there has been evidence for the involvement of hSNF5/INI1 in

hSNF5/INI1/SMARCB1 Tumor Suppressor Gene. Figure 3 hSNF5/INI1 gene and protein structure. Interacting proteins are indicated in front of the Rpt2 domain. a. Structure of the gene: 9 exons encompassing ~50 kb. b. Structure of the protein with the highly conserved hemology domain.

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hSNF5/INI1/SMARCB1 Tumor Suppressor Gene. Figure 4 hSNF5/INI1 and the G1–S transition. Arrows in green and red indicate respectively a positive and negative regulation. Dot lines indicate not fully consensual links. SWI/SNF has a physical interaction with RB and directly coregulates E2F1 transcription. hSNF5/INI1 on its own represses the G1 to S transition. This effect can be due either to CCND1 downregulation or to p16/INK4a over expression. hSNF5/INI1 acts upstream RB.

dynamic regulation of the cytoskeleton. The restoration of the hSNF5/INI1 gene in rhabdoid cells alters the expression of Rho-family genes, likely to modulate the cytoskeleton, and obviously modifies the organization of actin stress fibers. hSNF5/INI1 might therefore play a role in cellular adhesion and migration properties. Finally, taking into consideration the critical role of chromatin remodeling in the activation of celltype-specific genetic programs, the role of hSNF5/INI1 in driving cell differentiation has also been investigated. INI1-dependent differentiating effect has been observed in hepatocytes. Conversely, reexpression of hSNF5/INI1 in rhabdoid cell lines drives distinct differentiation phenotypes according to the cell anatomic origin. A critical impairment of INI1-dependent differentiation programs may account for the highly undifferentiated phenotype of hSNF5/INI1-deficient tumors. Genetic Alterations of hSNF5/INI1 in Human Malignancies Deletions encompassing the hSNF5/INI1 locus are encountered in many tumor types, including ▶rhabdoid tumors (RTs), ▶proximal epitheliod sarcomas (PES), meningiomas, schwannomas. Biological effects of hemizygous INI1 deletions are questionable, since loss of one allele results only in a 15–20% reduction in total INI1 mRNA levels due to transcriptional compensation by the remaining allele. Hence, “two-hit” events only are likely to drive transformation. Strikingly, biallelic inactivation is encountered in about 80% of rhabdoid

tumors. Karyotypic analyses in RTs usually show no or few other alterations than deletions or translocations of 22q11. Altogether, this indicates a very strong association between hSNF5/INI1 extinction and RTs. Both homozygous deletions and hemizygous deletions with point mutations are encountered. In RTs cell lines, mitotic recombinations of chromosome 22q and nondisjunction/duplication, lead to partial or complete uniparental disomy. This mechanism may account for most homozygous changes and result in the total or partial loss of one chromosome associated with the duplication of the remaining chromosome carrying the deleted or mutated allele. The association of a point mutation with a whole gene deletion is less frequently encountered, but seems to be preponderant in ▶brain tumors. Point mutations, consisting in nucleotides replacement, deletion or insertion and leading to truncating nonsense codons, are spread all over the nine exons with no obvious hotspot. Missense mutations are much rarely reported. Studies using immunohistochemistry with antiSMARCB1 antibody have confirmed the total loss of protein expression in a high proportion of RTs as a direct consequence of the genetic alterations. Whether hSNF5/INI1 inactivation is specific for RT or could be involved in miscellaneous other tumors remains not fully consensual (see next section). Congenital multifocal rhabdoid tumors and familial cases strongly supported the existence of constitutional mutations in a predisposing syndrome. Indeed, about 40


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hSNF5/INI1/SMARCB1 Tumor Suppressor Gene. Figure 5 pathological aspects of hSNF5/INI-deficient ▶rhabdoid tumor. (a) “Rhabdoid” cells: discohesive polygonal or round cells with abundant cytoplasm, juxtanuclear globular eosinophilic cytoplasmic inclusion, large nucleus with one prominent nucleolus. (b) negative staining of rhabdoid cells with anti-hSNF5/INI1 antibody, resulting from a complete loss of the protein and the biallelic inactivation of hSNF5/INI1 gene. Positive staining is retained in normal stromal cells.

germline mutations have been reported so far, associated to the development of a tumor during early childhood in almost all patients. Hence, the penetrance is very high, though not full. Nucleotides or whole gene deletions, point mutations and splice site changes have all been observed at the constitutional level. Pathological and Clinical Aspects of hSNF5/ INI-Deficient Tumors RTs were initially described as highly aggressive variants of Wilm tumors occurring in infants (▶Nephroblastoma). “Rhabdoid” cells were defined by key morphologic histological and immunophenotypical features: polygonal or round shape with abundant cytoplasm, juxtanuclear globular eosinophilic cytoplasmic inclusion, large nucleus with one prominent nucleolus (Fig. 5a), coexpression of vimentin and epithelial markers such as epithelial membrane antigen and/or cytokeratins. Such features were then observed in other pediatric malignancies, in brain (Atypical Teratoid/Rhabdoid Tumors) (Brain tumors) and miscellaneous soft-tissues. Presently, the cell origin of RTs remains unknown. The actual incidence of RTs may still be underestimated since those rare tumors are frequently misdiagnosed. However, immunohistochemistry with a monoclonal anti-hSNF5/INI1 antibody is very sensitive and highly specific for the detection of hSNF5/INI1 loss-of-function (Fig. 5b), and should now facilitate the diagnosis of most RTs. hSNF5/INI1 biallelic genetic inactivation has also been reported in other types of ▶childhood cancers such as central PNET (▶Medulloblastoma, deleted in malignant brain tumors), choroid plexus carcinomas, and in a small subset of undifferentiated malignant tumors without evidence of “rhabdoid” morphological features. In adults

interestingly, in which RTs are thought to be much rarer, hSNF5/INI1 complete defect has also been observed in some “proximal-type” of epithelioid sarcomas (▶Proximal-type epithelioid sarcomas), and, marginally, in composite tumors with “rhabdoid” component. hSNF5/ INI1 deficiency might therefore account for a wider spectrum of tumors. Nevertheless, it still remains unclear whether these different malignancies should be considered as phenotypical variants of a same biological entity. In accordance with this hypothesis, they usually share with typical RTs a highly aggressive clinical behavior. Conclusion There are increasing evidences of chromatin remodeling being a critical process in oncogenesis. hSNF5/INI1 offers a convincing example as a tremendously potent tumor suppressor gene. In humans, hSNF5/INI1 inactivation is strongly associated with rhabdoid tumors, but may not be as restricted to these children malignancies as previously thought. Identification of hSNF5/INI1 involvement in human cancers has given helpful diagnostic tools, based on molecular screening and specific anti-hSNF5/INI1 immunohistochemistry. Better knowledge on hSNF5/INI1 roles in oncogenesis should lead to more efficient targeted therapies, and improve the dramatically poor prognosis of hSNF5/ INI1-deficient tumors.

References 1. Roberts CW, Orskin SH (2004) The SWI/SNF complexchromatin and cancer. Nat Rev Cancer 4:133–142 2. Robert CW, Leroux MM, Fleming MD et al. (2002) Highly penetrant, rapid tumorigenesis through conditional inversion of the tumor suppressor gene Snf5. Cancer Cell 2:415–425

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3. Imbalanzo AN, Jones SN (2005) Snf5 tumor suppressor couples chromatin remodelling, checkpoint control, and chromosomal stability. Cancer Cell 7:294–295 4. Versteeg I, Sevenet N, Lange J et al. (1998) Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer. Nature 394:203–206 5. Sevenet N, Lellouch-Tubiana A, Schofield D et al. (1999) Spectrum of hSNF5/INI1 somatic mutations in human cancer and genotype-phenotype correlations. Hum Mol Genet 8:2359–2358

HSP Definition Heat shock protein. HSP89α, HSP90β; the HSP89α and HSP90β genes are composed of 11 and 12 exons, respectively. The regulation of HSP gene expression in eukaryotes is mediated by the conserved heat shock transcription factor (HSF). HSF acts through heat shock elements (HSEs) composed of three contiguous inverted repeats of a 5 bp sequence, nGAAnnTTCn; upon heat stress, HSF binds to HSEs as a trimer. ▶Hsp 90

Hsp60 Definition Heat shock proteins that acts as molecular chaperones in complex in bacteria and eukaryotes; the Hsp60/ Hsp10 complex is important for import and folding of key proteins into mitochondria. ▶Molecular Chaperones

Hsp90 P HILIP J. T OFILON Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

Synonyms Heat shock protein 90

Definition

Hsp90, the 90-kDa heat shock protein, is a ▶molecular chaperone that regulates the degradation, folding and/or transport of a diverse set of critical cellular regulatory proteins. Most Hsp90 clients, i.e. those proteins that require its “chaperoning” activity for appropriate function, participate in some aspect of signal transduction including a wide variety of protein kinases, hormone receptors, and transcription factors. Moreover, Hsp90 can stabilize mutated proteins allowing them to maintain normal function despite genetic abnormalities. This ability to buffer genetic changes and serve as a capacitor of phenotypic variation has implicated Hsp90 in evolutionary and oncogenic processes.

Characteristics Hsp90 is an ATP-dependent molecular chaperone and is one of the most highly expressed proteins in eukaryotic cells. There are two major isoforms of Hsp90, the major form Hsp90α and the minor form Hsp90β. The two hsp90 genes differ primarily in their non-coding and regulatory regions. Both the α and β proteins consist of four basic structural domains. The N-terminal domain is the site of ATP binding and is essential for its chaperone function. Inhibitory agents, at least those developed to date, specifically target this site preventing ATP binding and consequently Hsp90 function. The N-terminal domain is connected to the middle domain by a small highly charged linker region that is thought to be the site for co-chaperone binding. The larger middle domain contains the site for client protein binding and the C-terminal domain provides the dimerization site, which is essential for Hsp90 activity. The Hsp90 proteins function as homodimers of α/α and β/β. Whereas there does appear to be functional differences between the isoforms with β being associated with development, there is also considerable overlap. Although the majority of Hsp90 is located in the cytoplasm, this molecular chaperone can also be found in the nucleus, albeit at considerably lower levels, where it has been associated with the regulation of gene expression and the ▶DNA damage response to radiation. In addition, Hsp90 is located on the cell surface where it plays a role in antigen processing and the immune response. Finally, recent studies have shown that Hsp90 can be secreted into the extracellular space. Although the specific function has not been clearly defined, in this location Hsp90 has been suggested to play a role in blood clotting, cell migration and tumor metastatic processes. Mechanism Hsp90 chaperone function is mediated by an ATPdependent cycling between two conformations, which regulates its interactions with specific co-chaperones

Hsp90

and co-factors and drives the loading and off loading of client proteins. The Hsp90 dimer typically exists in a multi-protein chaperone complex that includes the co-chaperones Hsp70 (70-kDa heat-shock protein) and Hsp40 (40-kDa heat-shock protein), the adaptor protein HOP (hsp co-chaperone organizing protein) and the cofactor p23. In addition, there are specific immunophilins and other co-chaperones that regulate substrate binding. For example, the co-chaperone ▶Cdc37 is involved in Hsp90-mediated stabilization of protein kinases. A client protein initially binds to the Hsp70/Hsp40 complex, which links via HOP to an ADP-bound Hsp90. Exchanging ADP with ATP alters Hsp90 confirmation such that HOP and Hsp70/Hsp40 are released and p23 and other co-chaperones such as Cdc37 are recruited to the complex. In the ATP-bound confirmation and associated with these co-chaperones Hsp90 folds and stabilizes a client protein maintaining it in a responsive conformation. Although the specific processes have not been completely defined, in the absence of the appropriate stimulus or ligand binding to the client protein, Hsp90 through its ATPase activity cycles back to its ADP-bound conformation recruiting the initial set of co-chaperones, which ultimately leads to client protein degradation. Considerable insight into the mechanism of Hsp90 chaperone activity has been generated through the use of the inhibitor ▶geldanamycin. This benzoquinone ansamycin binds to the nucleotide binding site of Hsp90 resulting in a conformation that resembles the ADP-bound conformation. The inability of Hsp90 to cycle to its ATP-bound conformation then maintains the chaperone complex in a state that favors client protein degradation. Studies to date indicate that in cells treated with geldanamycin or one of its analogs the half lives of the Hsp90 client proteins are uniformly and significantly reduced. Clinical Aspects As a Single Modality Hsp90 has received considerable attention as a potential target for cancer therapy. Because of an increased understanding of the mechanisms and molecules that mediate malignant transformation, recent strategies in cancer therapy have begun to focus on a target-based approach. The putative advantages of this strategy include tumor selectivity sparing normal cells and the availability of markers indicative of tumor susceptibility. Most attempts to apply target based therapy have entailed the use of agents that target a single molecule. For tumors in which their phenotype is driven by a single oncogenic molecule, such as the targeting of Bcr-Abl by ▶STI571 in chronic myelogenous leukemia and ▶Her2/neu by ▶Herceptin is certain breast tumors, this approach has demonstrated clinical activity. However, most tumors, especially solid neoplasms, contain multiple genetic abnormalities and are subject to a high degree of genomic

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instability. The result is that their malignancy/survival is driven by variety of molecules existing in a number of different survival pathways. Under these circumstances, targeting a single molecule is likely to be of limited consequence. As an alternative to targeting a single molecule, inhibiting Hsp90 provides a multi-target approach to cancer treatment. Hsp90 client proteins include a large number kinases, receptors and transcription factors that have been implicated in transformation and maintenance of the malignant phenotype. Examples of such proteins dependent on Hsp90 include ErbB2 (▶Her2/Neu), Src, ▶Akt, ▶c-Raf-1, cyclin dependent kinases Cdk4 and Cdk6, HIF1-α, estrogen and androgen receptors and hTERT. Thus, Hsp90 inhibition provides a means of simultaneously targeting multiple proteins critical to a malignant cell. In addition, Hsp90 can stabilize mutant proteins (e.g. ▶p53) allowing for at least some functional activity. Given the level of genomic abnormalities and instability of tumor cells, the ability to buffer against such genetic variation suggests another avenue through which Hsp90 contributes to tumor cell survival. In laboratory studies exposure of tumor cells to Hsp90 inhibitors such as geldanamycin and its analogs 17-allylaminogeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) results in the combinatorial decrease in its client proteins mentioned above as well as additional client proteins, although there is some cell type specificity. With respect to a potential cancer therapy, inhibition of Hsp90 induces tumor cell death or significantly slows their proliferation in a number of in vitro and in vivo experimental systems, although the critical target or targets (i.e., client proteins) for the most part have not been defined. An additional characteristic that supports the application of Hsp90 inhibitors in cancer treatment is their relative selectivity for tumor cells over normal cells. The molecular basis for this selectivity has not been clearly defined. However, Hsp90 is typically expressed at higher levels in tumor cells than normal cells suggesting that tumors may be more dependent on its chaperoning activity. Regardless of the specific mechanisms involved, the ability of Hsp90 inhibitors to preferentially kill tumor cells over normal has led to the ongoing evaluation of 17AAG and 17DMAG in clinical trials as single modality agents. In Combination with Radiotherapy Hsp90 has also been identified as a determinant of tumor cell ▶radiosensitivity. Among Hsp90 clients are included a number of proteins (e.g. Raf-1, Akt and ErbB2/Her2/ Neu) that have been associated with protection against radiation-induced cell death; a reduction in their individual activities has been shown to result in radiosensitization in some, but not all tumor cell lines. Tumor cell radiosensitivity is regulated by a wide variety of signaling

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molecules with their specific contributions often determined by cell type. Because Hsp90 inhibitors induce the simultaneous loss of a number of these molecules that can potentially affect radiosensitivity, the use of these agents allows for implementing a multi-target approach to ▶radiosensitization. The putative advantages of such a multi-target strategy are increases in the degree and probability of radiosensitization. Indeed, inhibitors of Hsp90 such as geldanamycin, 17AAG, 17DMAG and radicicol have been shown to significantly enhance the radiosensitivity of a number of tumor cell lines derived from a variety of histologies. As for Hsp90 inhibitor treatment alone, the inhibitors have little to no effect on the radiosensitivity of normal cells evaluated in vitro. This lack of normal cell radiosensitization occurs despite a similar reduction in client proteins suggesting that it is not the difference between Hsp90 per se in tumor and normal cells, but the actions of its client proteins. Mechanistic studies of the tumor cell radiosensitization induced by 17DMAG have implicated Hsp90 in two components of the DNA damage response – DNA double strand break repair and ▶cell cycle checkpoint activation. The inhibition of double strand break repair could be traced to the loss of ErbB2 (Her2/neu) in 17DMAG treated cells and the consequent reduction in ErbB1 (EGFR) activity, which leads to a reduction in the ErbB1 interaction with DNA-PKcs and the subsequent attenuation of ▶DNA-PK activation after irradiation. The abrogation of cell cycle checkpoint activation by 17DMAG was associated with a reduction in radiation-induced activation of ▶ATM, which was the result of a reduced interaction between ▶NBS1 and ATM. Whereas most studies regarding Hsp90 as a target for cancer treatment have focused on its cytoplasmic activities, these radiation studies indicate that this chaperone has a critical role within the nucleus. The contribution of Hsp90 to double strand break repair and cell cycle checkpoint activation in tumor cells suggests that its inhibition may also be of benefit in combination with chemotherapeutic agents that kill tumor cells through DNA damage.

References 1. Dote H, Burgan WJ, Camphausen K et al. (2006) Inhibition of Hsp90 compromises the DNA damage response to radiation. Cancer 66:9211–9220 2. Eustace BK, Jay DG (2004) Extracellular roles for the molecular chaperone, hsp90. Cell Cycle 3:1098–1100 3. Sangster TA, Lindquist S, Queitsch C (2004) Undercover: causes, effects and implications of Hsp90-mediated genetic capacitance. BioEssays 26:348–362 4. Sharp S, Workman P (2006) Inhibitors of the Hsp90 molecular chaperone: current status. Adv Cancer Res 95:323–348 5. Sreedhar AS, Kalmar E, Csermely P et al. (2004) Hsp90 isoforms: functions, expression and clinical importance. FEBS 562:11–15

HSPG Definition Heparan sulfate proteoglycan; extracellular matrix protein containing heparan-sulfate polysaccharide chains. ▶Wnt Signaling ▶Proteoglycans

HSR Definition Homogeneously staining region. ▶Amplification

HSV-TK/Ganciclovir Mediated Toxicity D ONNA S HEWACH Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

Synonyms Suicide gene therapy; Gene directed enzyme-prodrug therapy; GDEPT

Definition

A form of ▶suicide gene therapy in which the cDNA for a viral enzyme, the herpes simplex virus thymidine kinase (HSV-TK), is transferred to tumor cells followed by treatment with the antiviral drug ganciclovir (GCV). Expression of HSV-TK enables cells to phosphorylate GCV to a monophosphate derivative. Cellular enzymes convert the monophosphate to GCV triphosphate, which elicits toxicity through incorporation into DNA.

Characteristics HSV-TK/GCV mediated killing of tumor cells, and indeed suicide gene therapy in general, has been developed as a mechanism to improve the selectivity of cancer chemotherapy. Since traditional antitumor drugs target rapidly dividing tissues, such as tumor cells, they also can destroy normal dividing host tissue

HSV-TK/Ganciclovir Mediated Toxicity

such as cells in the bone marrow, gastrointestinal tract, or hair follicles. Normal tissue toxicity is the major impediment to traditional chemotherapy. With HSVTK/GCV therapy, a foreign gene (encoding HSV-TK) which can activate a normally innocuous ▶prodrug (GCV) to a toxic product is transferred selectively to the tumor. When the patient is treated with the prodrug, only the tumor cells expressing the foreign gene will be affected (thus the designation of “suicide” gene therapy); since normal host tissues cannot activate the prodrug, they are spared from toxicity. Toxicity to HSV-TK-Expressing Cells GCV is an acyclic analog of 2′-deoxyguanosine that requires phosphorylation for biologic activity (Fig. 1). It was originally discovered as an antiherpesvirus agent, and it is used clinically in the treatment of cytomegalovirus infection. When herpes simplex virus infects human cells, a number of proteins are expressed from the viral genome to facilitate virus replication and spread. One of these proteins, HSV-TK, contrasts with mammalian thymidine kinase in that it can phosphorylate purine as well as pyrimidine nucleoside substrates and their analogs. GCV serves as a substrate for HSV-TK with a Km value of 50 μM, but it is not an efficient substrate for any of the mammalian nucleoside kinases thus accounting for its selectivity in herpesvirus-infected cells. Following subsequent phosphorylation by mammalian kinases to its triphosphate metabolite, the drug is incorporated into the viral DNA leading to cessation of replication. Based on this mechanism of selectivity, it was proposed that tumor cells genetically engineered to express HSV-TK would be killed when treated with GCV. Proof of this principle was first shown in murine sarcoma cells, and since then numerous reports have demonstrated similar results in many different cell types. At the triphosphate level, GCV can compete with the endogenous 2′-deoxyguanosine 5′-triphosphate (dGTP)

HSV-TK/Ganciclovir Mediated Toxicity. Figure 1 Structure of GCV.

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for incorporation into DNA by mammalian DNA polymerases. Since GCV has the equivalent of both the 5′ and 3′ hydroxyls of deoxyguanosine, it can be incorporated into DNA and allow further extension of the DNA strand (internucleotide addition). In contrast, the structurally related ▶acyclovir lacks the equivalent of a 3′ hydroxyl group, and thus is an obligate DNA chain terminator. GCV and its metabolites do not interfere with RNA or protein synthesis. The incorporation of GCV monophosphate into DNA is the primary lesion that results in cell death. For some cell types, GCV induces cell death through ▶apoptosis (Fig. 2). Compared to other substrates for HSV-TK, such as acyclovir, GCV is significantly more toxic and more mutagenic to cells that express HSV-TK. The action of acyclovir is primarily ▶cytostatic, whereas GCV induces cell killing at low, clinically achievable concentrations. Although GCV triphosphate accumulates in cells to a relatively low level of 10–20 μM, this is sufficient to produce several logs of cell death. This high toxicity may be attributable to the avid incorporation and lengthy retention of GCV into DNA. GCV triphosphate and its incorporation into the nascent DNA strand do not produce strong inhibition of DNA synthesis, so cells incorporate high levels of this drug, complete DNA replication and go on to divide. Daughter cells become irreversibly blocked when they enter S-phase, suggesting that GCV monophosphate cannot serve as a template for DNA replication. A

HSV-TK/Ganciclovir Mediated Toxicity. Figure 2 Mechanism of Cytotoxicity for GCV in HSV-TKExpressing Cells and Bystander Cells. GCV is selectively phosphorylated to the monophosphate in HSV-TK expressing cells. Further phosphorylation can be accomplished by cellular enzymes. GCV triphosphate competes with the endogenous dGTP for incorporation into DNA, leading to cell death. GCV at the mono-, di- or triphosphate level can be transferred directly to bystander (non-HSV-TK-expressing) cells via GJIC channels, resulting in its incorporation into DNA of bystander cells with subsequent cytotoxicity.

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strong G2/M block has also been observed in some cell types after GCV treatment. The cell cycle position in which cells become blocked may depend on the concentration of GCV. Toxicity to Non-HSV-TK-Expressing Cells (▶Bystander Effect) With current gene transfer technologies, only a small percentage of tumor cells will express the foreign gene. For this approach to be successful in cancer treatment in patients, there must be a mechanism by which cells that do not express the ▶transgene (bystander cells) can be killed. It was noted early on that when only a fraction of the cell population expressed HSV-TK, treatment with GCV resulted in killing of both the HSV-TK-expressing and HSV-TK-nonexpressing bystander cells. The strong cell killing ability of GCV in HSV-TK-expressing and neighboring bystander cells has resulted in complete regressions of experimental tumors in animals, spurring clinical interest in this approach. In suicide gene therapy, bystander cell killing generally occurs through the transfer of a toxic metabolite, produced in the transgene-expressing cell, to bystander cells. The bystander cell killing with HSVTK/GCV was an unexpected finding since the toxic metabolite, GCV triphosphate, is negatively charged and therefore would not readily pass through cell membranes to kill bystander cells. However, GCV mono, di- and triphosphate accumulate in bystander cells when co-cultured with HSV-TK-expressing cells and GCV. The primary mechanism that appears to account for transfer of GCV phosphates is ▶gap junctional intercellular communication (GJIC). GJIC allows the direct exchange of small molecules (90% accuracy in detecting bladder cancer. Furthermore, HYAL-1 mRNA levels measured in exfoliated cells are elevated in patients with invasive and poorly differentiated carcinoma. These studies show that HAase is a highly accurate marker for detecting high-grade bladder cancer, and when it is combined with HA, it detects both low-grade and high-grade bladder cancer with 90% accuracy. The prognostic potential of HYAL-1 has been explored in prostate cancer. By performing immunohistochemistry on radical prostatectomy specimens, on whom there was a minimum 5-year follow-up, Posey et al. and Ekici et al. found that HYAL-1 staining in radical prostatectomy tissues is an independent predictor of prostate cancer progression (local recurrence or distant metastasis). Furthermore, HA-HYAL-1 staining has an 87% accuracy in predicting disease progression. It is noteworthy that in prostate cancer specimens, while HYAL-1 is exclusively expressed by tumor cells, HA expression mainly results from the tumor-associated stroma. In a limited number of studies, HAase expression has also been studied in other carcinomas. For example, there is some evidence that HYAL-1 may be a marker for head and neck squamous cell carcinomas, as salivary HAase levels are elevated in head and neck cancer patients. PH20 mRNA levels are elevated in primary and lymph node metastatic lesions of laryngeal carcinoma when compared to normal laryngeal tissues. In contrast to the observations in many carcinomas, increased HYAL-2 expression inversely correlates with invasion in B-cell lymphomas and may serve as a prognostic indicator (▶Chemotherapy of cancer; Progression and perspectives). HAase and Cancer Therapeutics. Testicular HAase has been added in cancer chemotherapy regimens to improve drug penetration. Tumor cells growing in threedimensional multicellular masses, such as spheroids

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in vitro and solid tumors in vivo acquire resistance to chemotherapeutic drugs (i.e., multicellular resistance). But this acquired chemoresistance can be abolished by the addition of testicular HAase. In limited clinical studies, HAase has been used to enhance the efficacy of vinblastin in the treatment of malignant melanoma and Kaposi’s sarcoma, boron neutron therapy of glioma, intravesical mitomycin treatment for bladder cancer and chemotherapy involving cisplatin and vindesine in the treatment of head and neck squamous cell carcinoma. It is noteworthy that the HAase concentrations (1 × 105– 2 × 105 IU) used in these clinical studies far exceed the amount of HAase present in tumor tissues, and therefore, it is unlikely that at these concentrations the infused HAase will act as a tumor promoter. Summary HAase appears to be an important molecular determinant of tumor growth, infiltration and angiogenesis. At concentrations that are present in tumor tissues, HAase acts as a tumor promoter. HAases either alone, or together with HA are potentially accurate diagnostic and prognostic indicators for cancer detection and tumor metastasis. We are only beginning to understand the complex role that this enzyme plays in cancer. Nonetheless, it is already proving to be a useful target for developing novel cancer therapeutics and diagnostics. ▶Hyaluronidases ▶Early Detection

References 1. Stern R (2005) Hyaluronan metabolism: a major paradox in cancer biology. Pathol Biol (Paris) 53(7):372–382 2. Stern R, Jedrzejas MJ (2006) Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev 106:818–839 3. Lokeshwar VB, Schroeder GL, Carey RI et al. (2002) Regulation of hyaluronidase activity by alternative mRNA splicing. J Biol Chem 277:33654–33663 4. Lokeshwar VB, Cerwinka WH, Isoyama T et al. (2005) HYAL1 hyaluronidase in prostate cancer: a tumor promoter and suppressor. Cancer Res 65:7782–7789 5. Lokeshwar VB, Cerwinka WH, Lokeshwar BL (2005) HYAL1 hyaluronidase: a molecular determinant of bladder tumor growth and invasion. Cancer Res 65:2243–2250

Hybrid Genes ▶Fusion Genes

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Hybrid Positron Emission Tomography/Computed Tomography

Hydrogen Peroxide M IGUEL LOPEZ -L AZARO

▶Positron Emission Tomography

Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain

Synonyms Dihydrogen dioxide; Hydrogen dioxide

Definition

Hybridomas Definition Are fusion cells consisting of antibody-producing B cells and non-secreting cultured myeloma cells. After isolation of antibody-producing B cells from the spleen of immunized animals these polyclonal B cells are fused with myeloma tumor cells by permeabilization of the cell membranes. Hybridoma cells grow and multiply rapidly and produce large amounts of the desired antibodies. ▶Monoclonal Antibody Therapy ▶Bispecific Antibodies

Hydrogen Dioxide ▶Hydrogen Peroxide

Hydrogen Nuclei Definition Contain a single proton with a net charge and spin. They therefore possess a magnetic moment. They are abundant in the human body in water and fat and are the Magnetic Resonance Imaging (MRI) active nuclei used in all clinical and most research MRI. ▶Dynamic Contrast-Enhanced Magnetic Resonance Imaging

Hydrogen peroxide (H2O2) is a ▶reactive oxygen species (ROS) generated from molecular oxygen (O2). Although the controlled cellular production of H2O2 plays an important physiological role, high cellular levels of H2O2 can produce carcinogenic effects and induce cell death.

Characteristics H2O2 is a pale-blue liquid first isolated in 1818 by Louis Jacques Thénard. H2O2 has industrial and domestic uses (e.g. paper bleaching, chemical synthesis, laundry detergents, antiseptic for wound cleaning, etc.) and it is manufactured today through an autoxidation reaction using O2 from the air. Cells of aerobic organisms also generate H2O2 from O2. Most of the energy (ATP) that aerobic cells need to live is obtained through a process called ▶oxidative phosphorylation (oxphos). In this process, ATP generation is coupled with a reaction in which O2 is reduced [▶reduction/oxidation] to water (H2O) by a mitochondrial protein complex called cytochrome oxidase. In this reaction, four electrons and four protons are added to O2 to form two molecules of H2O. But when a molecule of O2 gains only one electron to form superoxide anion (O2•−), this highly reactive oxygen species tends to gain three more electrons and four protons to form H2O; this process involves several reactions and results in the production of other ROS such as H2O2 (Fig. 1). ROS are generated in multiple compartments within the cell (e.g. mitochondria, cytosol, plasma membrane, peroxisomes, endoplasmic reticulum, etc.) and by numerous enzymes (e.g. cytochrome oxidases, NADPH oxidases, cyclooxygenases, cytochromes P450, xanthine oxidase, etc.). ROS can be eliminated by endogenous antioxidant systems. For instance, glutathione and thioredoxin systems decrease the cellular levels of H2O2 by catalyzing a reaction in which H2O2 is reduced to H2O. Likewise, the enzyme catalase eliminates H2O2 by transforming two molecules of H2O2 into two molecules of H2O and one molecule of O2. Antioxidant agents can reduce the cellular levels of ROS by preventing their generation or by favoring their elimination. For instance, some polyphenols (e.g. ▶flavonoids) can prevent the generation

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Hydrogen Peroxide. Figure 1 Aerobic cells generally use O2 to generate energy (ATP) through a process called oxidative phosphorylation (oxphos), but they can also use O2 to generate reactive oxygen species such as hydrogen peroxide (H2O2).

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Hydrogen Peroxide. Figure 2 While the controlled generation of H2O2 has an important physiological role, a sustained increase in the cellular levels of H2O2 can produce carcinogenic effects, and an excessive increase in the levels of H2O2 can induce cell death. Antioxidant agents can reduce the cellular levels of H2O2 and prevent the carcinogenic effects induced by H2O2; these agents may therefore exert a cancer-preventive activity. Prooxidant agents can increase the cellular levels of H2O2 and may induce carcinogenic effects. A sufficient increase in the cellular levels of H2O2 induced by prooxidant agents may trigger cell death and be therapeutically useful.

of H2O2 by scavenging O2•−; and selenium compounds can favor the elimination of H2O2 by providing selenium atoms, which are essential components of the H2O2detoxifying enzyme glutathione peroxidase. Prooxidant agents, on the contrary, can increase the cellular levels of ROS by increasing their generation or by reducing their elimination. For instance, arsenic can increase ROS generation by activating the enzyme NADPH oxidase, and buthionine sulfoximine (an inhibitor of γ-glutamylcysteine synthetase, the rate-limiting enzyme of glutathione synthesis) can reduce ROS elimination by decreasing glutathione-mediated H2O2 decomposition. The controlled generation of ROS plays an important role in the physiological control of cell function. For instance, cells under hypoxic conditions generate H2O2 and use it to activate hypoxia-inducible factor 1 (HIF-1) [▶hypoxia and tumor physiology], a transcription factor that codes for many proteins that help cells adapt to low oxygen levels. An uncontrolled or excessive cellular production of H2O2, however, can produce carcinogenic effects and cell death (Fig. 2).

Carcinogenic Effects Cancer cells from different tissues have been observed to produce high amounts of H2O2. High cellular levels of H2O2 have been associated with DNA alterations, including DNA damage, mutations, and genetic instability. For instance, transition metals such as iron or copper can react with H2O2 to produce hydroxyl radical (OH•) via the Fenton reaction; OH• is a highly reactive species known to produce ▶oxidative DNA damage. The production of DNA alterations by H2O2 may play an important role in ▶carcinogenesis, as cancer is considered to be a genetic disease caused by DNA alterations. Cell proliferation, ▶apoptosis resistance, ▶angiogenesis, invasion and ▶metastasis are key events of the carcinogenesis process. It is well known that, in order for cancer to develop, tumor cells must proliferate. Accumulating data have shown that H2O2 stimulates cell proliferation; in fact, the cell proliferation induced by different stimuli can be decreased by H2O2-detoxifying enzymes (e.g. catalase). Under

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physiological conditions, cells with irreparable damages usually commit suicide by triggering a programmed process called apoptosis. Since tumor cells have important damages in many of their components, the formation of a cancer requires that tumor cells develop apoptosis resistance. Interestingly, it has been found that non-cytotoxic concentrations of H2O2 can produce apoptosis resistance in cancer cells. Angiogenesis – the generation of new blood vessels – is also necessary for the formation of a solid tumor; without vascular growth, the tumor mass is restricted to within a tissue-diffusion distance of approximately 0.2 mm. Recent results support that H2O2 has an important function in angiogenesis. For instance, angiopoietin-1 plays an important role in angiogenesis and it has been found that its angiogenic effect is mediated by H2O2. It is recognized that the metastatic spread of primary tumors accounts for approximately 90% of all cancer deaths. The process by which cells from a localized tumor invade adjacent tissues and metastasize to distant organs is therefore the most clinically relevant processes involved in carcinogenesis. H2O2 has been found to modulate the activity of several processes (e.g. cell ▶motility, migration, adhesion) and molecules (e.g. ▶MET, ▶matrix metalloproteinases) involved in tumor invasion and metastasis. Indeed, studies carried out in animal models have revealed that the targeted delivery of catalase can inhibit tumor metastasis. Although the transcription factor HIF-1 plays an important role in the physiological control of cell function, HIF-1 overexpression is commonly observed in most human cancers and has been associated with increased patient mortality in several cancer types. HIF-1 increases the transcription of a variety of genes that code for proteins involved in processes intimately related to cancer, including apoptosis resistance, angiogenesis or invasion and metastasis. H2O2 can both activate HIF-1 and mediate the activation of HIF-1 caused by different stimuli; in fact, the presence of enzymes that reduce the cellular levels of H2O2 prevents the activation of HIF-1 caused by different triggers (e.g. hypoxia, TNF-alpha). The key role of H2O2 in carcinogenesis is also supported by experimental data that have demonstrated that H2O2 can cause and mediate cell malignant transformation. For instance, it has been reported that the expression of Nox1 (a homologue of gp91phox, the catalytic moiety of the O2•−-generating NADPH oxidase of phagocytes) in normal NIH3T3 fibroblasts resulted in cells with malignant characteristics that produced tumors in athymic mice. These transformed cells showed a 10-fold increase in H2O2 levels. When catalase was overproduced in these transformed cells, H2O2 concentration decreased, and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice.

Clinical Relevance Despite some important advances in cancer therapy, the number of cancer deaths has not decreased in the last three decades. New strategies to control this disease are required. It is recognized that cancer chemoprevention (the use of chemicals to prevent, stop or reverse the process of carcinogenesis) is an essential approach to controlling cancer. Since H2O2 seems to have an important role in carcinogenesis, a chemical capable of preventing or decreasing excessive cellular levels of H2O2 might be useful in cancer chemoprevention. Some complementary and alternative medicine practitioners have used H2O2 and other “hyperoxygenation” therapies for the treatment of cancer. In 1993, the American Cancer Society studied the available literature and found no evidence that treatment with H2O2 and other “hyperoxygenation” therapies was safe or resulted in objective benefit in the treatment of cancer. Today it is accepted that the direct administration of H2O2 is not an appropriate strategy for the treatment of cancer, as H2O2 can produce toxicity by oxidizing macromolecules such as DNA, proteins or lipids. Indeed, as discussed above and represented in Fig. 2, a large body of research strongly supports that H2O2 can produce carcinogenic effects. However, an increasing number of reports indicate that a sufficient increase in the cellular levels of H2O2 may be an effective therapeutic strategy. For instance, it is recognized that many anticancer drugs currently used in the clinic produce their antitumor activity by inducing apoptosis in cancer cells, and H2O2 is an effective inductor of apoptosis in cancer cells. In addition, some studies have shown that specific concentrations of H2O2 can kill cancer but not normal cells; it has been proposed that the increased levels of H2O2 found in tumor cells may account for their increased susceptibility to H2O2. This selectivity for cancer cells is in accordance with animal experiments that have shown that the use of H2O2generating systems can deliver H2O2 to sites of malignancy and produce anticancer effects with little toxicity to the host. The therapeutic potential of H2O2 is also supported by the fact that the anticancer activity of several drugs commonly used in the clinic (e.g. paclitaxel [▶taxol], arsenic trioxide) is mediated, at least in part, by an increase in the cellular levels of H2O2. Recent data have also shown that high concentrations of vitamin C (only achievable through the i.v. route) can produce selective killing of cancer cells through a H2O2-dependent mechanism. Therefore, although the direct administration of H2O2 does not seem an appropriate anticancer approach, any strategy capable of increasing the levels of H2O2 in cancer cells might be therapeutically useful. Accumulating evidence suggests that the modulation of the cellular levels of H2O2 may be an important

5-HydroxyIndoleacetic Acid (5-HIAA)

approach for the development of cancer chemopreventive and therapeutic strategies. Chemicals with antioxidant properties may reduce the cellular levels of this oxidant and produce cancer chemopreventive effects; indeed, most cancer chemopreventive agents have antioxidant properties. Chemicals with prooxidant properties may induce a sufficient increase in the cellular levels of H2O2 and produce cell death in cancer cells; these drugs may produce a chemotherapeutic effect. Many chemicals (e.g. vitamin C, vitamin E, β-carotene, ▶curcumin, ▶sulforaphane, ▶epigallocatechin, etc) induce either an antioxidant or prooxidant effect mostly depending on the concentration at which they reach the cells. Low concentrations of these agents would therefore produce chemopreventive effects, while high concentrations would produce chemotherapeutic effects. However, these agents may produce carcinogenic effects when used at concentrations that increase the cellular levels of H2O2, but not sufficiently to induce cell death (Fig. 2). Antioxidant/prooxidant drugs may act as cancer preventive, therapeutic or carcinogenic agents mostly depending on their dose and route of administration. ▶Oxidative Stress

References 1. Burdon RH (1995) Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med 18:775–794 2. Szatrowski TP, Nathan CF (1991) Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 51:794–798 3. Arnold RS, Shi J, Murad E et al. (2001) Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci USA 98:5550–5555 4. American Cancer Society (1993) Questionable methods of cancer management: hydrogen peroxide and other ‘hyperoxygenation’ therapies. CA Cancer J Clin 43:47–56 5. Lopez-Lazaro M (2007) Dual role of hydrogen peroxide in cancer: possible relevance to cancer chemoprevention and therapy. Cancer Lett 252:1–8

Hydrophilic

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Hydroquinone Definition Product of two-electron (C6H4(OH)2).

reduction

of

quinone

▶Mitomycin C

Hydroxyapatite Definition Hydroxyapatite crystals constitute the major and essential component of normal bone and teeth. Hydroxyapatite crystals represent the mineral matrix of bone and teeth and give rigidity to bones and teeth. The chemical formula of basic hydroxyapatite is Ca10(PO4)2OH6 and these crystals can be physiologically substituted with magnesium, fluor, or carbonate. ▶Zoledronic Acid ▶Bisphosphonates

Hydroxybutyrate Dehydrogenase ▶Serum Biomarkers

5-HydroxyIndoleacetic Acid (5-HIAA) Definition

Definition Literally means water loving. These are drugs that much prefer to dissolve in water than in fat.

A breakdown product of serotonin that is eliminated in the urine. Urinary levels of 5-HIIA are used as a diagnostic and monitoring tool in carcinoid tumors.

▶ADMET Screen

▶Neuroendocrine Carcinoma

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Hydroxyl Group

Hydroxyl Group Definition Functional group consisting in an atom of oxygen joined to a hydrogen atom by a single bond. ▶Bisphosphonates

2-Hydroxyoleic Acid Definition

Synonym α-hydroxyoleic acid and 2-hydroxy-9-cisoctadecenoic acid. Synthetic derivative of oleic acid with anticancer activity against a wide variety of cancer types, oral administration and negligible side effects. ▶Membrane-Lipid Therapy

Hydroxylapatite 7-Hydroxystaurosporine Definition

Synonym ▶hydroxyapatite Ca5(PO4)3(OH).

14-Hydroxyldaunorubicin ▶Adriamycin

▶UCN-01 Anticancer Drug

7-Hydroxystaurosporine, NSC 638850 Definition

▶UCN-01 Anticancer drug

4-Hydroxynonenal Hydroxysteroid Dehydrogenases Definition An unsaturated hydroxyalkenal that is generated by the breakdown of arachidonic acid during stress in cells.

▶Reductases

▶Glutathione Conjugate Transporter RLIP76

Hyperalgesia 13-Hydroxyoctadecadienoic Acid (13-HODE) Definition A primarily growth stimulatory signaling molecule resulting from the metabolism of LA by 15-lipoxygenase-1 in cancer cells. ▶Melatonin

Definition Type of pain that is frequently associated to cancer chemotherapy or cancer itself. Hyperalgesia is an increased sensitivity and lowered threshold to a stimulus – such as burn of the skin – that is normally painful. Allodynia is caused by a stimulus – such as touch, pressure and warmth – that does not normally provoke pain. ▶Cannabinoids and Cancer ▶Allodynia

Hypericin

Hypercalcemia

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Hyperglycemia

Definition

Definition

An excess of calcium in the blood (normal range: 9– 10.5 mg/dL or 2.2–2.6 mmol/L). Can be due to excessive skeletal calcium release, increased intestinal calcium absorption, or decreased renal calcium excretion. Symptoms of hypercalcemia include fatigue, vomiting, depression, confusion, anorexia. Severe hypercalcemia (range above 15–16 mg/dl) may lead to coma and cardiac arrest.

Is a condition of elevated glucose levels in the bloodstream. It is a hallmark of metabolic syndrome and diabetes.

▶Vitamin D ▶Bone Loss, Cancer Mediated

▶Adiponectin

Hypericin C ONSTANCE L. L. S AW, PAUL W. S. H ENG Department of Pharmacy, National University of Singapore, Singapore, Singapore

Hyperchlorhydria

Synonyms Hypericum extract; Hypericum red

Definition Is the increased gastric acid secretion. ▶Gastrinoma

Hyperdiploidy Definition Having a chromosome number that is more than the normal diploid number. ▶Acute Lymphoblastic Leukemia

Hypergastrinemia Definition Is increased serum gastrin. ▶Gastrinoma

Definition

Hypericin is a natural ▶photosensitizer present in the plant, Hypericum perforatum, commonly known as St John’s wort. It belongs to the class of phenanthroperylenequinones and napthodianthrone, has a molecular weight of 504.45. St John’s wort has been used for centuries to treat mental disorders and nerve pain. In ancient times, herbalists wrote about its use as a sedative and a treatment for malaria, as well as a balm for wounds, burns, and insect bites. Today, St John’s wort is used by some for depression, anxiety, and/or sleep disorders. Hypericin is used in photodynamic diagnosis (▶PDD) and photodynamic therapy (▶PDT) for diagnosis and treatment of cancers.

Characteristics Physical Properties Hypericin yields red fluorescence when excited with a specific wavelength of light by lasers such as 442 nm (He-Cd), 488 nm (Ar), or 543 nm (He–Ne), and light by xenon-arc lamp with a band-pass filter of 380–450 nm or 375–400 nm. It has a high extinction coefficient near 600 nm. The two main maximum absorption and ▶photoactivation peaks of hypericin occur near 550 and 600 nm. The two fluorescence peaks of hypericin are near 600 and 650 nm. Hypericin has very low water solubility. The aggregated hypericin is not fluorescent. It generates a high quantum yield of singlet oxygen and superoxides.

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Hypericin

Clinical Applications PDD of Cancers The initial presentations of bladder cancer in 70–80% of the cases are superficial and limited to the urothelial lining of the bladder mucosa and submucosa. Visual differentiation between normal tissue and transitional cell carcinoma is relatively easy but not for carcinoma in situ or nonmalignant diseases such as cystitis due to radiotherapy, chemical or bacterial origins. They are often invisible to the naked eye. ▶Fluorescence cystoscopy is a form of PDD of cancer and has significantly improved the diagnosis and early detection of cancer. In bladder cancer detection, hypericin has several advantages over conventional photosensitizer, ▶5-aminolevulinic acid (▶5-ALA, a prodrug) (Table 1). A human clinical trial had found that the use of hypericin showed higher sensitivity (82%) than just white-light cystoscopy (62%). This report justifies the use of hypericin-PDD for bladder cancer in clinical settings. Hypericin was also shown to accumulate in patients with stomach cancer, and detected stomach cancer with 85% specificity. ▶PDT of Cancers ▶Basal cell carcinoma and ▶squamous cell carcinoma are the most common cancers among the Caucasians. The effect of hypericin-PDT had been investigated in these skin cancers. It was found that 1 mg or more of hypericin was acceptable as an alternative treatment for basal cell carcinoma with clinical remission achieved in 2 out of 11 patients. One out of eight patients with squamous cell carcinoma achieved clinical remission without sign of recurrence observed after 5 months of treatment with 1.5–3 mg of hypericin. Mild to transient erythema and edema at the tumor lesions were observed when the cancers were treated with hypericin and there appeared to be a correlation between the degree of tumor reduction and occurrence of erythema and edema at the tumor lesions. As this is the first reported clinical trial on the use of hypericin-PDT in the reported skin

Hypericin. Table 1

cancers, the PDT treatment protocol and dosage were transposed from results with animal models and the trial was restricted to 4–6 weeks because of ethical reasons. Thus, long-term remission rates are needed to further evaluate the clinical usefulness of hypericin with additional trials. Another hypericin-PDT use in cancer treatment was for local application of hypericin in a patient with recurrent malignant mesothelioma. The patient had previously been treated with radiotherapy, chemoimmunotherapy and subsequently PDT using hematoporphyrin derivatives (Photosan III). Topical hypericin was applied to the patient and a month later, the combination of hypericin and Photosan III was applied. It was found that complete remission was achieved histologically with a light dose at 90 J/cm2 together with PDT using combined systemic Photosan III and topical application of hypericin. Preclinical Investigations The chemical groups reported to cause the photodynamic activities of hypericin are semiquinone, singlet oxygen, and superoxide anion radicals. Light triggers the photosensitization (photoactivation) of hypericin for photodynamic reactions. Hypericin-PDT effect is a function of drug, light, and oxygen present. There are three PDT effects of the photosensitizers on biological systems. Firstly, it can damage the vasculature, causing thrombosis and blood flow stasis. Secondly, PDT kills tumor cells via generation of free radicals that damage mitochondria, plasma membrane, and other organelles, hence inducing apoptosis or necrosis of the tumor. Thirdly, PDT modulates the immune system against cancer cells. Successful cancer treatment is usually a result of the combination of all these effects. With the appropriate use of light and adjuvant therapy such as heat or ▶antiangiogenesis agents, better therapeutic outcomes can be expected. Table 2 shows the reported methods to improve the efficiency of hypericin for PDD and PDT in preclinical investigations. As

Comparison of 5-ALA and hypericin for detecting human superficial bladder carcinoma

Characteristics Form

5-ALA

Stability

Prodrug, need to be converted to the active form 75–100% Very low specificity, 43–68.5% (with many false-positives) Easily photobleached during process

Permeability across biological membrane

Charged molecule – difficult to cross biomembranes

Sensitivity Specificity

Hypericin Active form 82–94% High, 91–98.5% Greater stability, no significant photobleaching Hydrophobic – good permeability

Hypericin Hypericin. Table 2

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Methods for enhancement of hypericin-PDD/PDT efficacy

Concept Chemical modifications Physical methods Pharmaceutical formulations Combination with adjuvant therapy

Methods Improve physicochemical properties of hypericin. Analogs with better water solubility, absorption and fluorescence properties, and singlet oxygen quantum yield Optimize light conditions (fractionated light dose), oxygen perfusion, hyperthermia Prepare liposomes, nanoparticles, topical applications Add oxygen carrier, antiangiogenesis agents, drugs that are able to synergize with hypericin for cancer treatment

administration of hypericin to patients remains a challenging task due to its poor aqueous solubility and lipophilic nature, the most popular approach is by a formulation approach to enhance the efficacy of hypericin-PDT. There is a trend to develop formulations of hypericin without plasma proteins. Despite the use of cutting-edge technology approaches, such as preparing transferrin-mediated targeted delivery liposomes and nanoparticles of hypericin, such preparations did not always confer the desired enhanced treatment effects. It appeared that the use of an adjunct with hypericin, especially those prepared without using plasma proteins, was more successful in enhancing the delivery of hypericin with both in vivo and in vitro systems. Using a pharmaceutical solvent and penetration enhancer, N-methyl pyrrolidone, delivery of hypericin was shown to be better than the conventional formulation of hypericin with albumin. Using chick chorioallantoic membrane implanted with human bladder cancer cells, N-methyl pyrrolidone was demonstrated to be an excellent alternative to plasma proteins as adjuvant with hypericin. The improved N-methyl pyrrolidone–hypericin formulation enabled more efficient delivery and uptake of hypericin at the target site. This will not only allow a lower drug dose to be used potentially but will also significantly reduce patients’ waiting time. The exact mechanism for the tumor selectivity uptake of hypericin has not been fully understood. The uptake/ transport/delivery of hypericin is dependent on several competing or interrelated factors, which include the type of incubation medium (with or without serum proteins), cell type, delivery system, and biological testing method. It is likely that both active and passive transport mechanisms contribute to the overall uptake of hypericin but passive diffusion is likely to be the more dominant mechanism. Apart from its photosensitizing properties, hypericin was found to be a ▶protein kinase C inhibitor. It was also reported to involve in the modulation of immune system such as expression of cytokines. Hypericin-PDT triggered the expression of angiogenic factors, ▶matrix metalloproteinases, and various signaling pathways.

The rapid advancement in genetic research had shown that the effects of hypericin-PDT could be independent of the status of ▶p53, the tumor suppressor gene. Many reports have attributed the failure of conventional chemotherapy and radiotherapy to the mutated ▶p53, due to apoptosis failure caused by the nonfunctional p53. Therefore, hypericin-PDT provides an alternative pathway in treating cancers. In the absence of light, toxicity of hypericin was found to be very low. Apart from its potent photosensitizing properties that are light-dependent, hypericin shows unique activities even in the dark. In complete darkness, hypericin has cytostatic activities that were able to prolong the survival of mice with high-grade squamous carcinoma tumors. This is attributed to hypericin inhibiting several key steps of the angiogenesis. Many different types of cancers including leukemia, glioblastoma, and childhood rhabdomyosarcoma have been treated with hypericin-PDT and in vitro results were promising in many of the cases. Continual effort in basic and translational research will lead to a better understanding of the contributing factors responsible for effective hypericin-PDT applications. ▶Photodynamic Therapy

References 1. Kiesslich T, Krammer B, Plaetzer K (2006) Cellular mechanisms and prospective applications of hypericin in photodynamic therapy. Curr Med Chem 13:2189–2204 2. Saw CL, Olivo M, Soo KC et al. (2006) Delivery of hypericin for photodynamic applications. Cancer Lett 241:23–30 3. Saw CL, Olivo M, Chin WW et al. (2007) Superiority of N-methyl pyrrolidone over albumin with hypericin for fluorescence diagnosis of human bladder cancer cells implanted in the chick chorioallantoic membrane model. J Photochem Photobiol B 86:207–218 4. Dets SM, Buryi AN, Melnik IS et al. (1996) Laser-induced fluorescence detection of stomach cancer using hypericin. Proc Soc Photo Opt Instrum Eng 2926:51–56 5. Koren H, Schenk GM, Jindra RH et al. (1996) Hypericin in phototherapy. J Photochem Photobiol B 36:113–119

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Hyperlipidemia

Hyperlipidemia

Hypersensitivity Reaction

Definition

Definition

Refers to an elevation of lipids (fats) in the bloodstream.

A serious and sometimes life-threatening reaction to a drug or other chemical or venom.

▶Cachexia

▶Paclitaxel

Hypermethylation Hyperthermia Definition

Increased ▶methylation of cytosine residues in cytosine-guanine pairs in regulatory regions of DNA of specific genes or of global DNA within a cell or tissue. ▶Fragile Histidine Triad ▶Methylation-Controlled J Protein (MCJ)

A SHLEY A. M ANZOOR , M ARK W. D EWHIRST Department of Radiation Oncology, Duke University, Durham, NC, USA

Definition Hyperthermia refers to the elevation of temperature above physiological levels, typically to values of 40–45°C.

Characteristics

Hyperplasia Definition Consists of an increase in the number of tissue cells, without changes in their volume. This alteration, generally secondary to prolonged growth stimuli, is reversible and does not evolve to malignancy.

Hyperthermia utilizes elevated tissue temperatures, typically between 40°C and 45°C, to alter the tumor and normal tissue environment. The goal of hyperthermia in cancer treatment is to create an environment that will aid in eradicating tumor while sparing normal tissue. Hyperthermia accomplishes this by causing direct cytotoxic effects and a variety of physiologic effects, including the alteration of blood flow and oxygenation status. Clinically, hyperthermia can work synergistically with both radiation and chemotherapy.

▶Preneoplastic lesions

Hyperplastic growth Definition Tissue overgrowths that still retain their normal characters and ability to differentiate.

Hypersensitivity ▶Allergy

Cytotoxic Effects Hyperthermia elicits cytotoxic effects in tissue through a variety of mechanisms, inducing both ▶apoptosis and ▶necrosis. Above 40°C, protein is a dominant molecular target, with protein denaturation exhibiting a similar heat of inactivation as thermal cell kill and damage (130–170 kcal/mole). Other cellular targets include the cytoskeleton, which controls many signal transduction pathways, cellular respiration enzymes, and DNA repair processes. These components of the cell are particularly heat sensitive and lead to increased cytotoxic effects. Physiologic Effects Hyperthermia also has an intricate relationship with tumor physiology, as the degree of physiologic response varies with temperature. An initial temperature increase to 41–41.5°C will result in elevated blood flow and increased vascular permeability.

Hyperthermia

Generally, muscle and skin perfusion increase by at least tenfold, while tumor perfusion increases only 1.5to 2-fold. This difference in response between normal and cancerous tissue is one of the most exploited benefits of hyperthermia in cancer treatment. At these initial hyperthermia temperatures, edema may result from the increase in vascular permeability, and as higher temperatures are reached, vascular stasis and hemorrhage may develop. Additionally, higher temperatures may result in vascular damage, although typically these temperatures are neither utilized nor often achievable in the clinic. With respect to normal tissue, thermal damage from hyperthermia exhibits varying degrees of severity depending on temperature and tissue type. Some normal tissues are more heat sensitive than others, yet the sensitivity is not easily predicted by classical cytotoxicity or radiotherapy principles. While these principles correlate proliferative potential with radiation or chemo-sensitivities, this is not predictive of thermal sensitivity. For example, the brain and testes both exhibit high thermal sensitivities, yet the brain has almost no proliferative potential and the testes are highly proliferating. Furthermore, in addition to showing no correlation between thermal sensitivity and proliferative potential, there is also no tissue-specificity. For instance, the spinal cord, peripheral nerves, and brain all demonstrate varying sensitivity to heat, yet these are all nervous tissue. In addition to physiologic changes in blood flow and vascular permeability, ▶tumor metabolism and oxygenation can also be altered substantially with hyperthermia. As mentioned earlier, certain enzymes may be highly heat sensitive. Along metabolic pathways, enzymes involved in aerobic tumor metabolism exhibit much greater heat sensitivity than those involved in anaerobic metabolism. This lends itself to a theory that hyperthermia treatment may cause reductions in tumor metabolism and respiration. Reported effects in support of this theory have been decreased ATP production and increased lactate concentration following hyperthermia. Furthermore, alteration in tumor metabolism has additional downstream effects, particularly with respect to tumor oxygenation status. As a tumor shifts from aerobic to anaerobic metabolism following hyperthermia, the tumor may experience decreased oxygen consumption rates; this decrease in oxygen consumption may consequently lead to significant improvement in the tumor oxygenation status. Studies have shown oxygenation increases following hyperthermia in rodent and canine tumors, as well as human tumors. However, this oxygenation effect is most pronounced at temperatures between 41°C and 43°C, decreasing at higher temperatures where vascular damage occurs.

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Immunological Effects Hyperthermia can augment the body’s innate immunity as well as increase immune activity specifically towards tumors. In general, the body increases in temperature as a response to infection; many bacterial or viral pathogens are heat sensitive and thus an elevation in body temperature serves as a primary method of defense against invading agents. Furthermore, as body temperature rises, cellular metabolism does as well, which can aid in accelerating immunologic responses. With regards to immune response towards tumor, hyperthermia can induce maturation of ▶dendritic cells, cause increased lymphocyte trafficking, and engage in ▶heat shock protein 70 (HSP70) mediated immune response, which in turn increases T-cell specific antitumor activity and further stimulates dendritic cells. These physiologic responses augment the body’s natural immune response against tumors. Hyperthermia Physics Delivery Modalities Hyperthermia may be delivered using either invasive or non-invasive sources. Non-invasive techniques are generally preferred, and consist of two main methods of delivering heat to tissues: ▶electromagnetic (EM) heating or ▶ultrasound. While the underlying physics principles of heat deposition differ between EM and ultrasound modalities, they are similar in a variety of ways. Both are sensitive to tissue heterogeneity as well as blood flow geometry, and are susceptible to difficulties of energy coupling into tumor tissue. Materials with finite electrical conductivity experience energy dissipation with the flow of electric current; this is the principle behind ohmic, or resistance electromagnetic heating. Delivery of EM heating is dependent on both frequency and wavelength. As EM frequency increases over the radio frequency (RF) and microwave (MW) range, electrical conductivity also increases. Since energy deposition in tissue is proportional to the local tissue conductivity and the magnitude of the electric field, these higher frequencies result in greater energy deposition and shorter penetration depths. Additionally, in the design of antennae or electrodes to deliver the EM field, there is a general constraint that limits the ability to localize heating at depths greater than 2–5 cm. This arises from the relation between EM frequency and wavelength to the physical size of the antenna or electrode source. Longer wavelengths will penetrate deeper into tissue, but require physically larger heating antennae, thus preventing localization of the EM field. This tradeoff between localization and penetration yields a critical limitation in electromagnetic heating: localized heating is limited to depths less than 2–5 cm. At depths greater than 2–5 cm EM heating will result in regional energy deposition, potentially involving large volumes of normal tissue.

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Hyperthermia

In accordance with these depth limitations, electromagnetic heating devices are typically divided into two categories: superficial applicators which provide focused heating at depths less than 2–5 cm and deep heating devices which distribute heat to a deeper penetration but over a broader range. Superficial devices consist of waveguides and microstrip or patch antennas which operate in typical microwave band frequencies (433, 915, or 2,450 MHz) and usually require a water bolus to couple energy into the tissue. The deep heating devices typically used are magnetic induction, capacitive coupling, and phased array fields. These devices operate at lower frequencies, between 5 MHz and 200 MHz. A schematic illustration of a phased array device for deep heating is shown in Fig. 1. Similarly to electromagnetic heating, ultrasound also experiences decreased penetration with increasing frequency. As an advantage over EM, however, ultrasound operates with much lower wavelengths and consequently avoids the severity of problems associated with focusing at greater depths. Unfortunately, ultrasound is limited by resolution, since it is particularly sensitive to the reflective and absorptive effects of air and bone, respectively. This imposes severe limitations of ultrasound heating in regions of tissue heterogeneity and changing geometry. Ultrasound devices are also separated into categories of superficial and deep heating. Single and multiple transducer plane wave devices operating with frequencies between 1 MHz and 3 MHz have been designed for superficial tumors, while at depths greater than 2–5 cm scanned focused transducers, phased arrays, or multiple scanned focused transducers in the 0.5–2 MHz

range are used. Ultrasound devices require a temperature controlled water bolus for energy coupling. Thermometry One of the greatest challenges facing clinical hyperthermia is the difficulty in obtaining accurate and noninvasive thermometry. Currently, most thermometry is performed through placement of ▶catheters into the tumor via ultrasound or CT guidance; thermometers are then placed within the catheters. Temperature readings are attained statically with multipoint thermometers or dynamically by moving a single point thermometer through the catheter during heating. There are many disadvantages to the common method of invasive thermometry. For one, there is a greater risk to the patient. Invasive methods are uncomfortable and present a risk for infection and hemorrhage. Additionally, the procedure is lengthy, requiring image registration and extra time of the physician. Perhaps most crucially, the data obtained from invasive methods is limited and often does not provide much spatial data, which in turn limits the control and uniformity of the hyperthermic treatment. In an attempt to gain better uniformity of treatment, noninvasive thermometry methods have been developed; these methods rely mostly on ▶Magnetic Resonance Imaging (MRI) techniques, although a variety of technologies are being pursued. MR imaging has many temperature sensitive parameters that can be exploited for thermometry purposes, including relaxation times T1 and T2, bulk magnetization, and proton resonance frequency. Of these four parameters, the proton resonance frequency is the most commonly

Hyperthermia. Figure 1 Illustration of an annular phased microwave array for heating deep situated tumors.

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normalizing thermal data between different patients or treatments. This is expressed as Cumulative Equivalent Minutes at 43°C (CEM 43°C) and is given by the equation: CEM 43 C ¼ tRð43TÞ Hyperthermia. Figure 2 Example of noninvasive thermometry data acquired with phase difference (chemical shift) MRI in a sarcoma of the lower leg. This method can yield temperature data with a resolution of 1.0°C.

used, and has demonstrated superior temperature sensitivity and resolution. Clinical studies have illustrated resolutions of 0.5–1°C with MRI thermometry. Figure 2 shows a temperature distribution image obtained from an MRI of a sarcoma in the lower leg. Despite the many advantages hyperthermia affords, clinical implementation has proved challenging. As mentioned above, hyperthermia heating regimens can be difficult to reproduce, utilizing invasive thermometry methods that have limited spatial coverage. Additionally, the definition and calculation of thermal dose used in hyperthermia is not well defined, making clinical trial comparisons between institutions difficult. However, the technology associated with clinical hyperthermia continues to improve, and many of these challenges are being addressed in ongoing research. Thermal Dosimetry The Arrhenius relationship is used to describe the rate of cell kill for a given temperature over time, and provides the basis for thermal dosimetry. Hyperthermia cell survival curves decrease exponentially, with higher rates of cell kill at increased temperatures. Arrhenius plots can be determined from in vitro data by taking the log of the slope of cell survival curves as a function of temperature. A typical graph of surviving fraction with temperature and its resulting Arrhenius plot is given in Fig. 3. Arrhenius plots are characterized by their biphasic nature, with the temperature at which the slope changes commonly referred to as the “breakpoint;” this phenomenon is derived from tissues developing thermal tolerance. For human tissue, the “breakpoint” occurs at 43.5°C. Below the breakpoint, the rate of cell kill decreases twofold to fourfold for each 1.0°C decline in temperature. Above the breakpoint, the rate of cell kill doubles for each 1.0°C elevation of temperature. Achieving constant temperatures over time is difficult in tumor tissue, leading to non-uniformity in hyperthermia delivery between patients. Since the in vivo slopes of the Arrhenius plots are practically identical to the in vitro slopes, and they provide a correlation between rate of cell kill and temperature, the Arrhenius relationship is used as a method for

Where t = time of treatment in minutes, T = average temperature in Celsius during treatment, and R is a constant, equal to 0.5 above the breakpoint and 0.25 below. In more complex situations with large temperature variations, the thermal data may be broken into shorter time intervals of 1–2 min where the temperature remains relatively constant. In these instances the total CEM 43°C for the treatment is attained through summation of CEM 43°Cs for each interval period. Also referred to as the thermal isoeffect dose, the CEM 43°C has been a valuable predictor of treatment efficacy and standardization of heat treatment in clinical trials. Clinical Hyperthermia Hyperthermia has been used to treat superficial tumors as a lone method of treatment. However, while the occasional response is observed, in general heat treatment alone is not associated with long term tumor control. While the practice of using solely hyperthermia is still performed in some clinics, this use is not supported by peer-reviewed published scientific data. Instead, hyperthermia is best utilized as a synergistic adjunct modality to oncology treatments, including radiation and chemotherapy. Figure 4 underscores the important physiological processes of hyperthermia in augmenting radiation and chemotherapy efficacy. Radiation Therapy and Hyperthermia The rationale for combining hyperthermia with radiation therapy is based on a variety of synergistic effects. For one, the areas of radiation resistivity and heat sensitivity are complimentary. While cells in ▶S-phase are typically radioresistant, this is the most heat sensitive phase of the cell cycle. Also, hypoxic cells are typically three times more resistant to radiation than normoxic cells, whereas there is no difference in thermal sensitivity based on cellular oxygenation status. Furthermore, hyperthermia can augment radiation response by causing reoxygenation of tumor tissue and inhibiting sublethal repair through inactivation of critical DNA repair pathways. Clinically, the therapeutic gain of combining radiation therapy and hyperthermia is determined by the Thermal Enhancement Ratio, or TER. The TER is defined as the ratio of radiation dose to achieve an isoeffect for radiation divided by the radiation dose to achieve the same effect with combined radiation and hyperthermia. Typical TER values for local tumor control are greater than one, and are typically smaller

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Hyperthermia

Hyperthermia. Figure 3 Example of thermal cell survival curves (on the left), plotted as surviving fraction over time at a specific temperature. On the right, the Arrhenius plot is shown for the data. The breakpoint is observed at 43°C.

Hyperthermia. Figure 4 Physiologic benefits of low temperature (40–43°C) hyperthermia and the rationale behind synergistic radiation and chemotherapy uses.

for normal tissue damage, suggesting therapeutic gain for a combined hyperthermia and radiation course over radiation alone. Chemotherapy and Hyperthermia Hyperthermia also exhibits synergism with some chemotherapeutic agents, through increasing cellular uptake, oxygen radical production, and DNA damage, while inhibiting DNA repair. Chemotherapeutic drugs that have been shown to have heightened efficacy when combined with hyperthermia include ▶cisplatin, ▶nitrogen mustards, doxorubicin (▶Adriamycin), ▶nitrosoureas, ▶bleomycin, ▶mitomycin C, and hypoxic cell sensitizers. While this list of drugs that are synergistic

with hyperthermia are many, there are also those that have not shown interaction when combined with heat, such as ▶etoposide and the ▶vinca alkaloids. In addition to showing additive effects with traditional chemotherapeutic agents, hyperthermia also offers an avenue for exploitation of innovative drug delivery strategies. Liposomes (▶Liposomal Chemotherapy) are nanoparticle lipid vesicles that can be loaded with a variety of high concentration chemotherapeutics. These liposomes show enhanced extravasation following hyperthermia due to increased permeability of the vasculature. Furthermore, this enhanced accumulation is tumor specific, due to the discrepancy in degree of permeability increase between

Hypertrophy of Male Breast

Hyperthermia. Figure 5 Illustration of the principles underlying thermosensitive liposome efficacy. Thermosensitive liposomes release their contents upon heating to a desired temperature, and release drug both within the tumor vasculature and after extravasation into tumor tissue.

tumor and normal blood vessels. Not only does hyperthermia increase overall liposomal extravasation, but novel thermosensitive liposomes have been developed with lipid bilayers that degrade upon reaching a thermal transition temperature, typically designed to release in a range of 40–43°C. This allows for both targeted and triggered drug release into tumor tissue. Figure 5 shows an illustration of thermosensitive liposomes extravasating into tumor tissue and releasing their contents upon heating. There are a variety of factors that should be considered when combining hyperthermia and chemotherapy. For one, response may vary based on the hypoxic and pH parameters of the tumor. Additionally, a very notable effect of hyperthermia is its influence on tumor resistivity to certain drugs; hyperthermia can induce at least partial reversal of drug resistance with a variety of drugs, most notably cisplatin, ▶melphalan, nitrosoureas, and doxorubicin. Finally, sequencing of hyperthermia and drug delivery may greatly influence efficacy of treatment. Most drugs exhibit optimal results when heat and drug are delivered concomitantly, or when drug is given immediately prior to heating; some exceptions include 5-FU (▶Fluorouracil) and a few other antimetabolites. Clinical Trials Phase III clinical trials, particularly those comparing radiotherapy (RT) alone to RT + HT, have yielded significant positive results for a variety of cancers. Both superficial and deep tumor sites have been investigated, as well as both palliative and curative treatment goals. Statistically significant improvement in complete response (CR) rates with HT + RT versus RT alone have

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been demonstrated with many cancers, most notably chest wall recurrences of breast cancer, stage III and IV head and neck, metastatic melanoma, bladder, and cervical carcinoma. Most importantly, improvements in survival have also been shown in cervix, head and neck, glioblastoma, esophageal cancers. While many trials have been performed validating the use of hyperthermia with radiotherapy, fewer exist for chemotherapy (CT) and hyperthermia. However, phase II trials have illustrated improved treatment efficacy of CT + HT in sarcomas and breast carcinomas, compared with historical controls. Additionally, the use of tri-modality (CT, RT, and HT) treatments are being investigated, with encouraging results in phase II trials of locally advanced rectal and cervical cancers and a phase III trial for esophageal cancer.

References 1. Dewhirst MW, Jones E, Samulski T et al. (2006) Hyperthermia. In: Kufe DW, Bast RC Jr, Hait WN, Hong WK, Pollock RE, Weichselbaum RR, Holland JF, Frei III E (eds) Cancer medicine. BC Decker, Hamilton, pp 549–561 2. Jones EL, Samulski TV, Vujaskovic Z et al. (2004) Hyperthermia. In: Perez CA, Brady LW, Halperin EC, Schmidt-Ullrich RK (eds) Principles and practice of radiation oncology. Lippincott Williams and Wilkins, Philadelphia, PA, pp 699–735 3. Horsman MR, Overgaard J (2007) Hyperthermia: a potent enhancer of radiotherapy. Clin Oncol 19(6):418–426 4. Ponce AM, Vujaskovic Z, Yuan F et al. (2006) Hyperthermia mediated liposomal drug delivery. Int J Hyperthermia 22(3):205–213 5. Van der Zee J (2002) Heating the patient: a promising approach. Ann Oncol 13:1173–1184

Hypertrophy Definition Increase in size of an organ or tissue due to an increase in the size of cells while the number stays the same. ▶Peroxisome Proliferator-Activated Receptor

Hypertrophy of Male Breast ▶Gynecomastia

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Hypervariable Region

Hypervariable Region Definition

Regions of the heavy or light chains of ▶immunoglobulins in which there is considerable sequence diversity within that set of immunoglobulins in a single individual. These regions specify the antigen affinity of each antibody.

Hypomethylation of DNA M IGUEL A. P EINADO Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain

Synonyms DNA demethylation; DNA undermethylation

Definition

Hypodiploidy Definition Having a chromosome number that is less than the normal diploid number. ▶Acute Lymphoblastic Leukemia

DNA hypomethylation refers to the loss of the methyl group in the 5-methylcytosine nucleotide. Methylation is a natural modification of DNA, and mainly affects the cytosine base (C) when it is followed by a guanosine (G) in mammals ▶(Methylation). The term hypomethylation can be applied to describe the unmethylated state of most CpG sites in a specific sequence that is normally methylated, or as a general phenomenon affecting the bulk of the genome; this is a decrease in the proportion of methylated versus unmethylated cytosines.

Characteristics

Hypoechoic Definition The echo of the tissue is low compared to the adjacent area. ▶Hepatic Epithelioid Hemangioendothelioma

Hypogammaglobulinemia Definition Abnormally low levels of immunoglobulins.

Hypomethylation Definition

A reduced level of CpG island ▶methylation relative to the normal tissue specific pattern. ▶Methylation-Controlled J Protein (MCJ) ▶CpG Islands

In human, DNA methylation mainly occurs at CpG sites. Up to 80% of all CpG sites in human DNA are methylated. However, this methylation occurs primarily in areas where CpG density is low, or at repeat DNA sites, such as Alu elements. CpG islands are regions where CpG density is high and most of them are unmethylated. Patterns of DNA methylation have been linked to control of gene expression, maintenance of chromosomal integrity, and in regulation of DNA recombination in mammals. Methylation in a gene promoter region is generally associated with gene silencing. Heavily methylated DNA replicates later than nonmethylated DNA, and late replication is associated with the formation of inactive chromatin, which facilitates transcriptional silencing of noncoding regions. Global hypomethylation is typical in aging cells, as well as in neoplasia, where it is an early event. Early studies in the eighties already identified a depletion of the methyl-cytosine content as a landmark in colorectal cancer and other types of tumors. DNA hypomethylation has been shown to promote tumorigenesis in murine colon and liver and was included as an early event in a genetic model for colorectal tumorigenesis. Different investigations sustain a causal link between DNA hypomethylation and genetic instability, reporting an association between defects in DNA methylation and aneuploidy in human colorectal cancer cell lines, increased chromosomal rearrangements in hypomethylated centromeric regions in mitogen-stimulated cells from individuals affected with immunodeficiency, centromere instability and facial anomalies (ICF

Hypoxia

syndrome), and an increased mutation rate owing to DNMT1 deficiency in murine embryonic stem cells and in murine somatic cells. Moreover, DNMT1 deficiency also results in constitutive chromosomal instability in a human colon cancer cell line. Decreased methylation levels in LINE sequences correlate with losses of heterozygosity on discrete chromosomal loci in colorectal carcinomas and other studies have demonstrated that DNA hypomethylation precedes genomic damage in human gastrointestinal cancer.

References 1. Ehrlich M (2002) DNA methylation in cancer: too much, but also too little. Oncogene 21:5400–5413 2. Feinberg AP, Tycko B (2004) The history of cancer epigenetics. Nat Rev Cancer 4:143–153

Hypospadias Definition A congenital defect of the ventral surface of the penis which causes abnormal urethral opening proximal to its normal location. ▶Nephroblastoma

Hypothalamus Definition A region of the brain below the cortex and the cerebrum that regulates numerous important body functions and makes hormones that impact pituitary function. ▶Prolactin

Hypoxia L AWRENCE B. G ARDNER The NYU Cancer Institute, New York University School of Medicine, New York, NY, USA

Synonyms Anoxic; Anaerobic

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Definition Being oxygen deprived.

Characteristics Cellular hypoxia is a common stress in normal development and numerous pathological conditions, including cancer. By the time a tumor has grown to a detectable size, poor and disordered ▶angiogenesis, leaky vessels, and high interstitial tumor pressure all result in significant tumor hypoxia. Studies in human tumor ▶xenografts reveal a mean pO2 of T mutations at codon 249 in ▶hepatocellular carcinoma of ▶HBV chronic carriers; ▶ultraviolet radiation and CC to TT tandem mutations in nonmelanoma skin cancer; and cigarette smoke and G>T transversions in ▶lung cancer of smokers (▶tobaccorelated lung cancer). Specific mutation patterns in TP53 may thus help identify environmental exposures that may be involved in ▶carcinogenesis.

Database Structure and Content The IARC TP53 Database contains data that are extracted from peer-reviewed publications or bioinformatics databases and curated manually. The core database is a relational database that integrates data on somatic mutations in sporadic cancers, on germline mutations in familial cancers, on TP53 gene status of cell-lines, on polymorphisms in human population and on functional assessment of mutations. It is organized around a “gene variation module” to which five modules are connected: a “somatic module,” a “germline module,” a “function module,” a “polymorphism module” and a “cell-line module” (Fig. 1). The database is a relational database in which different sets of data are integrated in a single scheme. The structure and content presented corresponds to the R11 (October 2006) release of the database that is maintained in SQL sever. This database design and content allow, for every single gene variation, to retrieve data on its functional and structural impact, and data related to its expression in a somatic, cell-line or germline context. Details on database contents and annotations are available at http://www-p53.iarc.fr/ Help.html. The “gene variation module” contains all possible single nucleotide substitutions in the coding sequence and splice sites of TP53, plus all other sequence alterations that have been reported in human cancers, each alteration being a unique entry. Gene variation are described at the DNA and protein levels and are annotated with structural and functional information related to the position of the mutation in the protein sequence (functional domain, residue function, structural motif, solvent accessibility of residue). For missense mutations, classifications based on the predicted or experimentally assessed functional impacts are also available. The “▶polymorphism” includes common gene variations reported in publications or extracted from ▶SNP databases, with links to databases that provide information on population frequency or disease associations. The “somatic and germline modules” contain data related to the somatic or germline expression of mutations respectively, with common set of dictionaries used to annotate pathological, clinical and patient information. The “cell-line module” contains TP53 gene status of human cell-lines with links to the ATCC catalog (cell-line provider). The “Function module” contains experimental data obtained on the activities and properties of p53 mutant proteins when transfected and over-expressed in human or yeast cells. Each entry in this module corresponds to the results of a set of experiments performed with one mutant in a specific cell-type. Experimental assays that have been included were performed in yeast or human cells and were designed to (i) measure the transactivation activities of mutant proteins on reporter genes placed

IARC TP53 Database

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IARC TP53 Database. Figure 1 Structure and contents of the IARC TP53 database (R11, 2006).

under the control of various p53 response-elements, (ii) test the capacity of mutant proteins to induce cell-cycle arrest or apoptosis, (iii) exert dominant-negative effect over the wild-type protein, be temperature sensitive, or display various activities that are independent and unrelated to the wild-type protein (gain of function). A specific vocabulary has been implemented to describe experimental results in a standard format. The detailed annotation system used in all modules is described in the online user guide at http://www-p53. iarc.fr/Help.html. Web Site and New Search Tools The IARC TP53 web site (http://www-p53.iarc.fr/) provides a search interface for the core database and includes a comprehensive user guide, a slide-show on TP53 mutations in human cancer, protocols and references for sequencing TP53 gene, and links to relevant publications and entries to bioinformatics and cancer databases. The database interface allows the download of all modules and propose various tools for the selection, analysis and downloads of specific sets of data according to user’s query (Fig. 2). Mutations reported in specific types of cancers and/or population groups can be analyzed with graphs that display their type by base substitutions (mutation patterns), codon distribution,

position within the 3D structure of p53 DNA-binding domain, and predicted or observed effect on protein (function patterns). Other graphs can be drawn to display the types of tumor associated with specific mutations expressed in a somatic or germline context, and to display the prevalence of mutations found in specific tumor types and/or population groups. Other tools include: a mutation validation tools that allows the retrieval of all data available in the database for a specific DNA variation (frequency as a somatic or germline mutation, predicted and observed functional impact); a search option to retrieve experimental data on functional properties of mutant proteins; a search option to retrieve cell-lines for which TP53 gene status is known. Several options and tools are available to retrieve and analyze specific sets of data from the core database according to user defined queries. Different types of graphs are implemented to analyze data and raw data can be downloaded as tables. All datasets can be entirely downloaded as tab-delimited text files. The search page is available at http://www-p53.iarc.fr/ P53main.html. Recommendation to Users The IARC TP53 database is exclusively based on peer-reviewed publications. Trends in reporting and

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IARC TP53 Database

IARC TP53 Database. Figure 2 Search system for the analysis of the IARC TP53 database.

publishing mutations have thus a strong influence on the data included in the database. Studies from which data are extracted have diverse designs and diverse way of reporting mutations and related information. This diversity requires an effort of standardization of

annotations and affect database analysis. It thus important that users are aware of the limitations and possible biases that may affect their analysis of the database. These bias are described in details at http:// www-p53.iarc.fr/Help.html#Recommendations.

Id Proteins

The IARC TP53 database is a free service to the scientific community, and contributions from researchers and journal editors are most welcome to help us develop this important resource for cancer research.

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IC50 Definition

References 1. Hainaut P Hollstein M (2000) p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77:81–137 2. Hernandez-Boussard T, Montesano R, Hainaut P (1999) Sources of bias in the detection and reporting of p53 mutations in human cancer: analysis of the IARC p53 mutation database. Genet Anal 14:229–233 3. Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M (2007) Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat 28:622–629 4. Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310

50% inhibitory concentration; Refers to the concentration of a drug that reduces a biochemical activity (e.g. an enzymatic activity) or cellular parameter (such as cell multiplication) to 50% of its normal value in the absence of the inhibitor. ▶Small Molecule Screens

ICAMs I Definition

IkB Definition Are initially described as proteins that inhibit the function of transcription factor ▶NF-κB (inhibitor of κB). Meanwhile, IκBs extended to a protein family of seven members primarily structural related by the presence of several ▶ankyrin repeats in their central domain. Through these motifs, they interact with and regulate NF-κB function in various ways.

Intercellular ▶adhesion molecules are cell-surface ligands for the ▶leukocyte integrins and are crucial in the binding of lymphocytes and other leukocytes to certain cells, including ▶antigen-presenting cells and ▶endothelial cells. They are members of the immunoglobulin superfamily. ICAM-1 is the most prominent ligand for the integrin CD11a:CD18 or LFA-1. It is rapidly inducible on endothelial cells by infection, and plays a major role in local ▶inflammatory response. ICAM-2 is constitutively expressed at relatively low levels by endothelium. ICAM-3 is expressed only on leukocytes and is thought to play an important part in adhesion between T cells and antigen-presenting cells, particularly ▶dendritic cells. ▶Integrin Signaling

▶BCL3 ▶Nuclear Factor kappa-B

Id1 IkBa

Definition

Member of the group of ▶Id Proteins.

Definition MAD-3, pp40, RL/IF-1 and ECI6; Is one of a family of proteins that are the natural inhibitors of ▶NF-κB. It acts by complexing to NF-κB in the cytoplasm of unstimulated cells. When IκBα is inactivated, NF-κB is allowed to enter the nucleus. ▶Hodgkin Disease, Clinical Oncology ▶Nuclear Factor kappa-B

Id Proteins Definition Inhibitor of DNA (Id) binding proteins are a family of related nuclear ▶helix-loop-helix-proteins implicated

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Idarubicin

in the control of differentiation and cell cycle progression. Id nuclear proteins interact with ▶transcription factors and prevent them from binding to DNA. The primary targets of Id proteins are the basic helix-loophelix (bHLH) transcription factors, which regulate celltype-specific gene expression and expression of ▶cell cycle regulatory genes. Generally, they act as positive regulators of cell growth and as negative regulators of differentiation. Id proteins lack a basic DNA-binding domain; therefore, heterodimers between Id and bHLH proteins cannot bind to DNA. This mode of regulation is referred to as dominant-negative. Id proteins act as dominant-negative antagonists of other helix-loop-helix transcription factors; Id1, Id2, Id3, Id4, E-box. . Id1, inhibitor of DNA binding 1, is a protein of 154 amino acids and 16 kD. The gene maps to chromosome 20 band q11. . Id2, inhibitor of DNA binding 2, is a protein of 134 amino acids and 14 kD. It is expressed in most early fetal tissues but not in the corresponding mature tissues. The gene maps to 2p25. . Id3, inhibitor of DNA binding 3, also known as Heir1 is a protein of 119 amino acids and 12 kD. It is expressed abundantly in lung, kidney, and adrenal gland, but lacking in adult brain. The gene maps to 1p36, a region frequently deleted in human cancers including neuroblastoma. . Id4, inhibitor of DNA binding 4, is a protein of 161 amino acids and 16 kD. The gene maps to 6p22-21. ▶E-box ▶Helix-Loop-Helix Domain

Idiopathic Myelofibrosis ▶Primary Myelofibrosis

Idiotype Definition Is the collection of all antigenic determinants (idiotopes) contained within the variable regions of both heavy and light chain of an antibody molecule. The sum of the antigenic determinants of epitopes that are encoded in the variable regions of the ▶immunoglobulins heavy and light chain. ▶Idiotype Vaccination ▶B-cell Tumors

Idiotype Vaccination M AURIZIO B ENDANDI University Clinic and Center for Applied Medical Research, University of Navarra, Pamplona, Spain

Synonyms Anti-idiotype vaccination; Idiotypic vaccination

Idarubicin Definition

Is a semisynthetic ▶anthracycline antibiotic derived from ▶daunorubicin. ▶Adriamycin

Idiopathic Bone Infarction

Definition

▶Idiotype vaccination is an ▶immunotherapy procedure based on the fact that save for its very early stage of differentiation, each clone of B lymphocytes features a specific antibody on the cell surface. The most variable portion of this antibody is a unique feature of the corresponding clone. Its natural function is that of specifically recognizing an antigen, but it can also be used as an antigen (idiotype) itself, that is as a potential vaccine target and tool. The most relevant context in which this second function is exploited is idiotype vaccination as a treatment for human ▶B-cell lymphoma.

Definition

Characteristics

▶Bone Tumors

Field of Application The vast majority of B-cell lymphomas consist of the clonal expansion of neoplastic B-cells, all featuring the same surface antibody and consequently the same

Localized bone ▶necrosis and its associated bone marrow for unknown reasons.

Idiotype Vaccination

idiotype. However, each patient’s tumor presents with a different, patient- and tumor-specific idiotype. Therefore, an individualized, custom-made idiotype vaccine must be produced for each patient. Most of what we know about idiotype vaccination in human B-cell lymphoma derives from studies conducted in an indolent subset called ▶follicular lymphoma. Far less has been instead concluded so far as to whether or not the same approach could be successful in other B-cell malignancies, such as ▶mantle cell lymphoma, ▶multiple myeloma, and ▶chronic lymphocytic leukemia, or even in certain solid tumors, whenever an anti-idiotype ▶monoclonal antibody structurally mimics tumor-associated antigens other than antibodies. Nevertheless, idiotype vaccination stands out as the most successful human cancer vaccine, since over the last 20 years has provided the first formal proofs of principle concerning biological efficacy, clinical efficacy, and clinical benefit of such a procedure. Formulation Although idiotypic vaccination has been tested in humans with different formulations, such as soluble protein idiotype associated with an immunogenic carrier and an immunologic adjuvant, ▶dendritic cells pulsed with the soluble protein idiotype, or idiotype ▶DNA vaccine, most clinical results and all proofs of principle have been obtained using the first of these three options. In particular, the most successful idiotype vaccine formulation consists of soluble protein idiotype, that is the patient- and tumor-specific antigen, which is conjugated with keyhole limpet hemocyanin (KLH), the immunogenic carrier, and administered together with granulocyte-macrophage colony-stimulating factor (GM-CSF), the immunologic adjuvant. Production While KLH and GM-CSF are commercially available, soluble protein idiotype needs to be produced one patient at the time through either hybridoma or ▶recombinant technology. In the former case, each patient’s tumor cells, which are capable of producing the idiotype, but not to release it, are fused with a cell line that vice versa can release an idiotype, but is unable to produce it. The ▶hybridoma resulting from the fusion process features both functions, can be cultivated in vitro, and releases therapeutic amounts of the patient- and tumor-specific, soluble protein idiotype. In the latter scenario, the genetic information encoding for the idiotype is introduced by means of a vector inside mammalian, insect, plant, or bacteria cells, which will ultimately replace the hybridoma as a factory for soluble protein idiotype production. Clinical Aspects Idiotype vaccination has provided the first formal proof of biological efficacy in 1992, when scientists at Stanford University showed that patients with follicular lymphoma were capable of developing a specific, anti-idiotype

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immune response after receiving idiotype vaccination. Seven years later, researchers at the National Cancer Institute proved clinical efficacy of idiotype vaccination by showing that in most follicular lymphoma patients who had received it, the immune system had become capable of killing in vivo tumor cells that had survived prevaccine ▶chemotherapy. Finally, in 2006 clinical efficacy of idiotype vaccination was proved by showing that all follicular lymphoma patients who respond to the vaccine from an immunologic standpoint experience a significant prolongation of their disease-free survival. Idiotype vaccination’s procedure typically consists of a monthly, subcutaneous injection of 0.5 mg of idiotype conjugated with 0.5 mg of KLH, administered together with 125 mcg of GM-CSF. The same dose of GM-CSF alone is then given daily over the following 3 days at the same site of the complete vaccine formulation delivery. After 5 or 6 monthly doses, further boosts every 2–3 months are becoming increasingly popular and recommended. Idiotype vaccination’s side effects are mild and mostly local, if at all present. Flu-like symptoms are rare and self-limiting. Open Questions To date, a number of questions remain open with respect to idiotype vaccination. For instance, from a clinical standpoint, we do not know whether this form of active ▶immunotherapy has the potential to cure or just to control the disease in lymphoma patients who respond to it. Nor do we know whether patients with B-cell malignancies other than follicular lymphoma may benefit from it. The former question implies that it remains currently impossible to determine whether the administration of boosts should or could be stopped in patients who have responded to the vaccine from an immunologic point of view, irrespective of whether that immune response persists detectable, and remain free of disease. The latter question implies instead that only through welldesigned ▶clinical trials it might be possible to test idiotype vaccination in settings other than follicular lymphoma. In particular, it has to be reminded that typical randomized studies may fail to serve the purpose of proving the clinical benefit of a customized form of immunotherapy. In this respect, surrogate endpoints such as those defining minimal residual disease also need to be used with caution. For instance, in the very case of follicular lymphoma, both ▶bcl-2 rearrangement assessed by molecular fingerprinting through qualitative and quantitative polymerase chain reaction and tumor cell phenotype assessed through ▶flow cytometry not always correlate with immune responses and clinical outcome. All in all, it is possible that the currently ongoing randomized clinical trials will be at least able to shed some light on whether it may be possible to match the financial concerns of pharmaceutical companies

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with the peculiar logistic requirements of a customized treatment like idiotype vaccine. As for the adequate timing for treating patients with this immunotherapeutic approach, it seems to have become ever clearer that the best clinical setting to use idiotype vaccination is a good quality clinical complete response. This prevaccine result can be achieved nowadays through a variety of old and new therapeutic approaches such as standard or high-dose chemotherapy, cold or passive immunotherapy using ▶radioimmunoconjugates. However, it is likely that in lymphoma patients selected to later undergo idiotype vaccination, prevaccine treatment should privilege agents and procedures with the lowest immune suppressive potential. At the opposite side of the widening spectrum of possible applications of idiotype vaccination stand the anecdotal attempts conducted with healthy donors undergoing this totally safe procedure with the myeloma-specific soluble protein idiotype obtained from their sibling recipient before ▶hematopoietic stem cell allotransplant. Clinical and biological results remain incompletely understood, though extremely appealing from a purely scientific point of view. Another crucial context in which idiotype vaccination specialists still struggle is that of defining the relevance of idiotype vaccine-induced humoral and cellular responses. In fact, it is not yet clear whether, though both desirable, just either of them is required for patients to experience clinical benefit. The question is of no little importance, considering that in nearly half of the patients with any vaccine-induced, idiotype-specific immune response, either type of immune response is not detectable. Currently, humoral responses, that is those featuring vaccine-induced anti-idiotype antibodies, are assessed and monitored in the lab by a single, relatively standardized method, whereas at least half a dozen of nonstandardized techniques are used in different labs worldwide to assess and monitor vaccine-induced cellular responses

Idiotypic Vaccination ▶Idiotype Vaccination

IDO ▶Indoleamine 2,3-Dioxygenase

I-FLICE ▶FLICE Inhibitory Protein

IFN Definition

▶Interferon are ▶cytokines that can induce cells to resist viral replication. Interferon-α (IFN-α) and interferon-β (IFN-β) are produced by ▶leukocytes and ▶fibroblasts, respectively, as well as by other cells, whereas interferon-γ (IFN-γ) is a product of CD4 TH1 cells, ▶CD8 T cells, and ▶NK cells. IFN-γ has its primary action the activation of ▶macrophages.

References 1. Kwak LW, Campbell MJ, Czerwinski DK et al. (1992) Induction of immune responses in patients with B-cell lymphoma against the surface-immunoglobulin idiotype expressed by their tumors. N Engl J Med 327:1209–1215 2. Bendandi M, Gocke CD, Kobrin CB et al. (1999) Complete molecular remissions induced by patientspecific vaccination plus granulocyte-monocyte colonystimulating factor against lymphoma. Nat Med 5: 1171–1177 3. Inogés S, Rodríguez-Calvillo M, Zabalegui N et al. (2006) Clinical benefit associated with idiotypic vaccination in follicular lymphoma. J Natl Cancer Inst 98:1292–1301 4. Bendandi M (2006) Clinical benefit of idiotype vaccines: too many trials for a clever demonstration? Rev Recent Clin Trials 1:67–74

iFOBT ▶Fecal Immunochemical Test

IgE-Mediated Hypersensitivity ▶Allergy

Illegitimate Recombination

IGF Definition

▶Insulin- Like Growth Factors. ▶Insulin Receptor

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IISRE Definition

A type IIS “stretch” restriction endonuclease (IISRE) is a restriction enzyme that binds double-stranded DNA at a particular sequence and then cleaves double-stranded DNA at a fixed distance and direction from its binding site without regard to sequence specificity. Examples include BsgI, BpmI, Eco57I, FokI, and HphI. ▶Combinatorial Selection Methods

IGF-2 Definition Insulin-like growth factor 2. ▶Insulin Receptor ▶Insulin-Like Growth Factors

IL-1 Definition

▶Interleukin-1 ▶Photodynamic Therapy

IGF-I Definition Insulin-like growth factor-1. ▶Insulin Receptor-1 ▶Insulin-Like Growth Factors

IGs Definition

IL-12 Definition

▶Interleukin-12 is the product of ▶monocytes such as ▶macrophages, ▶neutrophils and ▶dendritic cells. It is an early component of the innate immune response and serves to activate ▶natural killer cells and assist in the differentiation of Th cells to the Th1 phenotype. Since both these cells are a source of ▶IFN-γ, IL-12 is a key regulator of IFN-γ production by the immune system. ▶Immunoediting

▶Immunoglobulin Genes.

Illegitimate Recombination IHC ▶Immunohistochemistry

Definition Recombination with nonhomologous regions of DNA, each of the polynucleotide strands of dsDNA being

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Image Cytometry

religated with DNA from different regions of the same or different chromosomes. ▶Chromosomal Translocation ▶Fusion Genes

Characteristics

Image Cytometry Definition Involves measurement of the shape and density of cellular structures to assess anatomic pathology of a given cell. Modern approaches to image ▶cytometry incorporate computational analysis to infer disease state based on hundreds of measurements from each individual cell. ▶Malignancy-Associated Changes

Imaginal Discs Definition Monolayer sac-like epithelial tissues in Drosophila larvae that later give rise to adult structures. ▶Lats in Growth Regulation and Tumorigenesis

Imatinib B RIAN J. D RUKER Oregon Health & Science University Cancer Institute, Portland, OR, USA

Synonyms Imatinib mesylate; Gleevec; ▶STI-571; CGP57148

(▶Platelet-derived growth factor receptor). It has significant anti-tumor activity in chronic myeloid leukemia and ▶gastrointestinal stromal tumors.

Glivec;

STI571;

Definition Is a small molecular weight compound that inhibits ▶tyrosine kinases including ▶ABL, ▶KIT (▶Kit/stem cell factor receptor in oncogenesis) and PDGFR

Imatinib (Gleevec, Glivec, formerly STI571 or CGP57148) is a tyrosine kinase inhibitor with activity against all of the ABL tyrosine kinases including ▶BCR-ABL, ABL, v-ABL, and ARG (Abelson-related gene). Besides the ABL tyrosine kinase, other kinases inhibited by imatinib are the platelet-derived growth factor receptor alpha (PDGFRA) and beta (PDGFRB) and KIT. Given the critical role of tyrosine kinases in the regulation of cellular growth and known activating mutations that cause several cancers, it was hypothesized that specific inhibitors of these protein kinases might be effective anticancer agents. Beginning in the late 1980s, scientists at Ciba Geigy (now Novartis), under the direction of N. Lydon and A. Matter, performed high throughput screens of chemical libraries searching for compounds with kinase inhibitory activity. From this time-consuming approach, a lead compound was identified. Its inhibitory activity against the PDGFR was optimized by synthesizing a series of chemically related compounds and analyzing their relationship between structure and activity (▶Drug design). The most potent molecules in the series were dual inhibitors of the PDGFR and ABL kinases. Of the several compounds generated from this program, imatinib emerged as the lead compound for clinical development based on its superior in vitro selectivity against ▶CML cells and its drug-like properties, including pharmacokinetic and formulation properties (▶Pharmacokinetics; ▶pharmacodynamics). BCR-ABL and CML as a Target for Imatinib CML is characterized by the presence of the BCR-ABL ▶fusion gene and protein. It arises from a ▶reciprocal translocation between the long arms of chromosomes 9 and 22 (▶Chromosome translocations) that generates a shortened chromosome 22 termed the ▶Philadelphia chromosome. This was the first consistent chromosome abnormality associated with a human cancer. The (9;22) translocation results in fusion of the ABL ▶oncogene from chromosome 9 with sequences from chromosome 22, the breakpoint cluster region (BCR), giving rise to a chimeric BCR-ABL gene. This fusion gene is transcribed and translated into a protein that functions as a constitutively activated tyrosine kinase. BCR-ABL induces a CML-like syndrome when introduced into bone marrow cells of mice (▶Mouse models), confirming its causative role in the human disease. All of the transforming activities of BCR-ABL are dependent on its tyrosine kinase activity, thus a specific inhibitor of

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counts, ▶Myelosuppression, fluid retention, diarrhea, nausea, muscle and joint aches, and skin rashes.

Imatinib. Figure 1 Schematic representation of the mechanism of action of the BCR-ABL tyrosine kinase and its inhibition by STI571 (imatinib).

this kinase would be predicted to be an effective and selective therapeutic agent for CML (Fig. 1). In a pivotal set of ▶preclinical testing, imatinib was shown to suppress the proliferation of BCR-ABLexpressing cells in vitro and in vivo. In colony-forming assays of peripheral blood or bone marrow from patients with CML, imatinib caused a 92–98% decrease in the number of BCR-ABL colonies formed, with minimal inhibition of normal colony formation. Activity of Imatinib in CML Given the central role of the BCR-ABL tyrosine kinase in causing CML and the favorable preclinical profile of imatinib, CML was selected as the initial disease in which to test imatinib. In a standard dose-escalation Phase I study (▶Clinical trial) conducted in patients with CML, imatinib was well tolerated. Significant clinical benefits were observed at doses of 300 mg per day and above. In patients with chronic-phase disease who were interferon resistant or intolerant, 53 of 54 (98%) had their previously abnormal blood counts return to normal, typically within 4–6 weeks after beginning imatinib. Ninety percent of these responses lasted beyond 1 year. In patients with myeloid ▶blast crisis, the most advanced phase of the disease, 21 of 38 (55%) patients responded, with 18% having responses lasting beyond 1 year. Imatinib rapidly advanced through Phase II and III testing for patients with CML and was FDA-approved in May 2001. It is now the standard initial therapy for patients with CML. A 5-year update of imatinib as initial therapy for patients with CML demonstrates an overall survival of 89%. At 5 years, an estimated 93% of patients remain free from disease ▶progression to advanced phases of the disease: accelerated phase or blast crisis. Most of the side effects of imatinib are classified as mild to moderate and include low blood

Mechanisms of Relapse/Resistance to Imatinib Response rates to imatinib in CML patients with chronic-phase disease are quite high and, thus far, have been durable. Response rates are also quite high in patients with advanced-phase disease, but relapses, despite continued therapy with imatinib, are common. In the largest studies of resistance or relapse, several consistent themes have emerged. In the majority of patients who respond to imatinib but subsequently relapse while remaining on therapy, the BCR-ABL kinase has been reactivated. BCR-ABL kinase activity was analyzed by assessing tyrosine phosphorylation of CRKL, a direct substrate of the BCR-ABL kinase, and the major tyrosine phosphorylated protein in samples from patients with CML. In these studies, between 50 and 90% of relapsed patients have a BCR-ABL point mutation located in one of over 40 different amino acids scattered throughout the ABL kinase domain. Some other patients have amplification of BCR-ABL at the genomic or transcript level. In contrast, in patients with primary resistance – that is, patients who do not respond to imatinib therapy – BCR-ABL-independent mechanisms are most common. In patients who relapse as a consequence of reactivation of the BCR-ABL kinase, the BCR-ABL kinase remains a good target. Alternate ABL kinase inhibitors are already in clinical trials (▶Nilotinib, ▶dasatinib) and dasatinib is FDA-approved for patients with CML with imatinib resistance. There remains at least one imatinib-resistant mutation, T315I, that is not inhibited by either drug. Activity of Imatinib in Other Diseases Given the success of imatinib in CML, it was logical to try imatinib in other diseases where activated tyrosine kinases targeted by imatinib have causative roles. Thus, imatinib also has significant activity in patients with ▶acute lymphoblastic leukemia who are BCR-ABLpositive. Another tumor in which imatinib has shown activity is ▶gastrointestinal stromal tumor (GIST). GISTs are mesenchymal neoplasms that can arise in any organ in the gastrointestinal tract or from the mesentery or omentum. The majority of GISTs express KIT, and in 90% of cases, KIT activation is linked to mutations, usually involving exons 9 or 11. Published data suggest that the response rate of GISTs to single- or multi-agent ▶chemotherapy is less than 5%. In contrast, the response rate to imatinib as a single agent in patients with advanced GIST was 53–65% with another 19–36% of patients having disease stabilization. Mutational status helps predict response to imatinib in GIST patients. Patients whose tumors contain the most common activating KIT mutation exon 11 have a significantly higher partial response rate (83.5%) to imatinib therapy

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than did patients whose tumors had no detectable mutations in KIT. Patients whose tumor harbored a KIT exon 9 mutation did less well than those with an exon 11 mutation but responded better (response rate of 48.7%) and had a longer time-to-treatment failure than those with no detectable mutation. Similar to the situation in CML, many imatinibresistant tumors have acquired kinase mutations in the kinase domain of KIT. However, resistance mechanisms in GISTs are more heterogeneous than those seen in chronic-phase CML as some tumors actually lose KIT expression and other tumors become imatinib-resistant without acquisition of secondary kinase mutations. Again, similar to the situation with CML, novel KIT kinase inhibitors are being tested in patient with imatinib-resistant GIST and one of them, sunitinib, has been FDA-approved for this indication. Imatinib also has activity in neoplasms that are caused by oncogenic activation of PDGFRs (Plateletderived growth factor receptor). This includes the subset of patients with chronic myelomonocytic leukemia that results from fusion of the ▶EVT6 (TEL) and PDGFRB genes. Similarly, dermatofibrosarcoma protuberans, a low-grade sarcoma of the dermis, is characterized by a (17;22) translocation involving the COL1A1 and PDGF-B genes, which results in overproduction of fusion COL1A1-PDGF-BB ligand and consequent hyperactivation of PDGFRB. Patients with this tumor also respond to imatinib. Hypereosinophilic syndrome is another example of an imatinib-sensitive disease. In this case a PDGFRA fusion gene product (FIP1L1-PDGFRA) is the activated target. Conclusion The development of imatinib and its clinical application demonstrate an emerging paradigm in cancer therapy where the tumor is defined by molecular genetic abnormalities instead of the tissue of origin. It further demonstrates that effectiveness of cancer therapy when treatments target events critical to the growth and survival of specific tumors. (▶Molecular therapy).

References 1. Druker BJ, Tamura S, Buchdunger E et al. (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566 2. Druker BJ, Talpaz M, Resta DJ et al. (2001) Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 344:1031–1037 3. Heinrich MC, Corless CL, Demetri GD et al. (2003) Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 21:4342–4349

4. Druker BJ, Guilhot F, O’Brien SG et al. (2006) Five-year follow- up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 355:2408–2417 5. Druker BJ (2006) Circumventing resistance to kinaseinhibitor therapy. N Engl J Med 354:2594–2596

Imatinib Mesylate ▶Imatinib

Imiquimod Definition A small molecular drug that activates TLR7 and/or TLR8 agonist that is used as a prescription medication to treat ▶skin cancer (▶basal cell carcinoma, ▶Bowen disease, superficial squamous cell carcinoma, some superficial malignant melanomas and actinic keratosis) as well as genital warts.

Immature B Cells Definition

Are ▶B cells that have rearranged a heavy- and a lightchain V-region gene and express surface IgM, but have not yet matured sufficiently to express surface IgD as well.

Immediate Early Genes Definition IEGs; A class of approximately 100 structurally and functionally unrelated genes, whose transcription is induced through the phosphorylation and activation of pre-existing transcription factors such as ▶ELK-1 by intracellular signaling pathways like ▶MAP kinase. A hallmark of immediate early genes (IEGs) is that their transcription is independent from de novo protein

Immune Escape

synthesis, which distinguishes them from delayed early genes. Examples for IEG products include ▶FOS, a component of the ▶AP-1 transcription factor complex, and several members of dual specificity phosphatases, which act as negative feedback regulators of MAP kinases. ▶B-Raf Signaling

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Immune Adjuvants Definition Substances that are typically added to vaccines to enhance their immunogenicity. ▶Peptide Vaccines for Cancer ▶Adjuvant

Immediate Early Stress Response ▶Stress Response

Immune Complex Definition

Immortalization Definition Describes the acquisition by a eukaryotic cell line of the ability to grow through an indefinite number of divisions in culture. Cells are capable of indefinite proliferation (or unlimited lifespan) without any other changes in the phenotype necessarily occurring. In long lived multicellular organisms, immortality may be thought of as an abnormal escape from cellular ▶senescence. ▶Telomerase ▶Senescence and Immortalization

The binding of antibody to a soluble antigen forms an immune complex. Large immune complexes form when sufficient antibody is available to cross-link the antigen; these are readily cleared by the reticuloendothelial system of cells bearing Fc and complement receptors. Small, soluble immune complexes that form when antigen is in excess can be deposited in and damage small blood vessels.

Immune Deviation Definition

Immortalized

Is the deliberate polarization of an immune response from one dominated by TH1 to one dominated by TH2, or vice versa.

Definition

Refers to the status of ▶immortalisation of cells.

Immune Escape Immortalized Cells Definition Transformed cells that can grow and divide indefinitely in vitro. ▶Adult Stem Cells ▶Immortalization

J IAHUA Q IAN , TAMARA F LOYD, Y UFEI J IANG , A NAT O HALI , R AED S AMARA , H ONG YANG , S AMIR N. K HLEIF Cancer Vaccine Section, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Definition Is one of the hallmarks of cancer development and ▶metastasis. It is characterized by the lack of ability of

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the immune system to eliminate transformed cells prior to and after tumor development.

Characteristics Several mechanisms have been proposed and tested to explain cancer immune escape. It is now evident that both the host as well as the tumor play important roles in this phenomenon. The host’s contribution is manifested by the host’s ability to recognize to antigens expressed by tumor cells, a phenomenon known as “Host Ignorance”. It happens because of defects in both the innate and adaptive arms of the immune system. The tumor’s contribution is manifested by the adaptation of tumor cells to evade the immune systems or developing a ▶microenvironment that suppresses the immune system. Host Ignorance The innate arm of the immune system forms a first line of defense against cancers. ▶Macrophages, ▶dendritic cells, and ▶monocytes are essential in eliminating cancers. Another type of cell involved in the elimination of cancers is ▶NK cells. These cells are capable of recognizing and attacking cells that have down-regulated ▶MHC class I expression. This phenomenon is known as “missing self-recognition.” NK cells can effectively eliminate cells that have been altered due to cellular transformation, infection, or other carcinogens and cannot present new antigens. If these players involved in the ▶innate immune system are defective, cancer cells may escape tumor ▶immunosurveillance from not only innate immunity but also ▶adaptive immunity as NK cells, macrophages, NKT cells, play a role in the induction and enhancement of ▶adaptive immune responses. The adaptive arm of the immune system has also been shown to play a crucial role in recognizing and eliminating cancer cells. However, several defects have been shown to hamper the ability of this arm to combat tumor cells. 1. Defects in ▶antigen-presenting cell (APC). To elicit an effective immune response, antigens must be processed first by the APCs and presented to the immune system. Dendritic cells (DCs) are professional APCs and play an important role in regulation of adaptive immune response. Many tumor patients have shown defects in the DC compartment in terms of cell number and/or altered function. Tumor infiltrating DCs have defective surface expression of HLA and B7 costimulatory molecules, thus are likely to induce anergy rather than to stimulate tumor-specific T cells. Additionally, the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO), which has been implicated as one mechanism that helps to maintain maternal ▶tolerance toward the fetus during pregnancy, was recently recognized

as a potential mechanism of tolerance in malignancies. Expression of ▶IDO on APCs was observed in tumor draining lymph nodes, which may be indicative of the creation of a tolerogenic microenvironment. The molecular mechanism by which ▶IDO suppresses T cells is still being elucidated. It was suggested to be mediated by depletion of the essential amino acid tryptophan and by the generation of pro-apoptotic metabolites. 2. Negative immune regulation by suppressive cells. There are several types of suppressive cells in the immune system, including ▶regulatory T cells, ▶myeloid suppressor cells (MSCs) and NKT cells. They exist to keep the immune response under control and prevent autoimmunity. In addition, these cells have been shown to inhibit immune responses against tumor cells. (i) Regulatory T (Treg) cells are a subpopulation of CD4+ ▶T cells. Tregs are crucial for controlling autoreactive ▶T cell responses. At least two distinct subsets of Tregs have been identified: natural and inducible Tregs. Natural Tregs are T cells that arise in the thymus. They require direct cell contact with their target cells in order to exert their regulatory functions. Inducible Tregs arise in the periphery and exert their regulatory functions by the soluble cytokines IL-10 and TGF-β. The exact mechanisms by which Tregs exert their regulatory functions remain largely unknown. However, there is sufficient evidence, both in humans as well as in rodents, demonstrating that Tregs play crucial roles in suppressing anti-tumor responses. (ii) Myeloid suppressor cells were first identified by the expression of surface marker CD11b and Gr-1. Their suppressive activity was shown by inhibiting lymphocyte proliferation and inducing apoptosis in CD8 T cells. (iii) NKT cells are a unique sub-lineage of T cells. However, in contrast to conventional and regulatory T cells that recognize peptides in the context of MHC class I and II molecules, NKT cells recognize glycolipids presented by CD1d, a non-classical antigen presenting molecule. This difference in recognition means that NKT cells can recognize a class of antigens that conventional T cells cannot recognize. NKT cells have been shown to promote as well suppress immune responses to cancer. Currently, it is still unclear what factors determine their function. 3. Defective anti-tumor T cells. Another factor that leads to a failed immune response to tumor is cells dysfunction in ▶tumor-infiltrating lymphocytes (TILs). In a number of studies, TILs have been shown to have defective anti-tumor killing effect. This dysfunction may result from the ineffective granule exocytosis. TILs also have been reported to have defective cytokine production and low expression of B7.1 and B7.2 costimulatory molecules, suggesting anergy could occur.

Immune Escape of Tumors

Tumor Cell Adaptation To escape immune surveillance, tumor cells deploy several strategies. 1. Downregulation of MHC class I expression on cell surface. The presentation of antigen by ▶MHC class I is crucial for immune responses. By downregulating MHC class I molecules on the surface, tumor cells may become elusive targets for T cells thereby escape the destruction by ▶cytotoxic T lymphocytes (CTLs). 2. Suppression of the Immune System. Tumor cells have several “counterattack” methods to fight the immune system. This include: (i) Secretion of immunosuppressive cytokines such as IL-10, TGFβ, IL-13, etc. IL-10 interferes with the induction of anti tumor responses. It has been shown to block dendritic cell-mediated priming of T cells into CTL effectors in vitro, and its expression in serum appears to be associated with negative prognosis in certain cancers. ▶TGF-β is another cytokine that inhibits activation, proliferation, and activity of T lymphocytes. This ▶cytokine is often found at high levels in malignancies and is associated with a poor prognosis as well as lack of response to tumor immunotherapy. Both cytokines can be produced by the tumor cells themselves or by infiltrating stroma cells. Interestingly, TGF-β has also been shown to drive expansion of regulatory T cells, thus bringing together two evasion mechanisms. (ii) Up-regulation of tumor cells of Fas ligand on the surface to induce apoptosis of tumor killing effector cells. The deathinducing ▶FAS/APO-1/CD95 ligand (FasL/ CD95L) has been reported to be expressed on many human tumors of various origins. Expression of FasL in tumors implies that cancer cells are resistant to Fas-induced cell death, which preserves them from extermination by cytotoxic T cells and actively induces death of effector T cells. In addition to local defense, accumulating evidence suggests that the FasL expression may also be relevant for tumor progression and formation of metastasis. It was further described that tumor cells are able to release membrane vesicles (MV) or exosomes containing FasL which induce ▶apoptosis of activated T cells. This apoptosis-inducing pathway, via the release of FasL-positive MV, may indeed play a significant role in eliminating the most effective component of the anti tumor T cell response, and provides an explanation for the observed spontaneous T cell apoptosis in the peripheral circulation of cancer patients. (iii) Inhibition of effector cells by inhibitory ligands including PD-L1, CTLA-4 and LAG-3. Evidence suggests that tumor cells may evade T and NK cell recognition by receptor ligand interaction. CTLA-4 is a receptor predominantly

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found on T lymphocytes interacting with CD86 ligand. CTLA-4 is related to CD28 on T lymphocytes, but plays an opposite role. CTLA-4 may act at the level of TCR or CD28 signaling to inhibit T cell activation. Several studies provide evidence that CTLA-4 can up-regulate TGF-β expression and can attenuate CD28 signals on Treg expansion and survival. The precise mechanism of T cell inactivation by CTLA-4/CD86 signaling, however, remains uncertain. The PD-1 – PD-L pathway has been postulated to regulate the immune response in both lymphoid and non-lymphoid organs. PD-Ls are expressed in many tumors such as ▶ovarian cancer, ▶esophageal cancer, ▶kidney cancer and ▶brain cancer. It is suggested that tumor associated PD-Ls may promote T cell apoptosis and thus affect immune responses. Another inhibitory ligand associated with ▶MHC class II interactions is LAG-3 (lymphocyte activation gene-3). LAG-3-MHC class II could enhance the cross-talk between T cell and DC. In human cells, LAG-3 serves as a negative regulator of activated T cells. A comprehensive understanding of these inhibitory pathways will facilitate the design of effective ▶immunotherapy in the future.

References 1. Lang K, Entschladen F, Weidt C et al. (2006) Tumor immune escape mechanisms: impact of the neuroendocrine system. Cancer Immunol Immunother 55:749–760 2. Munn DH (2006) Indoleamine 2,3-dioxygenase, tumorinduced tolerance and counter regulation. Curr Opin Immunol 18:220–225 3. Rivoltini L, Canese P, Huber V et al. (2005) Escape strategies and reasons for failure in the interaction between tumour cells and the immune system: how can we tilt the balance towards immune-mediated cancer control? Expert Opin Biol Ther 5:463–476 4. Gajewski TF, Meng Y, Harlin H (2006) Immune suppression in the tumor microenvironment. J Immunother 29:233–240 5. Diefenbach A, Raulet D (2002) The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol Rev 188:9–21

Immune Escape of Tumors Definition Many tumors grow progressively despite an ongoing tumor-specific immune response. It has been experimentally shown that tumor cells adopt a variety of

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strategies to “escape” both ▶innate immunity as well as ▶adaptive immunity. For example, tumor cells downregulate or loose tumor antigens or important components of the antigen-processing machinery so that ▶T cells are no longer able to recognize peptides bound to major histocompatibility complex molecules on the cell surface. Tumor cells may also express ▶cytokines (such as IL-10 or ▶TGFβ) or cell surface molecules (such as ▶FAS or PD-1) which actively inhibit cellular anti-tumor immunity including the induction of T cell ▶apoptosis, T cell anergy and regulatory T cells. ▶Melanoma Vaccines ▶T-Regulatory Cells

(iii) the escape phase, in which tumors actively disable immune recognition by co-opting immune cells for growth, ▶angiogenesis and ▶invasion. ▶Melanoma Vaccines ▶Immunoprevention ▶Allergy

Immune System Definition

Immune Response

Refers to the tissues, cells, and molecules involved in ▶adaptive immunity, or sometimes the totality of host defense mechanisms.

Definition Is the response made by the host to defend itself against a pathogen.

Immunoassay Definition

Immune Responses to Autoantigens

A test using antibodies to identify and quantify substances. Often the ▶antibody is linked to a marker such as a fluorescent molecule, a radioactive molecule, or an enzyme.

▶Autoimmunity and Prognosis

Immunoblotting Immune Surveillance of Tumors Definition Definition The concept that the immune system is able to recognize and eliminate cancerous or pre-cancerous cells was first postulated in 1909 by Paul Ehrlich and was further propagated by McFarlane Burnet in the 1950s. Studies in genetically modified, immunodeficient mice have led to a refinement of this theory by Robert Schreiber and co-workers and has been called “cancer ▶immunoediting.” It describes three phases: (i) The elimination phase in which nascent tumor cells are destroyed by elements of the ▶innate immunity and ▶adaptive immunity; (ii) the equilibrium phase, in which tumor cells persists but are “equilibrated” by the immune system which prevents tumor progression; and

Is a common technique in which proteins separated by gel electrophoresis are blotted onto a nitrocellulose membrane and revealed by the binding of specific labeled antibodies.

Immunochemical Fecal Occult Blood Test ▶Fecal Immunochemical Test

Immunoediting

Immunocompetent Definition

Capable of developing an ▶immune response.

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Immunoediting Y VONNE PATERSON Professor of Microbiology, University of Pennsylvania, Philadelphia, PA, USA

Definition

Immunocytochemistry Definition

synonym: ▶Immunohistochemistry; Is a method of identifying cell types based on the demonstration of a specific cytoplasmic or nuclear protein or antigen in situ. It does this by detecting specific antibodyantigen interactions where the antibody has been tagged with a visible label, most commonly an enzyme. It is very useful for differentiating and subclassifying ▶carcinomas, ▶sarcomas, ▶melanomas and ▶lymphomas. ▶Fine Needle Aspiration ▶Pathology

Immunodeficient Nude Mice Definition Are athymic hairless mice without T-cells, that are unable to generate an effective ▶immune response. This makes possible the transplantation of human cancer cells without rejection. ▶MIC-1 ▶T cell

Immunodiffusion Definition Is the detection of antigen or antibody by the formation of an antigen:antibody precipitate in a clear agar gel.

Changes in the immunogenicity of tumors due to the anti-tumor response of the immune system that result in the emergence of immune-resistant variants.

Characteristics History The concept of immunoediting is predicated on the insight that the immune system can recognize tumor cells. The notion that the immune system monitors the host, not only for pathogen invasion but also for neoplastic changes, arose early in the history of Immunology, was first proposed by Paul Ehrlich in 1909 and then resurrected 50 years later by Burnet and Thomas. These early immunologists proposed that the immune system recognizes cells that have undergone neoplastic changes and eliminates them before they can form tumors, a concept known as immune surveillance. However, although this notion has been around for nearly a century it was only quite recently unequivocally demonstrated in murine models when Schreiber and colleagues, in 2001, examined the incidence of adenomas and carcinomas in aging wild type mice compared to mice lacking the ▶recombinase activating gene-2 (RAG2). The RAG2 gene controls the ▶V(D)J recombination of genes required to generate ▶B cell and ▶T cell antigen-specific receptors. In its absence, or the absence of its partner in this process, RAG1, the development of lymphocytes that bear these receptors is aborted. Thus in mice that lack RAG1 or RAG2, T cells, B cells and NKT cells fail to develop and the mouse has no adaptive immune response. The Schreiber lab showed that these immunodeficient mice had a higher incidence of spontaneous cancer and were more susceptible to carcinogen induced sarcomas. These tendencies were increased in mice lacking not only the RAG2 gene but in addition the ▶STAT-1 gene, which controls the ▶interferon– gamma ▶signal transduction pathway. Thus both adaptive immune effector cells and the multifunctional lymphokine IFN-γ were shown to play a clear role in immunosurveillance. However, in the process of studying ▶immunosurveillance the Schreiber group made the interesting observation that a high proportion of tumors that emerged in carcinogen treated RAG2 deficient mice failed to grow in wild type mice indicating that within the population of tumor cells that

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arose in the immunodeficient mice were cells that could be recognized and eliminated by an intact immune system. In contrast tumors that arose in wild type mice were less immunogenic and would grow when transplanted into either wild type or RAG2 deficient mice. Schreiber termed this process by which the immunological environment alters the immunogenicity of tumors “immunoediting.” The Role of Interferons and Other Immune Components A large body of work provides evidence that both ▶adaptive immunity and ▶innate immunity play a role in controlling the immunogenicity of tumors that develop in the mouse. Most of these studies have focused on the formation of carcinogen induced sarcomas in mice genetically manipulated to lack the expression of genes required for the generation of lymphocytes and cytokines. Thus in terms of innate immune cells, ▶γδ (gamma delta) T cells, ▶Natural Killer cells and NKT cells have been shown to be involved in controlling tumor growth as have conventional αβ (alpha beta) ▶T cells, the hallmark of the adaptive cellular immune response. The importance of cytotoxic lymphocytes, such as T cells and ▶NK cells, is highlighted by the inability of mice that lack the specific toxic molecules ▶perforin and ▶Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), expressed by cytotoxic cells, to control both carcinogen induced and spontaneous tumors. That at least a subset of the lymphocytes that recognize and control tumor growth are ▶MHC class I restricted, tumor-specific, ▶cytotoxic T cells (CTLs) is evidenced by the fact that mice lacking the ▶LMP2 (Low Molecular Mass Protein 2) ▶proteasome subunit, involved in processing antigen for recognition by CTL, develop spontaneous ▶uterine tumors. The adaptive immune response has evolved to ignore its own tissues, by eliminating self-reactive lymphocytes during their development, in order to avoid ▶autoimmunity or “horror autoxicus” as Paul Ehrlich termed it. Thus the participation of CTLs in tumor immunoediting implies that tumors must express ▶tumor-associated antigens. The original observation of immune editing identified a key role for the type II interferon, IFN-γ, in the process. This pleiotropic cytokine is the product of lymphocytes of both the innate (γδ T cells, NK and NKT cells) and adaptive (CD4+, MHC class II restricted ▶helper T (Th) cells and CD8+ MHC class I restricted CTLs). It seems likely that both innate and adaptive lymphocytes are the source of the IFN-γ that shapes the immune response to tumors. The effects of IFN-γ on the immunogenicity of tumors could occur though multiple processes since it is known as a key regulator of adaptive and innate immune responses. There is a

great deal of evidence that some of its effects are mediated through host cells in addition to tumor cells. However, given that many tumor cells express IFN-γ receptors, it can directly interact with tumor cells and there is evidence that it stimulates tumor cells to increase MHC class I expression, downregulate ▶angiogenesis and promote the infiltration of CTLs into the tumor mass. Another cytokine, ▶IL-12, has also been shown to promote tumor immunoediting. IL-12 is intimately involved in the regulation of IFN-γ production early in the immune response and probably mediates its effects through its influence on IFN-γ expression. In addition to IFN-γ, the ▶type I interferons have a profound influence on tumor growth. Early studies in mice using type I interferons for tumor ▶immunotherapy have been translated into several clinical applications for these cytokines in the therapy of ▶melanoma, ▶CML, ▶follicular lymphoma, ▶hair cell leukemia and ▶Kaposi sarcoma. There are several important differences in mice and humans between the type I interferon, IFN-γ, which is the sole molecular species, and the type II interferons, IFN-α for which there are at least 12 variants, and IFN-β, which is unique. Unlike IFN-γ, which is the product only of cells of the lymphocytic lineage, type I interferons can be produced by all nucleated cells when stimulated by products of viral infection and also by infection with some bacteria. As such they are a first line of defense against pathogen invasion of the host. In addition, although tumor cells can express receptors for type I interferons, it appears, that unlike IFN-γ, the type I interferons do not act directly on tumor cells and mediate their anti-tumor effects though host cell responses. Exactly which cells are involved requires further study but they appear to be cells of the hematopoietic lineage. Similar to IFN-γ, there are several points at which type I interferons might influence the immune response to tumors since they can activate ▶dendritic cells, ▶macrophages and ▶NK cells and are involved in the priming and survival of T cells. In addition, similarly to IFN-γ they can act on stromal cells within and surrounding tumors to down regulate angiogenic factors. Finally, there is evidence that type I interferons may inhibit the transformation of normal cells by upregulating the expression of the tumor-suppressor molecule, ▶p53. The importance of this class of cytokines in shaping the immune response to tumors was confirmed recently by studies in mice lacking the IFN-α receptor. Carcinogen induced sarcomas were found to arise more frequently in these mice and were also shown to be more immunogenic when transplanted into wild type mice. The Role of Induced Immune Pressure The editing of tumors in response to immune pressure is exacerbated when the immune system has been specifically primed to a ▶tumor-associated antigen.

Immunoglobulin

In a ▶mouse model for ▶breast cancer in which ▶HER-2/neu, a member of the ▶epidermal growth factor receptor family, is overexpressed on breast tissue, resulting in the emergence of breast tumors, it was shown that mice immunized with ▶cancer vaccines expressing fragments of HER-2/neu could control tumor outgrowth for a significant length of time but eventually succumbed. When HER-2/neu was isolated from the emerging tumors they were found to have accumulated a significant number of mutations in the precise region of the molecule the particular vaccine targeted. In addition, it was verified for one of the fragments that each mutation occurred in a region recognized by CTLs and that this mutation abrogated CTL recognition. This is a clear demonstration of how the host immune system can sculpt a tumor-associated antigen to evade the immune system. Transplanted, syngeneic mouse tumors have also been shown to mutate in a region of an antigen expressed by the tumor and recognized by CTLs after adoptive transfer of T cells that recognize that region. Evidence of Immunoediting in Human Cancer Although there is an abundance of evidence for immunoediting in murine models of cancer, the evidence that this occurs in human cancer is largely circumstantial. It has long been known that cancer patients develop immune responses to their own tumors. Indeed, both tumor-specific Tcells and antibodies isolated from cancer patients have been harnessed to identify tumor-associated antigens by methods such as ▶SEREX. In addition the ability of a patient to mount a response to their tumors, particularly if there is evidence of tumor infiltration of immune effector cells, is a strong predictor of a favorable prognosis. That the immune system sculpts tumor immunogenicity in tumors that arise in cancer patients is supported by the emergence of tumors in patients undergoing antigen-specific immunotherapy that have down regulated the tumor antigen to which the therapy is directed, or components of the cellular machinery that generates the cell surface complex of ▶HLA I and antigen recognized by CTLs. These include LMP proteasome subunits and transporter molecules that chaperone antigen peptides from the cytosol to the Golgi apparatus for loading onto HLA class I molecules. Sometimes the HLA I molecule itself is lost, usually by mutation of the beta2 microglobulin subunit, common to all HLA class I molecular complexes. In some cases, however, lack of HLA class I on the surface of a tumor is due not to mutational events in the HLA class I genes themselves but to down regulation of expression of the protein. This can often be reversed by cytokines, such as IFN-γ but it is interesting to note that human tumors have been shown to arise that lack the IFN-γ receptor. The phenomenon of HLA class I loss from the surface of tumor cells has been documented in numerous clinical

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cancer vaccine trials for ▶melanoma, ▶prostate carcinoma and for HER-2/neu positive tumors. In addition, even in the absence of immunotherapy, HLA class I expression has been shown to be lost or down regulated in all types of tumors especially in patients with advanced disease. Indeed, the frequency of deletion or down regulation of these cell surface molecules has been found to be as high as 15% in primary melanoma lesions and 50% in primary ▶prostate carcinoma lesions. A final piece of evidence for immunoediting in human cancer is that patients that are seriously immunosuppressed, for example post organ transplant, have a higher incidence of cancer than healthy individuals.

References 1. Bui JD, Schreiber RD (2007) Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes? Curr Opin Immunol 19:203–208 2. Dunn GP, Koebel CM, Schreiber RD (2006) Interferons, immunity and cancer immunoediting. Nat Rev Immunol 6:836–848 3. Neeson P, Paterson Y (2006) Effects of the tumor microenvironment on the efficacy of tumor immunotherapy. Immunol Invest 35:359–394 4. Singh R, Paterson Y (2007) Immunoediting sculpts tumor epitopes during immunotherapy. Cancer Res 67:1887– 1892 5. Chang CC, Campoli M, Ferrone S (2005) Classical and nonclassical HLA class I antigen and NK Cell-activating ligand changes in malignant cells: current challenges and future directions. Adv Cancer Res 93:189–234

Immunogenicity Definition The ability of a foreign substance to confer a certain level of immunity to the host. ▶T-Cell Response

Immunoglobulin Definition Ig; The immunoglobulin molecule, composed of two light and two heavy protein chains, comprising the antibody. ▶Immunoglobulin Genes

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Immunoglobulin E (IgE)

Immunoglobulin E (IgE)

Immunoglobulins

Definition

Definition

An antibody characteristic of the atopic allergic immune response.

Immunoglobulins (Ig): All antibody molecules belong to a family of plasma proteins called immunoglobulins (Ig). Membrane-bound immunoglobulin serves as the specific antigen receptor on B lymphocytes. Different immunoglobulin isotypes are called IgM, IgD, IgG, IgA, and IgE. Immunoglobulin A (IgA): Immunoglobulin A is the class of immunoglobulin characterized by α heavy chains. IgA antibodies are secreted mainly by mucosal lymphoid tissues. Immunoglobulin D (IgD): Immunoglobulin D is the class of immunoglobulin characterized by δ heavy chains. It appears as surface immunoglobulin on mature naïve B cells but its function is unknown. Immunoglobulin E (IgE): Immunoglobulin E is the class of immunoglobulin characterized by ε heavy chains. It is involved in allergic reactions. Immunoglobulin G (IgG): Immunoglobulin G is the class of immunoglobulin characterized by γ heavy chains. It is the most abundant class of immunoglobulin found in the plasma. Immunoglobulin M (IgM): Immunoglobulin M is the class of immunoglobulin characterized by μ heavy chains. It is the first immunoglobulin to appear on the surface of B cells and the first to be secreted.

▶Allergy ▶Immunoglobulin Genes

Immunoglobulin Genes Definition Cluster in multimember gene families and are located on chromosome 14 (heavy chain gene segments), 2 (κ light chain) and 22 (λ light chain): V genes (variable region genes), D genes (diversity region genes) and J genes ( joining region genes) for the heavy Ig heavy chains, V and J region genes for κ and λ light chains. Ig gene rearrangement brings together one representative of these gene families (V, D and J for the heavy chain, V and J for κ and λ light chains). ▶V(D)J recombination is a remarkable process since it entails double strand DNA breaks, loss or DNA and re-ligation of DNA strands. Joining of the V-D-J segments is imprecise and involves insertion of non-template nucleotides (N-additions) and trimming back of the beginning/ends of the gene segments. Ultimately, the rearrangement leads to a unique sequence of VH-N-DN-JH, which generates diversity for antigen recognition, and which acts a marker for each individual B-cell and its progeny. ▶B-Cell Tumors

Immunohistochemistry M ARC B IRKHAHN , C LIVE R. TAYLOR , R ICHARD J. C OTE Departments of Urology and Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Synonyms

Immunoglobulin Libraries Definition Recombinant nucleic acid libraries of natural or synthetic origin that encode antiBody binding sites (Fab or scFv) for which the products (and corresponding genes) can be selected against an antigen of interest and subsequently modified to improve their affinity. Applications: Fast selection of new antiBody binding sites from different species. Modification of pre-existing binding site affinity.

IHC; Immunohistology; Immunocytochemistry; Molecular Morphology

Definition Immunohistochemistry (IHC) is a technique to detect and localize specific proteins in tissue sections by labeled antibodies that bind specifically to the investigated antigen.

Characteristics History Since the introduction of microscopy into the diagnosis of diseases by Bennett in 1842 and Virchow in 1858,

Immunohistochemistry

pathologists have searched for better and more specific stains. In 1934, Marrack was the first to employ modified antibodies to visualize cholera and typhoid agents. Nearly a decade later, Coons (1941) used a primary antibody that was labeled with a fluorescent dye, fluorescein isothiocyanate, and utilized it on human tissue. However, as a specialized dark-field microscope and use of frozen sections were needed for the fluorescent dyes, the technique remained mainly a research tool until other labels were developed. In 1974, Taylor and Burns demonstrated that IHC could be performed on routinely processed tissue using immunoperoxidase techniques with chromogenic dyes. This step enabled the use of IHC in routine clinical laboratories.

Techniques Immunohistochemistry combines the principles of immunology with histochemistry, and involves two basic steps: first, an ▶antibody binds to its specific target antigen at the cellular location in the tissue sample. The antigen-antibody binding is then detected by labeling techniques. IHC therefore not only enables pathologists to detect whether or not particular antigens are present within a given tissue, but also allows marking its cellular location. There are two major types of antibodies in use: polyclonal antibodies or antisera and monoclonal antibodies. Polyclonal antibodies are produced by an injection of an antigen or antigen fragments into a host animal. The most common host animals are rabbit, horse, mouse and goat. If a small antigenic molecule or antigen fragment (hapten) is used as immunogen, immunogenicity is typically enhanced by coupling the hapten with a compound such as keyhole limpet hemocyanin. In the resulting immunologic reaction, host lymphocytes are activated and ultimately plasma cells initiate production of antibodies. As antigens contain various immunogenic determinants, the resulting antiserum will be a heterogeneous mix that recognizes several epitopes. Such potentially crossreacting specificities are unwanted for use in IHC and must be removed by further purification. Monoclonal antibodies are developed by the hybridoma technique or through molecular engineering using bacteriophages. In the hybridoma technique, activated (antibody-secreting) B-lymphocytes are isolated from the immunized animal (in the past usually mouse, now increasingly common rabbit) and fused with cultured myeloma cells which have limitless proliferative potential. Each of the resulting hybrid cells (hybridoma) produces a single type of antibody, thus being ‘monoclonal’ in nature. The detection methods include the ‘direct’ and the ‘indirect’ alternatives. In the direct conjugate-labeledantibody method, the label (such as a fluorescent agent

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or an enzyme) is chemically attached to the primary antibody. The localization of this fluorescent label is detected by ▶immunofluorescence microscopy or if the label is an enzyme (typically, horseradish peroxidase) coupled to antibody, it is detected by its action on a chromogenic substrate. The indirect method uses an unlabeled primary antibody and a labeled secondary antibody (second layer) directed against the primary antibody. Several secondary antibody molecules bind to each primary antibody molecule resulting in signal enhancement, thus in increased sensitivity. Furthermore, methods involving a third layer for signal amplification can be employed. These include the PAP (peroxidase-antiperoxidase) method, the biotin-avidin mediated procedures (such as those employing the avidin-biotin conjugate or ABC procedure, biotin-streptavidin systems), immunogold or polymer based labels, catalyzed signal amplification and alkaline phosphatase-based methods. Several efforts have been made to improve the sensitivity, the reliability and the standardization of IHC. A major step was the development of antigen retrieval. The antigen retrieval process breaks protein cross linkages formed during formaldehyde fixation of a tissue and enables these proteins to be accessible for antibodies. Antigen retrieval is performed by exposing tissue to heat for various lengths of time in retrieval buffer solutions with specific pH. Several protocols are available but these often have to be adjusted for optimal results which can be done standardized for various temperatures and durations in a ‘test battery’ approach. Several automated immunostaining systems are commercially available. While they significantly increase the reproducibility and standardization, they certainly do not guarantee optimal results. Quality control issues as well as the need for the validation of the results apply for staining carried out with both the automated systems and manual IHC. Interpretation of IHC staining results necessitates a highly trained pathologist and even among experts is subject to inter-observer discrepancies. The development of automated image analysis systems addresses this problem. The currently available systems can both assess the intensity as well as the percentage positivity of antigenic markers. The limitations of the human eye to differentiate colors restrict the use of multiple markers on the same slide. The spectral imaging technology allows to differentiate different chromogens reporting on multiple markers by serially, digitally “erasing” a given chromogen image from the microscope image by a specially designed software. Uses IHC plays an essential role in modern pathology. Its most common use is the identification of tumor origin

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and tumor type by tissue-specific markers. By IHC a pathologist can specify tumor entities and evaluate tumors of unknown origin as well as stage lymph nodes and bone marrow for the presence of metastatic disease. More recently, several markers have been identified that can predict disease prognosis, treatment response and serve as therapeutic target. Use in Research In a research setting, the expression of relevant markers is investigated. This demands studies on well-defined samples across various tumor stages and clinical outcome. The tissue microarray technique allows an acceleration of such studies in a high-throughput manner. Tissue microarrays (TMA) consist of compendia of 0.6 mm cylindrical biopsy cores from paraffin-embedded tissue that are transferred to defined array coordinates in a recipient block. These multi-tissue-core blocks can then be processed similar to a routine single-tissue block. A difference from the conventional IHC is the high level of standardization since all slides in one TMA experiment are incubated together, ensuring identical reagent concentrations and incubation temperatures.

Clinical Uses Diagnosis Intermediate Filaments The presence of intermediate filaments, which function as cytoskeletal components in both normal and malignant cells, is useful in the initial classification and diagnosis of neoplasms. Five major classes of intermediate filaments exist: ▶Cytokeratin (CK), vimentin, desmin, neurofilament and glial fibrillary acidic protein (GFAP). Most neoplasms show a predominant expression of one or more of these intermediate filaments. Based on their molecular weight and isoelectric point, the cytokeratins are subdivided into more than 20 types. Carcinomas are typically CK positive, whereas sarcomas, melanomas, and lymphomas are generally vimentin positive. Tumors of myogenic origin characteristically express desmin and/or muscle actins and vimentin. Glial tumors are predominantly positive for GFAP. Nuclear Transcription Factors Transcription factors are proteins involved in the regulation of gene expression. By binding to promoter elements upstream of genes they either facilitate or inhibit transcription. Though not exclusively found in a specific tumor type, transcription factors are highly tissue specific and can be useful in determining the primary site of a tumor.

Carcinoma Essentially all cells of epithelial origin express cytokeratins, which are therefore highly sensitive markers for carcinomas. However, other tumors such as mesotheliomas and non-seminomateous germ cell tumors also stain positive for CK. More specific markers and sub-typing of keratins have to be applied to further differentiate CKpositive cells and to assess the site of origin of the carcinoma. The profile of cytokeratin sub-types is useful to determine the tumor type. As for example, hepatocellular carcinomas are positive for CK antigens that can be detected by antibodies AE3 and CAM5.2 but are negative for antibody AE1, which is directed against a different set of CK. Recently, the use of CK7 and CK20 profile has proven a useful tool in distinguishing cellular origin. Nuclear transcription factors are useful in the detection of carcinomas. Thyroid transcription factor-1 (TTF-1) is found in the thyroid and lung. CDX-2 is specific for colorectal epithelium. For some tissue types, tissue-specific tumor markers (e.g. prostate specific antigen (PSA) for prostate, or thyroglobulin for thyroid) or tissue-associated markers (e.g. gross cystic disease fluid protein 15 (GCDFP-15) and mammaglobin in breast, uroplakins for transitional urothelial cells, OC 125 for ovarian cells, synaptophysin for neuroendocrine lesions, etc.) are available. It is important to remember that these markers are often not uniquely found in the specific tissue. For instance, the detection of PSA has also been described for samples of salivary glands and breast. Melanoma For melanomas, which usually are cytokeratin-negative and vimentin-positive, a sensitive tissue marker, S-100, is available. HMB-45, Melan A and tyrosinase are used to confirm the diagnosis. Sarcoma The common IHC pattern for all forms of sarcoma includes vimentin positivity. Generally speaking, sarcomas are CK-negative, but some may yield positive reactions especially for low molecular CKs following antigen retrieval and highly sensitive labelling techniques. Additional stains are then employed to identify the tumor type. Rhabdomyosarcoma and Leiomyosarcoma express markers that are typical for muscular tissue such as desmin and muscle specific actin. Lymphoma Lymphomas show a wide variety of morphologic appearances, making IHC especially important in their diagnosis. Membranous staining for ▶CD45 can typically be seen in lymphoid tissue. CD3 and ▶CD20 are more specific markers and are used to


further confirm lymphomas. ▶CD antigens are essential in subclassifying lymphoid tissue. Infectious Agents Since the early days of its development, IHC has been used for the detection of infectious agents. Nowadays, microbiologic cultures are the gold standard but for pathogens that are difficult to culture such as cytomegalovirus, mycobacteria, toxoplasmosis, pneumocystis carinii, histoplasma capsulatum, ▶Helicobacter pylori and ▶human papillomavirus IHC methods are at least as effective. Tumor Stage and Occult Metastases The most important factor determining outcome of patients with cancer is the presence of regional and/or systemic dissemination (metastases). While routine pathologic work-up cannot detect small metastatic deposits in many instances, IHC is a highly sensitive way to detect such occult metastases in blood, lymph nodes and bone marrow. This technique is based on the fact that monoclonal antibodies can reveal cells of different histogenesis in the investigated tissue. As normal lymph nodes, bone marrow or blood do not contain cells with epithelial antigens, the finding of CKpositive cells suggests metastases of a carcinoma. The presence of occult metastases has been shown to influence clinical outcome for several cancer types such as breast, lung and prostate cancer. In breast cancer, IHC is able to show the presence of occult metastases in bone marrow in 10–40% of patients with low-stage disease. Lymph node metastases occult to routine pathologic work-up have been described for several tumors including breast cancer in up to 20% of patients and in more than 10% of patients with prostate cancer. The reported sensitivity of this technique ranges from the detection of 1 epithelial cell in 10,000 to 2–5 epithelial cells in a million hematopoietic cells. Prognosis Mutations and overexpression of oncogenes and tumor suppressor genes play a vital role in tumorigenesis. The presence or absence of their protein products may predict the biological behavior of tumors more accurately than clinical and pathologic criteria. Since antibodies are available for many of these gene products, tumors with different prognosis can be differentiated by means of IHC. The best investigated proteins include ▶p53, the protein product of the ▶Retinoblastoma gene (pRb), p27, ▶p21 and ▶p16. RB gene alterations resulting in reduced expression are known to characterize tumors with a higher risk for metastatic disease. p21, p16 and p27 are the three major cyclin-dependent kinase

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inhibitors which negatively regulate pRb activity and are normally required for cell cycle suppression. The loss of p27 expression is associated with colon, breast, prostate, and gastric cancer progression. Changes in protein expression of p21 have been associated with higher tumor grade and worse prognosis in patients with bladder cancer. p53 plays an important role in cell cycle progression, and apoptosis in response to DNA damage, and is expressed by all human cells. Normal (wild-type) p53 protein has a short half-life of only 6 to 30 minutes because of its ubiquitin action-mediated degredation dictated by its binding with another regulatory protein, ▶MDM2. Therefore p53 does not accumulate in normal cells. Overexpression of p53 (by altered p53 gene or because of ineffective MDM2 protein) in the nuclear compartment therefore indicates a dysfunctional p53 pathway, a characteristic of tumors. Alterations in p53, p21 and pRb act in cooperative or synergistic ways to promote cancer progression and simultaneous overexpression has been linked to worse prognosis. Treatment Response An increasingly important application of IHC is to identify specific targets for therapy. Consequently, this will allow better selection of patients who will benefit from certain treatment modalities. In several tumor types, treatment decisions already are influenced or determined by molecular findings. An early example is hormonal therapy for breast cancer patients depending on estrogen and progesterone expression of the tumor. Because of its vital role in the apoptotic pathway ▶p53 alterations are likely to influence response to chemotherapy. p53 alterations confer increased chemosensitivity on tumors such that combining agents with different actions may have synergistic effects on tumor cell killing. Targeted Therapy In breast cancer Her-2/neu overexpression was linked to resistance to tamoxifen therapy and commonly used adjuvant chemotherapy. However, it has been shown that the HER2/neu receptor tyrosine kinase can serve as a therapeutic target. Treatment with the monoclonal antibody ▶trastuzumab (brandname: Herceptin) which is directed against this protein is effective only for patients whose tumors show overexpression of Her2/ neu. In invasive ▶bladder cancer there is evidence to suggest that patients who harbor p53 alterations respond to adjuvant chemotherapy that contains DNA-damaging agents such as cisplatinum. Immunohistochemistry has allowed for identification of tumor origin, tumor prognosis, and likelihood of response to therapy for an increasing array of tumors. The ability to determine which tumors are most likely

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Immunohistology

to progress (and thus need further therapy), coupled with the ability to predict specifically the response of individual tumors to chemotherapeutic agents and the ability to identify specific targets of therapy will have a profound effect on the way treatment decisions for patients with cancer are made. It is not difficult to envision the day when drug selection is based on the presence of specific targets and on the resistance patterns of individual tumors to specific agents. Treatment decisions will become less organ based and will reflect the biology of the tumors. This has already helped us to approach patient-specific (as opposed to disease specific) management of care. ▶Gastrointestinal Stromal Tumor

References 1. Mitra AP, Lin H, Cote RJ et al. (2005) Biomarker profiling for cancer diagnosis, prognosis and therapeutic management. Natl Med J India 18(6):304–12 2. Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval immunohistochemistry and molecular morphology in the year 2001. Appl Immunohistochem Mol Morphol 9(2):107–116 3. Taylor CR, Cote RJ (2006) Immunomicroscopy: a diagnostic tool for the surgical pathologist, 3rd edn. Saunders, Philadelphia, PA 4. Hawes D, Taylor CR, Cote R (2003) Immunohistochemistry. In: Weidner N, Cote R, Suster S, Weiss L (eds) Modern surgical pathology, 1st edn. Saunders, Philadelphia, PA, pp 57–80

Immunohistology ▶Immunohistochemistry

Immunoliposomes R OLAND E. KONTERMANN Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany

Definition

Are ▶liposomal drug formulations possessing antibody molecules conjugated to the liposomal surface. This allows for a ▶targeted drug delivery to tumor cells or other tumor-associated structures (active targeting).

Characteristics

▶Liposomes are vesicular particles composed of a lipid bilayer enclosing a hydrophilic inner phase. Liposomes can be used as carrier systems for cancer drugs (▶Drug delivery systems) by encapsulating hydrophilic drugs into the interior or by incorporating lipophilic drugs into the lipid bilayer. Liposomes have normally a size (diameter) of 100–200 nm. Several liposomal formulations of therapeutic drugs are approved for cancer therapy, e.g., liposomal doxorubicin (Doxil/Caelyx, Myocet) (▶Liposomal chemotherapy). Delivery of these drugs to tumors is a passive process and efficacy depends on long circulation and enhanced permeability and retention in the tumor tissue (EPR effect). New formulations of liposomal drugs (e.g., Doxil/Caelyx) have polyethylene glycol (PEG) chains incorporated into the lipid bilayer to further increase stability and pharmacokinetic properties and to decrease elimination of the liposomes by phagocytic cells. The coupling of antibody molecules to the lipid surface allows for an active targeting by recognition of antigenic structures expressed by the tumor. Thus, immunoliposomes are designed to increase selectivity and efficacy of liposomal drugs. Immunoliposome Types and Antibody Formats Immunoliposomes are generated by chemically coupling antibodies or antibody fragments to the liposomal surface. Besides whole ▶antibody molecules (e.g., IgG molecules), antibody fragments such as Fab′ fragments or ▶single-chain Fv fragments can be utilized for the generation of immunoliposomes. The use of whole antibodies is less favorable since these immunoliposomes are recognized by phagocytic cells via Fc receptors. Consequently, Fab′ or scFv′ fragments are the formats of choice to generate immunoliposomes. Immunoliposomes can be classified depending on the position of coupled antibodies and the liposomal composition. In type I immunoliposomes, the antibodies are coupled directly to the lipid bilayer either in the absence (type Ia) or presence of PEG chains (type Ib). Coupling of the antibodies to the distal end of incorporated PEG chains results in type II immunoliposomes (Fig. 1a). Coupling of antibodies is facilitated by the use of functionalized lipids, e.g., lipids possessing an aminoreactive succinimidyl moiety or a sulfhydryl-reactive maleimide group. Antibody or antibody fragments can be generated by established ▶hybridoma technology and biochemical means or by genetic engineering as recombinant molecules. For example, Fab′ fragments can be produced by enzymatic cleavage of IgG molecules with pepsin resulting in F(ab′)2 fragments, which after reduction are separated in Fab’ fragments exposing a free sulfhydryl group at the end of the molecule (Fig. 1b). Furthermore, the implementation of antibody engineering allows for the generation of

Immunoliposomes

small antibody molecules with desired properties, e.g., scFv molecules exposing a genetically introduced cysteine residue at the C-terminus used for site-directed coupling (Fig. 1b).

Immunoliposomes. Figure 1 Classification of immunoliposomes and antibody formats. (a) Three types of immunoliposomes can be distinguished. Type I immunoliposomes have the antibody molecules coupled directly to the lipid bilayer, either in the absence (type Ia) or presence (type Ib) of polyethylene glycol (PEG) chains. In type II immunoliposomes, the antibodies are coupled to the distal end of PEG chains. (b) Antibodies formats used for the generation of immunoliposomes. The two variable domains (VH, VL) forming the antigen-binding site are shown in dark and light gray.

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Drugs and Targets Immunoliposomes can be combined with a wide variety of different drugs. Besides small compounds and chemotherapeutic drugs, immunoliposomes can also be used for the delivery of nucleic acids (e.g., ▶siRNA) or therapeutically useful peptides and proteins (Fig. 2). Encapsulation of drugs into liposomes alters their pharmacokinetic properties, e.g., reduces rapid renal elimination of small molecular weight drugs. In addition, drug encapsulation has been shown to reduce side effects and to increase stability of the drug within the body. Several modes of action have been described for immunoliposomes. Delivery to extracellular structures may lead to increased accumulation of liposomes in the tumor tissue and slow release of drug, which then can enter the target cell. Alternatively, binding of immunoliposomes to cell surface receptors results in internalization of immunoliposomes (▶Endocytosis) and intracellular release of the drug. Several studies have shown that internalization of immunoliposomes leads to increased cytotoxicity and may also bypass drug resistance mechanisms. Main targets of immunoliposomes are molecules expressed by tumor cells. Thus, various immunoliposomal formulations of chemotherapeutic drugs (e.g., ▶doxorubicin, ▶vincristine) have been generated using antibodies against tumor-associated antigens including CD19, ▶CD20, Her2/neu, ▶epidermal growth factor receptor (EGFR), and disialoganglioside GD2 for therapy of lymphoma and solid tumors. In addition, research has been focused on targeting of tumor blood vessels (▶vascular-targeting adjents) as well as targeting of extracellular tumor stroma components or tumor stroma fibroblasts, which are more easily accessible for circulating liposomes. Efficacy of immunoliposomes is influenced by several factors. Besides lipid composition, which has an influence on stability and release of drug, and the antibody format used, efficacy depends also on the kind of target molecule as well as its density on the cell surface and sensitivity of target cells for the encapsulated drug.

Immunoliposomes. Figure 2 Active compounds combined with immunoliposomes. Immunoliposomes allow for a targeted delivery of various kinds of active molecules, including small compounds, chemotherapeutic drugs, nucleic acids, and peptides and proteins.

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Immunologic Tolerance

References 1. Kontermann RE (2006) Immunoliposomes for cancer therapy. Curr Opin Mol Ther 8:39–45 2. Park JW, Benz CC, Martin FJ (2004) Future directions of liposome- and immunoliposome-based cancer therapeutics. Semin Oncol 31:196–205 3. Allen TM, Cullis PR (2004) Drug delivery systems: entering the mainstream. Science 303:1818–1822 4. Drummond DC, Meyer O, Hong K, et al. (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev 51:691–743 5. Sapra P, Tyagi P, Allen TM (2005) Ligand-targeted liposomes for cancer treatment. Curr Drug Deliv 2:369–381

Immunophenotyping Definition Determining the expression of antigens on or in cells. ▶Flow Cytometry ▶Leukemia Diagnostics

Immunophilins Immunologic Tolerance Definition

The lack of an ▶immune response.

Immunological Fecal Occult Blood Test ▶Fecal Immunochemical Test

Definition Are a group of intracellular proteins that bind to the anti-inflammatory agents ▶cyclosporine A and FK506. The complexes interfere with calcineurin-mediated transcriptional activation and inhibit the transcription of genes encoding several proinflammatory molecules. Proteins that bind immunosuppressant drugs.

Immunoprevention P IER-LUIGI L OLLINI , PATRIZIA N ANNI Section of Cancer Research, Department of Experimental Pathology, University of Bologna, Bologna, Italy

Immunophenotype

Synonyms Immunoprophylaxis of cancer

Definition The overall expression of proteins that identify a specific cell type as determined by using specific antibodies reacting against surface proteins on cell membrane. The collective repertoire of proteins expressed on the outer surface of a particular type of cell.

Definition

▶CD Antigens

Characteristics

Immunophenotypic Determinants ▶CD Antigens

Prevention of cancer onset or of early cancer development and ▶progression by means of immunological treatments, such as vaccines, antibodies or cytokines.

Immunoprevention of cancer can be applied to tumors caused by viruses (and other infectious agents) or to tumors unrelated to infectious agents. In both cases the aim is the same, however the underlying concepts and the advancement of clinical development are different. Prevention of viral tumors is based on vaccines against viral antigens, whereas immunoprevention of tumors unrelated to infectious agents targets antigens expressed by early neoplastic cells.

Immunoprevention

Immunoprevention of Viral Tumors About 18% of all human tumors are directly caused by infectious agents or indirectly by persisting inflammation accompanying chronic infection. In such cases the application of immunological strategies to prevent infection is a type of ▶primary cancer prevention because it aims at removing a risk factor that can cause cancer. The first success in this direction was the demonstration that vaccination programs implemented in the 1980s against ▶hepatitis B virus (HBV) significantly reduce the incidence of hepatitis virus associated ▶hepatocellular carcinoma. Current vaccines are highly effective (90–95%) in preventing chronic HBV infection. Studies in countries where HBV infection is frequent demonstrated that vaccination reduced by half infantile hepatocellular carcinoma incidence and mortality. HBV is responsible for one third (developed countries) to two thirds (less developed countries) of all cases of hepatocellular carcinoma, and ▶hepatitis C virus (HCV) causes about one fourth. HCV vaccines under development are expected to contribute a further substantial decrease in the worldwide incidence of hepatocellular carcinoma. ▶Human papillomaviruses (HPV) are the most prevalent carcinogenic viruses in humans; various types of HPV cause more than half a million new cases of ▶cervical cancer and other tumors worldwide. The first human vaccine against HPV was approved in 2006 in the US, EU, and various other countries. It is a quadrivalent vaccine against HPV types 6, 11, 16 and 18. A divalent vaccine against HPV 6 and 18 is undergoing approval in 2007. Both vaccines are made of ▶virus-like particles (VLP). Clinical trials of both vaccines demonstrated 89–100% protection of vaccinated women from persistent HPV infection, 100% protection from histologic evidence of cervical cancer, and no serious adverse events. These vaccines are thus expected to have a major impact on the incidence and mortality of HPV-related cancers. Four critical issues are (i) current vaccines do not include HPV types causing one third of cervical cancers worldwide, hence screening programs should not be abandoned until cross-protection by current vaccines has been demonstrated or novel multivalent vaccines have been developed; (ii) vaccines are currently very expensive, and only a few national or regional health systems are prepared to subsidize the costs; (iii) the matter is further complicated by the need to vaccinate prepuberal (9–12-year-old) girls against a sexually-transmitted virus, an issue that is causing considerable ethical debate in some countries; (iv) the follow-up of vaccinated persons is still too short to estimate the long-term duration of immunity and the need for periodic boosts. Attempts toward the development of vaccines against ▶Helicobacter pylori, the third major infectious risk

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factor of human cancer, until now did not produce a credible candidate for mass vaccination. On the whole it can be estimated that a full-scale deployment of the approaches described above for HBV, HCV and HPV could lead to the immunoprevention of two thirds of infectious human cancers, or more than 10% of all human cancers. This is a very important result which however leaves open the question of what immunoprevention can do for the majority of human cancer not caused by infectious agents. Immunoprevention of Tumors Unrelated to Infectious Agents Immunoprevention of non-infectious tumors is a relatively recent development and is currently at the level of ▶preclinical testing. This type of immunoprevention targets early neoplastic cells, hence it can be classified as a kind of ▶secondary cancer prevention because it aims at preventing the evolution of incipient tumors into clinically-evident, symptomatic masses. The ▶immune surveillance theory posits prevention of tumor onset as a fundamental function of the immune system, on a par with prevention of infection. In fact cancer incidence in knockout mice lacking ▶adaptive immunity and ▶non-adaptive immunity is much higher that in immunocompetent mice. However the very existence of progressive, malignant tumors demonstrates that the efficiency of spontaneous immune surveillance is lower than 100%. Thus the aim of cancer immunoprevention is to enhance immune surveillance of tumors by means of treatments that elicit protective antitumor immune responses and/or decrease immunosuppressive components. Immune targeting of ▶preneoplastic lesions or of early neoplastic cells has several advantages with respect to conventional cancer ▶immunotherapy, which instead must necessarily target advanced tumors. The efficacy of immune defenses is higher against smaller tumor deposits, a property shared by most antitumor approaches, including chemotherapy. Nascent neoplastic lesions are less protected from immune effectors by ▶stromagenesis and ▶angiogenesis, tumor progression caused by the accumulation of multiple genetic alterations is still at an early stage, and genomic instability has not yet generated a wide array of heterogeneous tumor variants, eventually leading to the selection of immunoresistant phenotypes. Demonstrations of cancer immunoprevention were mainly obtained in ▶cancer-prone genetically modified mouse models and in some ▶chemical carcinogenesis systems. Three different strategies were effective in inhibiting and/or delaying tumor onset in mice: (i) monoclonal antibodies directed against membrane tumor antigens; (ii) immunostimulants, such as recombinant ▶cytokines (▶interleukin-12), plasmids containing CpG sequences (▶CpG islands), or bacterial

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derivatives (α-galactosylceramide); (iii) vaccines containing whole cells, recombinant proteins, synthetic peptides or plasmids encoding the target antigen. Using standard categories borrowed from immunotherapeutic jargon, the first approach can be classified as passive immunoprevention, because it is based on the administration of a preformed immunological “drug” directly acting on tumor cells, the second as active, antigen nonspecific immunoprevention and the third as active, antigen specific immunoprevention. Early attempts provided proof-of-principle demonstrations that stimulation of the immune system in healthy, cancer-prone hosts can effectively delay or reduce tumor incidence later in life. Second generation studies aimed at increasing preventive efficacy through the combination of different strategies, much as it happened in chemotherapy (▶Chemotherapy of cancer, progress and perspectives). Various combinations of antigen specific vaccines and powerful immunostimulants completely prevented tumor onset in very aggressive mouse models of cancer development, thus demonstrating that cancer immunoprevention can effectively halt a genetic predisposition to cancer. Microscopic analysis of tumor-free aged mice showed that tumor progression is blocked at the stage reached when vaccination begins. For effective immunoprevention of tumor onset vaccination must start before tumor progression reaches specific critical stages, such as in situ carcinoma. Immune Mechanisms of Cancer Prevention Highly active vaccines like those used for complete cancer immunoprevention elicit simultaneously many overlapping immune responses in immunocompetent mice, hence vaccination of mice with selective immunodepressions is the only way to dissect protective mechanisms from less important immune components. The most important immune mechanisms at work in cancer immunoprevention comprised both ▶T cell responses, including the release of cytokines (interferon-γ, IFN-γ) and, less frequently, ▶cytotoxic T lymphocytes (CTL), along with antibodies whenever the target antigen was expressed on the cell surface. The importance of antibody responses clearly distinguishes cancer immunoprevention from cancer immunotherapy, the latter being mainly based on CTL rather than antibodies. The largely different time scales involved justifies this discrepancy, because cancer immunoprevention requires a long-term protection from tumor onset, ideally extending for the entire life of the host, whereas a rapid destruction of tumors or metastases is the goal of cancer immunotherapy. CTL activity must be of short duration to avoid severe toxicity for the host, whereas protective antibodies persisting for a long time are harmless. The same dualism applies to viral

immunity in which acute infection is mostly resolved by CTL, whereas long term immunity from reinfection and protection elicited by vaccination are provided by neutralizing antibodies. The direct interaction of cytokines like IFN-γ and of antibodies with early neoplastic cells results in multiple molecular blocks of cell growth and tumor progression combining cytostatic and cytotoxic mechanisms. Inhibition of cell proliferation is a logical part of the set of antiviral activities of IFN-γ that can directly block tumor cell growth, moreover it inhibits the release of ▶matrix metalloproteinases involved in tumor cell invasiveness (▶Invasion) and induces the production of chemokines with antiangiogenic activity. Antibodies binding to surface tumor antigens mediate tumor cell killing via ▶complement-mediated cytotoxicity and ▶antibody-dependent cell-mediated cytotoxicity (ADCC). Whenever the target antigen is involved in mitogenic signaling, antibodies can directly inhibit tumor cell proliferation without the need of further molecules or cells of the immune system, in practice by acting as “receptor antagonists.” In the case of the ▶HER-2/neu oncogene, specific antibodies induced by preventive cancer vaccines inhibit dimerization of surface HER-2/neu proteins (a key step required for the initiation of signal transduction) and induce HER-2/ neu internalization and recycling, eventually leading to a complete depletion of HER-2/neu surface expression. In HER-2/neu-addicted cells (▶Oncogene addiction) a prolonged loss of HER-2/neu expression and signaling blocks cell proliferation and can trigger ▶apoptosis. Target Antigens of Cancer Immunoprevention The inhibitory mechanisms targeting HER-2/neu can be ineffective against most other tumor antigens, either because the target is not a surface molecule and antibodies cannot bind tumor cells, or because tumor progression leads to a loss of antigen processing machinery (including major histocompatibility complex molecules), required for antigen recognition by T cells. The latter is a very frequent event affecting 80–90% of all human tumors. For cancer immunoprevention it was proposed that the immune recognition of ideal target antigens should persist even if defects in antigen processing impede T cell recognition, hence the antigen should be expressed on the cell surface and recognized by antibodies. A second desirable property is a direct involvement of the target antigen in oncogene addiction or in the maintenance of tumorigenicity, as is the case of HER-2/neu, because this prevents tumor escape from immune defenses through loss of antigen expression, again a frequent phenomenon for most other tumor antigens. Tumor antigens fulfilling both requirements were named ▶oncoantigens. Members of this new class of tumor antigens ideally suit the

Immunoreceptor

concepts of cancer immunoprevention, in particular for what concerns the need of persistent, lifelong antitumor responses, however oncoantigens make also attractive new targets for cancer immunotherapy because they are not prone to relevant mechanisms of immunoresistance and therapeutic failure. Clinical Developments The mass of preclinical data demonstrating the efficacy of cancer immunoprevention in mouse models warrants the translation of this approach to humans, however this will require a precise definition of the subjects who can benefit from this type of intervention and consequently of the design of clinical trials. The analysis of these issues reveals key scientific issues to be investigated and suggests possible developments. Immunoprevention of hereditary cancer syndromes would be a straightforward translation from preclinical models, however the definition of suitable target antigens in most hereditary tumors is currently lacking and will require adequate immunological studies. Analogous problems face the application of cancer immunoprevention to other human groups at increased risk of cancer due to exposure to carcinogens and/or presence of preneoplastic lesions, moreover the lower level of risk (as compared to hereditary cancer) implies the recruitment of a large number of subjects. Lessons for the clinical deployment of cancer immunoprevention might come from the development of ▶tamoxifen, which was first used for therapy of advanced ▶breast cancer patients, followed by ▶adjuvant therapy i.e. for prevention of tumor relapse and ▶metastasis; the finding that tamoxifen also prevented the appearance of additional primary tumors eventually led to large prevention trials that demonstrated its efficacy in preventing breast cancer. Early clinical testing of preventive vaccines in advanced cancer patients will also provide much needed data concerning safety and risks of adverse effects. The Risks of Cancer Immunoprevention The lack of severe adverse effect registered in the clinical trials leading to the approval of HPV vaccines, along with similar results from worldwide HBV vaccination programs, indicates that the immunoprevention of virus-related cancers will share with other vaccines for infectious diseases an intrinsic high level of biosafety. The main reason is that the target antigens are not expressed by normal tissues of the host, hence the risk of triggering autoimmune reactions is very low. On the contrary, virtually all tumor antigens unrelated to infectious agents are also expressed by some normal cells during life. In some cases normal cells express a cross-reacting molecule with minor aminoacidic differences from the tumor version, but more frequently the

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antigens are structurally identical, and the only differences are either quantitative or topographical. The implication is that some autoimmune responses will frequently accompany successful prophylactic or therapeutic vaccination, even though development of autoimmune diseases is not an automatic consequence. A major difference between cancer therapy and prevention is that both physicians and patients accept inherently high risks of serious adverse effects when dealing with an existing life-threatening disease, whereas preventive treatments to be administered for long periods to healthy individuals need to minimize not only severe, but also mild adverse reactions. Preclinical studies of cancer immunoprevention did not reveal major risks of ▶autoimmunity, however the use of transgenic mouse models implies that in most instances protective immune responses were actually directed against transgenic products rather than against endogenous molecules, thus minimizing “real” autoimmunity. Therefore early clinical trials of cancer immunoprevention in humans will require intensive analyses to discover early signs of autoreactive immune responses and a special attention to the long-term risks of triggering autoimmunity.

References 1. Lollini PL, Cavallo F, Nanni P et al. (2006) Vaccines for tumour prevention. Nat Rev Cancer 6:204–216 2. Wheeler CM (2007) Advances in primary and secondary interventions for cervical cancer: human papillomavirus prophylactic vaccines and testing. Nat Clin Pract Oncol 4:224–235 3. Stewart BW, Kleihues P (eds) (2003) World cancer report. Lyon, IARC, pp 144–150 4. Lollini PL, Forni G (2003) Cancer immunoprevention: tracking down persistent tumor antigens. Trends Immunol 24:62–66

Immunoprophylaxis ▶Immunoprevention

Immunoreceptor ▶Chimeric T Cell Receptors

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Immunoreceptor Tyrosine-based Activation Motif

Immunoreceptor Tyrosine-based Activation Motif

Immunostimulatory Molecules Definition

Definition ITAM; A common cytoplasmic motif, YxxL/I-6-8YxxL/I, that activates signaling pathways; it is found for example in adapter molecules (DAP12) that associate with NK activation receptors that lack cytoplasmic domains (for example NKp44), which is recognized by downstream signaling molecules of the tyrosine kinase family. This conserved amino acid sequence was originally discovered in receptors found in immune cells, but has also been found in other types of non-hematopoietic, non-receptor proteins. Phosphorylation of two tyrosine amino acids within this motif creates a binding site for other proteins and activates complex signaling pathways. ▶Natural Killer Cell Activation

Are molecules that provide signals for antigenic specificity and immune response of T cells. Examples include ▶T cell receptor (TCR), CD28, CD40, B7-1 and ▶cytokines. ▶T-Cell Response

Immunosuppressed Definition A state of reduced immune function. ▶Immunosuppression ▶Allergy

Immunoregulatory Aberrations ▶Autoimmunity and Prognosis

Immunosuppression Definition

Immunoscreening cDNA Expression Libraries

Is a state in which the ability of the body’s immune system to fight infections or disease is decreased. A process or act that leads to a reduced activation or efficacy of the immune system.

Definition This method utilizes the exquisite specificity of antibodies to recognize a given antigen in the midst of thousands of antigens in a cDNA library. It lends itself to study structure and function of the expressed autoantigen in cDNA expression libraries of the desired cancer, built into a virus such as λgt11, T7, or other virus which are grown in bacteria. The viral coat displays the universe of potential autoantigens selected by autoantibodies in sera which can be arrayed forming a library. The microarray can be probed with multiple sera from patients and controls and finally, associations are sought between positive phages, diagnosis, or other clinical parameters ▶Autoantibodies

Immunosuppressive Drugs Definition Compounds that inhibit adaptive immune responses are called immunosuppressive drugs. They are used mainly in the treatment of graft rejection and severe autoimmune disease. ▶Adaptive Immunity ▶Allograft Rejection ▶Graft Acceptance and Rejection

Immunotherapy

Immunotherapy M EHMET K EMAL T UR 1 , S TEFAN B ARTH 1,2 1

Department of Experimental Medicine and Immunotherapy, Helmholtz Institute for Biomedical Engineering, University Hospital RWTH Aachen, Aachen, Germany 2 Department of Pharmaceutical Product Development, Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany

Synonyms Biological therapy

Definition Immunotherapy is the treatment of cancer or inflammatory/autoimmune disease by inducing, enhancing or suppressing an immune response. Immunotherapy can be nonspecific or (antigen)-specific. Nonspecific immunotherapy aims to enhance the overall host immune response, whereas specific immunotherapy targets the immune system against a particular tumor or increases tolerance towards a specific allergen. There are four main categories of specific immunotherapy: ▶adoptive immunotherapy, antibody-based immunotherapy, cancer ▶vaccine therapy and ▶allergen-specific immunotherapy. From these, adoptive and antibody-based immunotherapies are passive approaches, whereas cancer vaccine therapy and allergen-specific immunotherapy are active approaches.

Characteristics Despite advances in oncological research, cancer remains a leading cause of death throughout the developed world. Nonspecific approaches to cancer treatment, such as surgery, radiotherapy and generalized chemotherapy, have been successful in the management of a distinct group of leukemias and slow-growing solid cancers. However, many solid tumors show considerable resistance to such approaches, and the prognosis in these cases is correspondingly poor. Immunotherapy is an emerging alternative area of cancer treatment. Cancer immunotherapy includes both passive and active strategies. Passive immunotherapy involves the ex vivo creation of established tumor-immune elements (antibodies, immune cells) that are administered to patients to mediate anti-tumor activity directly or indirectly, and which do not stimulate the host immune system. In contrast, active immunotherapy induces a tumorspecific immune response in the patient, leading to the production of specific immune effectors (antibodies and T-cells). Historically, cancer immunotherapy has

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focused on nonspecific immune stimulants. Pioneer work began more than 140 years ago, when Wilhelm Busch observed that tumors show temporary regression during an infection. Two decades later, Sir William Coley developed and improved this therapeutic concept by vaccinating a large number of sarcoma patients with attenuated mixed bacterial extracts (▶Coley toxin). Nonspecific Immunotherapy 1. ▶Bacillus Calmette-Guerin (BCG) is the most effective intravesical nonspecific immunotherapeutic agent, and is used for the prevention and treatment of superficial ▶bladder cancer. The proposed anti-tumor mechanism of BCG involves activation of the immune system and the promotion of a local acute nonspecific ▶inflammation in the bladder lumen. Immune cell activation in response to BCG is mediated by a family of transmembrane recognition receptors called ▶Toll-like receptors (TLRs). Intravesical, BCG-induced inflammation facilitates the infiltration of a broad range of immune cells (▶macrophages, ▶lymphocytes and ▶natural killer cells) and the activation of pro-inflammatory cytokines such as ▶interleukin-1 (IL-1), ▶interleukin-6 and ▶tumor necrosis factor-alpha (TNF-α). 2. ▶Cytokines are low-molecular-weight, soluble proteins that regulate the innate and adaptive immune systems. The anti-tumor activity of cytokines is mediated by one of two general mechanisms: first, a direct anti-tumor effect, and second, indirect enhancement of the anti-tumor ▶immune response. It has been hypothesized that both the cytokineactivated lymphocytes and their secretory products such as interferon-gamma and tumor necrosis factorbeta (TNF-β) may contribute to the lysis of tumor cells in vivo. The exogenous administration of ▶interleukin-2 (IL-2) is efficient in a broad spectrum of experimental tumors, including sarcomas, carcinomas, hemoblastoses, melanomas and hepatomas. In humans, IL-2 and interferon-α2b are approved for the treatment of advanced melanoma and for use with ▶adjuvant therapy. Specific Immunotherapy 1. Adoptive Immunotherapy involves the infusion of immunologically-competent, ex vivo-expanded, donor-derived lymphocytes (DLI), which specifically destroy tumor cells by ▶graft-versus-leukemia (GvL) or ▶graft-versus-tumor (GvT) effects. In addition, peripheral blood-derived ▶lymphokine-activated killer (LAK) cells and ▶tumor-infiltrating lymphocytes (TILs) derived from tumor sections have proven to be effective anti-tumor agents. To address MHC and exogenous cytokine-independent activation of anti-tumor effector functions, T cells can be engineered to express ▶chimeric T-cell receptors.

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Immunotoxin

Chimeric receptors are composed of a recognition unit (antibody fragment) and an intracytoplasmic signaling molecule. Such receptors can be used to target various types of effector cells including cytotoxic T-cells towards any tumor-associated antigen for which there is a suitable antibody. 2. Antibody-based immunotherapy exploits the highly specific binding between antibodies and their corresponding ▶tumor-associated antigens (TAAs), resulting in some significant clinical responses. Tumor-associated antigens are structures presented predominantly by tumor cells, thereby allowing antibodies to distinguish tumors from non-malignant tissue. Therapeutic ▶monoclonal antibodies can destroy tumor cells directly by inducing ▶apoptosis or indirectly through immunologic mechanisms such as ▶antibody-dependent cell-mediated cytotoxicity (ADCC) and/or ▶complement-dependent cytotoxicity (CDC). In addition, the natural function of antibodies can be enhanced by conjugating them to toxins (▶immunotoxins), radionucleotides (radioimmunoconjugates), liposomes (▶immunoliposomes) and cytotoxic drugs. Host immune responses can be enhanced through the induction of ▶anti-idiotypic antibodies or through the use of ▶bispecific antibodies containing arms with different specificities. Monoclonal antibodies are the largest class of biotechnology-derived proteins, with 19 monoclonal antibodies already approved for human use by the United States Food and Drug Administration (FDA). 3. ▶Cancer vaccine therapy represents an active, systemic, tumor-specific immune response of host origin. It is used either to treat existing cancers (▶therapeutic vaccines) or to prevent cancer development (▶prophylactic vaccines). There are several types of cancer vaccine: isolated whole cell cancer vaccines or tumor cell lysates, protein- or peptidecontaining vaccines, viral vector vaccines and antiidiotype vaccines. Following the administration of a vaccine-antigen that resembles a specific target, the patient’s humoral and T-cell-specific immune response induces defense mechanisms to combat the target in vivo. ▶Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy

Immunotoxin Definition A toxic substance that is attached to a cell binding ligand and used to destroy a specific target cell. ▶Cytokine Receptor as the Target for Immunotherapy and Immunotoxin Therapy ▶Monoclonal Antibody Therapy

Importin-Alpha-3 Definition Is a nuclear localization signal receptor subtype that helps in translocation of certain proteins from cytosolic compartment to the nuclear compartment. ▶Transglutaminase-2

Importins Definition A group of proteins involved in transporting molecules from the cytoplasm through the nuclear pores of the nuclear envelope into the cell nucleus. ▶Modular Transporters

Imprinted Gene Definition

References 1. Schuster M, Nechansky A, Kircheis R (2006) Cancer Immunotherapy. Biotechnol J 1(2):138–147 2. Waldmann TA (2003) Immunotherapy: past, present and future. Nat Med 9(3):269–277

A gene that is marked in the germline; this denotes its maternal or paternal origin and influences its expression in the developing embryo. Refers to the parent-oforigin-specific monoallelic expression of a gene. ▶Imprinting

Imprinting

Imprinting C HRISTOPH P LASS German Cancer Research Center, Toricology and Cancer Risk Factors, Heidelberg, Germany

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of imprinted genes encode for RNAs but do not have an open reading frame and are not translated. It is believed that these RNAs play a role in the regulation of the imprinting process. Interestingly, short GC-rich repeat sequences were identified in the vicinity of many imprinted genes usually located in or near so called differentially methylated sites, ▶CpG island-like sequences that are methylated on only one allele.

Definition

▶Genomic imprinting; Describes a phenomenon in which a gene is expressed either from the paternal or form the maternal ▶allele and thus discriminates these genes from the majority of genes that are expressed from both alleles.

Characteristics Normally genes are expressed from both the maternal and the paternal allele. Genomic imprinting results in allele specific expression of certain genes from either the paternal or the maternal allele. These genes are marked before fertilization in a way that either the maternal or the paternal allele is transcriptionally silenced in the offspring. One of the first indications that certain autosomal regions are subject to genomic imprinting came from mouse genetic studies using Robertsonian and ▶reciprocal translocations. In these studies, uniparental duplications or deficiencies for certain chromosomal regions were analyzed. The failure of a disomy or duplication from one parent to complement a corresponding nullisomy or deficiency from the other parent constituted the genetic evidence for the occurrence of imprinting effects. In addition, embryos that contain either two copies of the maternal or the paternal genomes fail to survive in early development indicating the complementary need for both the maternal and paternal genome. More than 25 imprinted genes have been identified in mice and humans and there are estimates for about 100 imprinted genes in the mammalian genome. Certain characteristic features have been identified for imprinted genes. Most of the imprinted genes have important roles in early development. Interestingly, imprinted genes tend to occur in clusters suggesting a common regulatory mechanism. One of the best studied cluster of imprinted genes is located on mouse distal chromosome 7 (human 11p15.5), encompassing 1.5 Mbp and including the maternally expressed genes p57KIP2, Kvlqt1, Mash2 and H19 as well as the paternally expressed genes Ins2 and Igf2. It is now well accepted that imprinting could be regulated in a tissue specific manner in a way that only some tissues express the gene from one allele while others show biallelic expression. Here, unknown mechanisms exist that allow to by-pass the regulation of imprinting. It is interesting to note that a number

Cellular and Molecular Aspects Regulatory mechanisms underlying genomic imprinting are under intense investigations in many laboratories but only incompletely understood. The features of the imprinting signal and the mechanism are unknown, but strong evidence suggests the involvement of DNA ▶methylation. Several requirements for the underlying mechanisms can be postulated. First, the imprinting signal, or imprint mark, in the imprinted region must be established before fertilization. Second, the imprint mark must be an ▶epigenetic modification and must directly or indirectly affect the transcription of a gene by silencing one allele and leaving the other active. Third, the imprint mark must be stable in ▶mitosis and must be transmitted during cell division. Finally, the imprint mark must be reversible in a passage through the opposite germline. At present, DNA methylation is the only mechanism that conforms with the above requirements. Several lines of experimental data support the assumption that DNA methylation plays an important role in imprinted regulation. In mammals, ▶DNA methylation occurs only at the cytosine residue of ▶CpG dinucleotides. It was shown that DNA methylation in promoter regions can turn off the transcription of a gene. Most genes are subject to a process of demethylation directly after fertilization with most of the CpG sites unmethylated at the 16 cell stage. However imprinted genes are exceptions in this demethylation process by maintaining small regions that show allele specific methylation. DNA methyltransferase generates methylation patterns that are transmitted correctly following DNA replication and cell divisions. Studies of the expression of imprinted genes in DNA methyltransferase-deficient mutant mice indicated that normal level of DNA methylation are required for the control of allele specific expression. Studies with transgenic mice suggested that methylation is the epigenetic modification underlying genomic imprinting. A direct correlation between paternal inheritance, ▶transgene, ▶hypomethylation and tissue specific expression of the transgene was shown, while the maternally derived copy is methylated and not expressed. Clinical Relevance Imprinted genes are involved in critical steps during normal embryonic development. A growing body of

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In Situ Breast Cancer

evidence implicates genomic imprinting in the pathogenesis of certain human disorders, inherited tumor syndromes and sporadic tumors. At least ten genetic disorders have been found to be associated with genomic imprinting effects. In some cases, the trait is transmitted exclusively (or mainly) from one parent (either father or mother) or the disease is particularly severe when transmitted from one parent. In other cases the disease is associated with uniparental disomies or parent-of-origin specific aberrations. The best studied examples of imprinted genetic diseases are the Prader–Willi-Syndrome (PWS) and Angelman syndrome (AS). PWS is characterized by mild to moderate mental retardation, individuals are slow moving and overweight due to severe hyperphangia. Patients with AS show severe mental retardation are thin, hyperactive and show disorders of movement and uncontrolled laughter. Both syndromes are linked to abnormalities on human chromosome 15q11–13. The first hint for a possible imprinting effect in these syndromes came from the finding that the deleted fragments in both syndromes are from opposite parental origins. In PWS the deletion occurred in the paternal copy and in cases of AS the maternal copy was deleted. Additional evidence came from the finding of maternal disomy of chromosome 15 in PWS patients and paternal disomies of chromosome 15 in AS. These data suggest that the PWS gene(s) is transcribed from the paternal allele only and the AS gene(s) is expressed from the maternal allele. Several imprinted genes were identified in the critical region for PWS/AS including paternally expressed SNRPN and maternally expressed UBE3A. There is also evidence that some of the imprinted genes have oncogenic or tumor suppressor function. Loss of tumor suppressor function of an imprinted gene could be achieved by ▶loss of heterozygosity (LOH) involving the usually active copy, as shown for the cyclin dependent kinase inhibitor, p57KIP2, in lung cancers, H19 in ▶Wilms tumor and NOEY2, a member of the ▶RAS superfamily, in breast and ovarian cancers. Alternatively, uniparental disomy including the normally silent allele could lead to inactivation of an imprinted tumor suppressor gene. Activation of a growth supporting gene such as IGF2 (▶Insulin-like Growth Factors) could occur by uniparental disomy involving the normally active copy. In addition, relaxation of imprinting control, also called loss of imprinting (LOI), could lead to biallelic expression and thus overexpression of an imprinted oncogene, as shown for IGF2 in Wilms tumor. The first evidence for the involvement of DNA methylation in LOI came from the finding of complete methylation of the CpG island located immediately upstream of H19 transcription start site. Usually this ▶CpG island shows allele specific methylation on the maternal allele. This epigenetic change correlated with LOI in IGF2 and silencing of H19.

Another human disease is the ▶Beckwith–Wiedemann Syndrome (BWS) that is characterized by a number of growth abnormalities including gigantism. Between 5% and 10% of BWS patients are prone to Wilms tumor, adrenocortical carcinoma, hepatoblastoma or embryonal rhabdomyosarcoma. Wilms tumors have been shown to exhibit preferential loss of maternal alleles at chromosome 11p. A cluster of at least seven imprinted genes was identified in 11p15.5 including the paternally expressed IGF2 and the maternally expressed H19. The most common abnormality in BWS patients is LOI of IGF2 without any detectable chromosomal abnormalities.

References 1. Bartolomei MS, Tilghman SM (1997) Genomic imprinting in mammals. Annu Rev Genet 31:493–525 2. Cattanach BM, Kirk M (1985) Differential activity of maternally and paternally derived chromosome regions in mice. Nature 315:496–498 3. Falls JG, Pulford DJ, Wylie AA, et al. (1999) Genomic imprinting: implications for human disease. Am J Pathol 154:635–647 4. Nicholls RD, Saitoh S, Horsthemke B (1998) Imprinting in Prader–Willi and Angelman syndromes. Trends Genet 14:194–200 5. Reik W, Maher ER 1997 Imprinting in clusters: lessons from Beckwith–Wiedemann syndrome. Trends Genet 13:330–334

In Situ Breast Cancer ▶Ductal Carcinoma In Situ

In Situ Cancer or Carcinoma ▶Dormancy

In Situ Carcinoma ▶Dormancy

Indian

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Incidence

Definition

Definition

Describes experiments undertaken outside of a living organism (e.g. in Petri dish).

The number of new cases of a disease or condition occurring in a specific population during a given period of time.

▶Malignancy-Associated Changes

▶Incidence Rate

In Vitro Genetics Definition Is an umbrella term encompassing a variety of ▶combinatorial selection methods that involve large pools of nucleic acid sequence variants, a selectable function (e.g. protein binding or ribozyme-catalyzed chemical transformation), and ▶PCR amplification.

Incidence Rate I Definition Is the number of new disease cases per population at risk measured over a given time interval (high incidence rate implies high disease occurrence). ▶Epidemiology of Cancer ▶Cancer Epidemiology

In Vivo Definition Inside of a living organism.

Independent Prognostic Factor Definition

Inbred Lines ▶Mouse Models

An indicator used to estimate the risk of disease recurrence and death in an individual patient. Multivariate statistical analysis determines whether a prognostic factor exhibits a new, independent value as compared to established prognostic factors. ▶Prognosis ▶Prognostic Biomaker

Inbred Strain Definition A strain of animal that has no genetic differences (▶polymorphisms) with other animals of the same strain. ▶Mouse Models

Indian Definition

Gene in ▶Hedgehog Signaling.

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Indirubin and Indirubin Derivatives

Indirubin and Indirubin Derivatives G ERHARD E ISENBRAND, K ARL -H EINZ M ERZ Department of Chemistry, Division of Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany

Synonyms 3-(1,3-Dihydro-3-oxo-2H-indol-2-ylidene)-1,3-dihydro2H-indol-2-one; 2′,3-Biindolinylidene-2,3′-dione: 3-(3Indolinone-2-ylidene)-indolin-2-one

Definition Indirubin is the parent compound of a spectrum of 2′,3-bisindoles synthesized to improve the biological activity of this natural 2′,3-bisindole lead structure. Indirubin and its isomers indigo and isoindigo are composed of two indolinone ring systems, linked through a double bond to make up 2,2′-(indigo), 3,2′(indirubin) and 3,3′-(isoindigo) bisindoles, respectively (Fig. 1).

Characteristics History While indigo is one of the oldest dyes known, with a history of use dating back to bronze age, the two other isomers, especially indirubin, have gained reputation as components of traditional medications used against a variety of human diseases. The discovery of the anticancer activity of indirubin can be traced back to a traditional Chinese medication, consisting of 11 ingredients, mostly of herbal origin, with the name of Danggui Longhui Wan. The preparation is used traditionally for a variety of chronic and acute diseases including chronic myelocytic leukemia (▶CML). Chinese scientists achieved the identification of the active ingredient of this medication, Quing Dai, which corresponds to natural indigo, prepared from the leaves of indigo-producing plants. Furthermore, the Chinese scientists discovered that not only the blue dye indigo, but a minor byproduct, the red colored trace constituent indirubin was the active antileukemic principle. In a clinical trial, synthetic indirubin was given orally to CML patients at dosages of 150–450 mg/day. A total of 314 patients participated in the study. Complete remissions were observed in 26%, partial remission in 33%, and some beneficial response in 28% of patients. Treatment was well-tolerated, without major side effects. Studies exploring the mechanism of action reported a spectrum of relatively unspecific biological effects, not really convincing to fully explain the respectable anti-CML activity of the compound.

Indirubin and Indirubin Derivatives. Figure 1 Structures and numeration of indigoid bisindoles. (a) Indigo, (b) Indirubin (E211), (c) Isoindigo.

Mechanism Research on indirubins gained momentum, when in 1999 it was discovered that such 3,2′-bisindoles act as potent inhibitors of serine/threonine kinases, especially of ▶cyclin-dependent kinases (CDKs) and of glycogensynthase kinase 3β (GSK3β). Later it was found that indirubins also inhibit ▶receptor tyrosine kinases, such as ▶vascular endothelial growth factor receptor (VEGFR) or ▶Src kinase and block Stat-3 signaling. Moreover, indirubins have also been discovered to activate the ▶aryl hydrocarbon receptor transduction pathway. Thus indirubins exert, in addition to their multimodal kinase inhibitory activity, further bimolecular effects contributing to anticancer activity. The relative kinase inhibitory profile of individual representatives of these 3,2′-bisindoles, as well as their pharmacokinetic properties is markedly influenced by the type and pattern of substituents attached to the 3,2′bisindole basic scaffold. Within cancer targets, VEGFR2 and c-Src, as well as specific CDKs are of prime interest, since their respective partner proteins, activators or inhibitors, are aberrantly expressed in many human malignancies.

Indirubin and Indirubin Derivatives

Structure/Activity Chemical improvement of the parent molecule initially was driven by the aim to improve solubility and bioavailability without compromising anticancer activity.

Indirubin and Indirubin Derivatives. Figure 2 Superposition of crystal structures of CDK2–E226 (yellow) and CDK2–AMPPNP (white). Green arrows point to 5- and 3′-position of indirubin scaffold. (Reprinted from Davies et al (2001), Structure 9:389; with permission from Elsevier).

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Indirubins act as ATP-competitive inhibitors of ATP-dependent kinases. Crystal structures of CDK2–indirubin complexes have been described in detail. It was shown that the flat disk shape of the 3,2′-bisindole molecule slides easily into the binding pocket and is tightly bound, mainly by virtue of hydrogen bonding and lipophilic interactions with the ATP-binding cleft, situated in the hinge region connecting the amino terminal with the carboxy terminal part of the kinase. The strong binding affinity of indirubin derivatives to the ATP site is practically not affected by additional binding of the activating partner cyclin A to CDK2. A superposition of the structures of the binding complexes of the indirubin derivative, E 226, and of a nonhydrolyzable ATP mimic, AMPPNP, with CDK2, unraveled molecular positions of the indirubin scaffold preferably to be exploited for chemical improvement of the molecule. Thus, positions 5 and 3′ (arrows in Fig. 2) were identified as those of first choice for molecular modifications by attaching appropriate substituents. The chemistry to achieve the synthesis of such derivatives has been described in detail. A selection of results from comprehensive structure/activity studies is summarized in Tables 1 and 2. The results show that the inhibitory activity of the parent molecule, indirubin (E211) on isolated

Indirubin and Indirubin Derivatives. Table 1 Inhibition of CDK/cyclin complexes (IC50-Werte, μM) by indirubins in comparison with roscovitine Substance

E-Nr E211 E226 E231

1

O O NOH

2

R

H So3− H

CDK1/Cyclin B a

CDK2/Cyclin A

CDK6/Cyclin D

7.5 0.15a 0.33 ± 0.05b 0.25a 0.09 ± 0.01b

– – 0.08 ± 0.03b

a

E729

OCH3

E804

H

1.7 ± 0.4b

0.5 ± 0.1b

0.2 ± 0.04b

0.06 ± 0.01b

7.9 ± 0.4b 0.65a

3.5 ± 1.3b 0.7a

1.0 ± 0.4b 0.7a

>10b

Method: Meijer et al. (1997). Method: Jakobs et al. (2005) Glc: β-D-glucopyranosyl –: not determined.

b

CDK2/Cyclin E

2.2 0.035a 2.2 ± 0.2b 0.44a 0.8 ± 0.1b

a

10 0.055a 5.1 ± 2.9b 0.18a 5.6 ± 1.3b

Roscovitine a

R

0.4 ± 0.1b

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Individual Susceptibility

Indirubin and Indirubin Derivatives. Table 2 derivatives a

Substance E211 E226 E231 E729 E804

Solubility in water and human tumor cell growth inhibition of indirubin

Solubility in water (mg/L) >100 0.12 42 1.6

LXFL-529L 9.9 ≥100 3.0 0.5 0.9

MCF-7 4.0 ≥100 3.3 1.2 0.1

HCT116 – – – 0.5 8 years) and anatomical extent of colitis [standardized incidence ratio (SIR): 1.7, 95% confidence interval (CI): 0.8–8.2 for proctitis; SIR: 2.8, 95% CI: 1.6–4.4 for left-sided colitis; SIR: 14.8, 95% CI: 11.4–19.9 for pancolitis], appear as the most important and reproducible from one study to another. Other factors like family history of sporadic CRC (twofold higher risk), young age of IBD onset, backwash ileitis, and more recently degree of inflammation in the involved colon, are also considered as risk factors. Finally, association to primary sclerosing cholangitis (PSC), although rare (approximately 2–7.5% of IBD patients, more frequently in UC patients), appears as a very important risk factor and has conducted to a specific surveillance strategy in these particular patients since the cumulative incidence of patients presenting both UC and PSC was 33% at 20 years in a population-based Swedish study. By contrast, a few other factors have been suggested to be protective, such as folate, ursodeoxycholic acid, and with the highest degree of evidence, treatment by 5-aminosalicylates (5-ASA). The goal of surveillance strategies (i.e., surveillance ▶colonoscopy) is to detect by using a safe and effective intervention, neoplasia at a curable stage (ideally as ▶dysplasia). Dysplasia is defined as an unequivocally

Inflammatory Bowel Disease-associated Cancer

but noninvasive (intraepithelial) neoplasia. Despite the theory proposing that IBD-associated colon carcinogenesis progresses from no dysplasia to indefinite dysplasia, and then to low grade dysplasia (LGD), high grade dysplasia (HGD), and finally invasive cancer, in reality, this conceptually useful model is by no means absolute. This uncertainty thrives on much of the controversy regarding treatment of LGD. Except in the case of dysplasia in DALM which usually is not too difficult to detect for experienced practitioners, macroscopic identification of flat dysplastic lesions (either of low or high grade) appears particularly difficult during conventional colonoscopic surveillance. Nevertheless, this early identification represents a major challenge, as the presence of HGD in the colonic epithelium is associated with concurrent, macroscopically-undetectable CRC in 42–67% of patients (in case of colectomy performed shortly after HGD diagnosis at colonoscopy), and to the finding of a synchronous CRC in up to 19% of patients in case of LGD diagnosis at colonoscopy. Considering these facts, recommendations for colonoscopic surveillance have been established. Screening ▶colonoscopy should be initiated after 8–10 years of disease, including biopsies of each macroscopic lesion (with additional biopsies in the flat mucosa surrounding the DALM) and random biopsies of the macroscopically normal appearing mucosa (four-quadrant biopsies every 10 cm, some authors considering sampling every 5 cm in the rectosigmoid). In patients with PSC, colonoscopic surveillance should begin at the time of diagnosis of PSC and IBD. At each surveillance examination, the whole colon should be examined, and biopsies processed in separate clearly identified specimen containers. Recently, new endoscopic technologies have been suggested to improve the diagnosis yield of early malignancy, especially dysplasia detection in flat mucosa. This is the case for high-resolution and magnifying endoscopy, chromo (or dye) endoscopy based on vital staining with methylene blue or contrast staining with indigo carmine, magnifying chromoendoscopy, narrow-band imaging, and confocal laser endoscopy. These techniques open a new world of endoscopic imaging, but their usefulness for improving dysplasia detection in IBD needs to be carefully assessed before they will be recommended in routine screening. Nevertheless, the most recent data indicate that dye endoscopy with indigo carmine or methylene blue have to be seriously considered in daily practice. If no dysplasia is identified, surveillance examination is recommended every 1–3 years (some authors proposing a subsequent screening at 3 years between 8 and 20 years of disease, every 2 years between 20 and 30 years, and each year after 30 years of IBD diagnosis), except for IBD patients with concurrent PSC (surveillance colonoscopy recommended each

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year regardless of IBD duration). In case of LGD detection in flat mucosa, sample analysis by a second pathologist is recommended, and if LGD is confirmed, a surveillance colonoscopy should be performed 6 months later, although some authors propose to perform colectomy. Detection of HGD in flat mucosa indicates colectomy. Decision after LGD or HGD diagnosis in a polypoid lesion should integrate additional information: (i) if the polypoid lesion has been completely removed and no dysplasia has been detected elsewhere in the colon, a control colonoscopy with multiple random biopsies is recommended 6 months later; (ii) if this is not the case (incomplete removal and/or dysplasia detected in other biopsies), colectomy is indicated. A number of studies have examined the chemopreventive potential of several medications. Until now, 5-ASA have been the most thoroughly studied as potential IBD-associated CRC preventing agents. In fact, at least in vitro but also in some in vivo studies, 5-ASA have been shown to inhibit cell proliferation, to induce apoptosis, to act as potent RONS scavengers, and to enhance DNA repair. Although the optimal dose and duration of 5-ASA treatment to prevent IBDassociated CRC are unclear, data suggest that chronic systemic administration of 5-ASA at a dose of at least 1.2 g/day is the most likely to prevent IBD-associated CRC development. Small Bowel Adenocarcinoma in Crohn Disease The incidence of small bowel adenocarcinoma (SBA) is very low. In CD, its relative risk has been reported to be up to 50 times higher than in the general population. In a recent study, its cumulative risk in patients with ileal CD has been estimated to be 0.2% after 10 years of disease, and 2.2% after 20 years, with a median age at diagnosis of 47 years compared with 68 years for patients with SBA de novo. It occurs in a median time of 15 years after CD diagnosis arising from long-standing inflammation. CD-associated SBA is difficult to diagnose and causes premature mortality in early-onset CD patients. Cancer risk Associated to Immunosuppressive Therapy in IBD In the recent years, the efficacy of “classical” immunosuppressive drugs [azathioprine, 6-mercaptopurine (6-MP), or methotrexate], as well as that of biological agents (i.e., in current practice, anti-TNFα antibodies such as infliximab) led to their increased use in IBD. This emphasizes the question of their potential carcinogenic effects, in particular considering the risk of lymphoma which has been previously reported in patients treated by azathioprine or 6-MP after renal or hepatic transplantation (although used at doses even higher than in IBD). This question is not definitively resolved, probably due to the heterogeneity

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Inflammatory Cytokines

of the different studies, in particular when considering population-based cohorts on one hand and hospitalbased cohorts on the other hand. Discrepancies may result from varying factors such as cohort sizes, duration and dosage of treatment, and/or duration of follow-up. Nevertheless, some general statements based on the most relevant studies in the field can be suggested: (i) the absolute risk of ▶lymphoma in the general IBD population appears extremely low, (ii) lymphoma risk in IBD patients treated by azathioprine or 6-MP is probably not more than two- to fourfold increased, although the respective responsibility of treatment and underlying disease has to be more accurately investigated, (iii) particular attention needs to be brought to the ▶Epstein–Barr virus positive lymphoma risk in azathioprine or 6-MP treated IBD patients, and finally, (iv) the issue of lymphoma risk is likely to become more relevant in the future with the growing number of immunosuppressive and/or biological agents being used (or tested) in IBD, sometimes concurrently. This has been emphasized by the report of hepatosplenic T-cell lymphomas in young petients treated both with purine analogs and infliximab.

manner. ▶Interleukins, ▶lymphokines, and ▶interferons are all cytokines. ▶Aging and Cancer ▶Inflammation

Inflammatory Response Definition Refers to the ability of the innate component of the immune system to react to an infection or irritation. An orchestrated cellular and biochemical response of the body to injury or infection. Can be classified into acute and chronic. The response consists of cellular and exudative components. These involve the movement of white cells and fluid containing proteins and antibodies into the tissue to repair damage and inactivate a foreign agent. ▶Inflammation ▶Inflammatory Response and Immunity

References 1. Farraye FA (ed) (2006) Dysplasia and cancer in inflammatory bowel disease. Gastroenterol Clin North Am 35:517–734 2. Itzkowitz SH, Yio X (2004) Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol 287:G7–G17 3. Kwon JH, Farrell RJ (2005) The risk of lymphoma in the treatment of inflammatory bowel disease with immunosuppressive agents. Crit Rev Oncol Hematol 56:169–178 4. Li HC, Stoicov C, Rogers AB et al. (2006) Stem cells and cancer: evidence for bone marrow stem cells in epithelial cancers. World J Gastroenterol 12:363–371 5. Palascak-Juif V, Bouvier AM, Cosnes J et al. (2005) Small bowel adenocarcinoma in patients with Crohn’s disease compared with small bowel adenocarcinoma de novo. Inflamm Bowel Dis 11:828–832

Infratentorial Primitive Neuroectodermal Tumor ▶Medulloblastoma

Inherited Human Polycystic Kidney Disease ▶Polycystic Kidney Disease

Inflammatory Cytokines Definition Small proteins that are released primarily by activated immune cells. Inflammatory ▶cytokines act by binding to specific cell membrane receptors that are involved in amplification of inflammatory reactions. Cytokines can act in an ▶autocrine, ▶paracrine, or ▶endocrine

Inhibin Definition Dimeric peptide hormones (designated inhibin A and inhibin B) secreted by the follicular cells of the ovary

Initiation and Promotion

and the Sertoli cells of the testis. Inhibits production and secretion of ▶follicle stimulating hormone (FSH) by the pituitary. ▶Granulosa Cell Tumors

Inhibitor of Apoptosis Family Definition IAPs are a family of proteins that are important regulators of ▶apoptosis. IAPs function by binding to and inhibition of activated ▶caspases. IAPS are characterized by having one or more protein domains of 70 amino-acids called baculovirus IAP repeat (▶BIR domain). ▶Apoptosis-Induction for Cancer Therapy

Inhibitor of Cyclin-Dependent Kinase Definition:

Protein that inhibits the kinase activity of ▶CDK. ▶Early B-cell Factors ▶Cyclic Dependent Kinases

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which require administration to cells and tissues by various routes. ▶Cystatins

Initial Area Under the Gadolinium Contrast Agent Concentration–Time Curve Definition IAUGC; A model-free biomarker derived from contrast agent concentration data that is used as a biomarker in clinical trials of angiogenesis inhibitors. ▶Dynamic Contrast-Enhanced Magnetic Resonance Imaging

Initiation Definition In carcinogenesis, the first step in skin carcinogenesis is initiation, which is a reversible process during genetic mutations, gene activation or inactivation occur. Examples of initiation are mutations in the ▶RAS oncogene or inactivation of the p53 tumor suppressor gene ▶Skin Carcinogenesis

Inhibitor of FLICE ▶FLICE Inhibitory Protein

Initiation and Promotion Inhibitors

Definition

Definition

In carcinogenesis, the process in which an animal is treated with a low dose of a carcinogen, then this is followed once per week with a tumor promoter, and leads to a synergistic induction of tumors.

Proteins or chemicals capable of blocking the activity of an enzyme. Endogenous inhibitors are natural products of cells and tissues as opposed to exogenous inhibitors,

▶Chemical Carcinogenesis ▶Tumor Promotion

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Initiation Codon

Initiation Codon Definition The mRNA sequence AUG, which specifies methionine, the first amino acid used in the ▶translation process.

p18INK4c, and p19INK4d) contain four tandemly repeated ▶ankyrin motifs and have similar biochemical phenotypes. The p16INK4a is an alternative reading frame product (▶ARF) of p16INK4a and p15INK4b, which are coded at the human chromosome 9p21 region. The significance of the p16INK4a, ARF, and p15INK4b in tumorigenesis are confirmed by the high frequency of the genetic and epigenetic modifications of these genes in tumor samples.

Characteristics

Initiation Factors Definition

Initiation of ▶translation involves the small subunit of the ribosome binding to 5′ end of mRNA with the help of initiation factors.

Initiator Caspases Definition Are activated independent of cleavage by dimerization of the monomeric zymogen at multiprotein complexes, to which the ▶caspase zymogens are recruited by virtue of their N-terminal recruitment domain. In the extrinsinc pathway, ▶caspase-8 and -10 are activated at the death-inducing signaling complex (▶DISC), whereas in the intrinsinc pathway, the site of activation of caspase-9 is the ▶apoptosome.

INK4a TAKEHIKO K AMIJO Division of Biochemistry, Chiba Cancer Center Research Institute, Chuoh-ku, Chiba, Japan

Definition

Inhibitor of ▶cyclin-dependent kinase 4; proteins are cyclin-dependent kinase inhibitors that block the action of cyclin-dependent kinase to induce cell cycle arrest. The four INK4 family proteins (p16INK4a, p15INK4b,

Structure of INK4b/ARF/INK4a Locus and Molecular Functions of INK4s/ARF The INK4b/ARF/INK4a gene locus is located at the human chromosome 9p21 region. Chromosome region 9p21 is involved in chromosomal ▶inversions, ▶translocations, and ▶deletions in a variety of malignant cell lines and primary tumor samples, including those from ▶melanoma, pancreatic adenocarcinoma, ▶non-small cell lung cancer, ▶leukemia, and ▶glioma. These findings indicate that 9p21 contains a tumor suppressor locus that may be involved in the tumorigenesis of several tumor types. In a region of less than 40 kb of the human genome, three related genes, p15INK4b, ARF, and p16IN4a are encoded (Fig. 1). The INK4 class of ▶cell cycle inhibitors, p15INK4b and p16IN4a are homologous inhibitors of the cyclindependent kinases, CDK4 and CDK6, which inactivate the tumor suppressor RB1 protein via phosphorylation of its c-terminal region. The association of the INK4 proteins to CDK4/CDK6 induces an allosteric modification that abrogates the binding of these kinases to ▶cyclin D, resulting in inhibition of CDK4/6-mediated phosphorylation of retinoblastoma family member proteins. Hence, the existence of p15INK4b and p16INK4a maintains retinoblastoma family member proteins in a hypophosphorylation state, which facilitates binding of ▶E2F to induce a cell cycle arrest in the G1 phase (Fig. 2). ARF and INK4a have different first exons, exon 1 beta and exon 1 alpha, respectively. These first exons are spliced to a common second exon and third exon. Although exons 2 and 3 are shared by p16INK4a and ARF, these proteins are encoded in alternative reading frames. The predicted 132-amino acid p14 (ARF) is shorter than the corresponding mouse protein, p19(ARF), and the 2 proteins share only 50% identity. However, both proteins have the ability to elicit a p53 response, manifest in the increased expression of both p21Cip1/Waf1 and several p53-downstream proapoptotic molecules, resulting in a distinctive cell cycle arrest in the G1/G2M phases and apoptotic cell death, respectively. Previous reports showed that ARF binds to ▶MDM2 and promotes the segregation of MDM2 in the nucleolus. This interaction is mediated by the exon

INK4a

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INK4a. Figure 1 Genomic stucture of INK4b/ARF/INK4a locus. Residing on chromosome 9p21 in humans and chromosome 4 in mice, the INK4b/ARF/INK4a locus includes 2 different genes. INK4b and INK4a/ARF. INF4a and ARF, which have the independent exon1 alpha and exon1 beta, respectively, and common exon2/exon3, encode different proteins via an alternative splicing mechanism. In this commentary, p14ARF (murine p19ARF) will be referred to as ARF.

I

INK4a. Figure 2 Physiological roles of INK4s/ARF. Mitogenic signals activated cyclin D-dependent kinases, which phosphorylate RB and RB family proteins to facilitate entry into S phase. Consititutive oncogenic signals can activate the INK4a/ARF locus. By antagonizing the activity of cyclin D-dependent kinases, p16INK4a and p15INK4b prevent entry into S phase. MDM2 is a p53-inducible gene that normally acts to terminate the p53 response. The ARF protein inhibits MDM2 to induce p53, leading either to p53-dependent apotosis or to induction of the CDK inhibitor p21Cip1, inhibition of cyclinE/Cdk2, and RB-dependent cell cycle arrest.

1-beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. Roles in Tumorigenesis Human cancers frequently harbor ▶homozygous deletions of the INK4b/ARF/INK4a locus that abrogate expression of all p15INK4b/ARF/p16IN4a. In a large number of human cancers, specific somatic loss of p16INK4a, through ▶point mutation or small deletion, has been reported. Furthermore, silencing of p16INK4a through promoter methylation is reported at high frequency in numerous types of human malignancies.

Therefore, p16INK4a is an important tumor suppressor in human malignancies. In the case of p15INK4b, specific ▶epigenetic gene silencing by ▶hypermethylation of the p15INK4b promoter has been described in ▶hematological malignancies including ▶leukemia and ▶myelodysplastic syndrome and rare cases of glial tumors. In myelodysplastic syndrome, hypermethylation of p15INK4b has been reported in the absence of p16INK4a hypermethylation. p15INK4b seems to be an important tumor suppressor in specific lineages of human malignancies, e.g., hematological malignancies.

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INK4a

Selective inactivation of ARF, in the absence of additive loss of p16INK4a and p15INK4b, has only been reported in a small number of human malignancies, e.g., familial melanoma/astrocytoma patients and somatic ARF-specific mutations and promoter methylations in colon carcinoma patients. Therefore, it seems that ARF cooperates with p16INK4a and p15INK4b in tumorigenesis of human malignancies and their relative and combinational importance in any given tumor types remains to be elucidated. Roles in Senescence Cellular ▶senescence is a fundamental cellular program that is activated in various situations of stress and acts to prevent further cell proliferation. Senescence induced by extrinsic stress, such as DNA damage or oncogene activation, occurs relatively rapidly, in a matter of days. As a population of cells is propagated in culture, cells are exposed to various extrinsic and intrinsic stresses and the population gradually stops dividing. These findings have led to a distinction between “stressinduced premature senescence,” a term referring to rapid senescence triggered by extrinsic stress, and “replicative senescence,” a term referring to senescence that occurs following extended proliferation, presumably triggered by various stresses. Cellular senescence is thought to play an important role in tumor suppression and contribute to organismal aging. In fact, the two definitive tumor suppressor pathways, ARF/MDM2/p53 and p16INK4a/Rb, have been shown to play critical roles in the induction of cellular senescence. In tissue culture of primary cells, the accumulation of one or more INK4b/ARF/INK4a locus genes can eventually lead to cell cycle arrest through the mechanisms presented in Fig. 2. The cellular senescence is cancelled by downregulation of expression caused by gene deletion or epigenetic regulation, by inactivation of the gene products by mutation, or by cellular resistance to those cell cycle inhibitors. Considering the significance of the INK4b/ARF/INK4a locus genes in the cellular life span, p16INK4a plays a central role in senescence in human cells, whereas ARF assumes a prominent role in mouse cells. For mice, p19ARF and p16INK4a both accumulate significantly after passage, but spontaneous escape from senescence occurs through deletion of INK4/ARF or p53 mutation in wild type MEFs. Consistent with this, ARF-null MEFs do not have proliferation failure, but p16INK4anull and p15INK4b-null MEFs indicate limited proliferation. Whereas senescence generally occurs in the setting of increased expression of p16INK4a, but not ARF, and enforced Ras–Raf pathway activation also appears to induce only p16INK4a, along with senescence in cultured human cells. Furthermore, only p16INK4a induction has been reported with human

aging, although ARF expression studies in human aging have not been reported. Activators of the p15INK4b/ARF/p16INK4a Ras–Raf–p38MAPK Pathway Involvement of the Ras signaling pathway into the regulation of the INK4b/ARF/INK4a locus-encoded genes has been reported in detail. Expression of oncogenic Ras in primary human or rodent cells results in a permanent G1 arrest of the cell cycle. The arrest induced by ▶Ras is accompanied by accumulation of ▶p53 and ▶p16, and is phenotypically indistinguishable from cellular senescence. Constitutive activation of MAPKK induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Ras signaling pathway activation can result in phosphorylation and enhance binding of ▶Ets transcription factor to the p16INK4a promoter. These results imply that premature senescence via INK4b/ARF/INK4a locus activation acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Whereas oncogenic Ras induces ARF transcription in MEFs, similar effects have not been detected in human cells. One of the key molecules activated by Ras signaling pathway is DMP1, which binds directly to the p19ARF promoter region. Although there are putative binding sites in the human ARF promoter region, the effects of DMP1 have not been demonstrated in human cells. E2F The ▶E2F transcriptional factors are divided into two groups; (i) transcriptional activating E2F1, E2F2, and E2F3; (ii) transcriptional repressing E2F4, E2F5, and E2F6. Several E2Fs have been detected at the endogeneous ARF promoter by chromatin immunoprecipitation assay and directly activate ARF transcription without association with DP-1. Since ARF transcription is not regulated by a cell cycle-dependent manner, the unphysiological level of E2F seems to be required to induce ARF. Some studies described the inverse correlation between pRb status and p16INK4a expression in tumor cells. However, the physiological relation remains to be elucidated in terms of the relationship between the pRb/E2Fs and INK4 pathway. Myc Oncogene ▶Myc Oncogene has the ability to control p16INK4a transcription in human cells. Myc protein binds to the promoter and first intron of human p16INK4a, which is in line with the reports that Myc upregulates p16INK4a transcription in human cells. However, Myc has little effect on p16INK4a expression in mouse cells. In mouse cells, establishment of MEFs as continuously growing cell lines is normally accompanied

INK4a

by loss of the p53 or p19ARF, which act in a common biochemical pathway. Myc rapidly activates ARF and p53 transcription in MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive Myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. These observations are consistent with the cooperation of -Myc and Bmi1 in mouse lymphomagenesis, suggesting that the ARF/p53 pathway is a physiological safeguard system against Myc-induced oncogenic stresses. Suppressors of p15INK4b/ARF/p16INK4a AML1/ETO ▶AML1/ETO chimeric protein results from the t(8;21) translocation in human ▶acute myelogenous leukemia. AML1/ETO can bind directly to the ARF promoter regions as well as the POZ/BTB domain protein, ZBT7B. The translocation seems to convert the wildtype AML1 from being an inducer of ARF to a repressor. p53 One of the most important tumor suppressors, p53 seems to have a significant role in ARF transcriptional suppression because ARF is generally transcriptionally upregulated in ▶p53-inactivated cells. However, the mechanism of the transcriptional repression of ARF by reintroduction of wild-type p53 into p53-null cells remains to be elucidated. ▶Polycomb Proteins Traditionally, cancer has been viewed as a genetic disease that is driven by the sequential acquisition of mutations, leading to the constitutive activation of proto-oncogenes and loss of function of tumor suppressor genes. However, it has become increasingly evident that tumor development also involves “▶epigenetic changes” patterns of altered gene expression that are mediated by mechanisms that do not affect the primary DNA sequences. The INK4b/ARF/INK4a region is known to be regulated by not only the genetic alteration but also by epigenetic modifications. ▶Polycomb group (PcG) genes were first identified in Drosophila as a group of genes required for maintenance of stable repression of Hox cluster genes during development. There are increasing lines of evidence that PcG proteins themselves affect cellular proliferation and replicative senescence. Targeted disruption of Bmi1, Mel18, rae28, and M33, which are members of the class II PcG complex, leads to proliferation defects in hematopoietic stem cells and mouse embryo fibroblasts, indicating that inactivation of these PcGs results in cell proliferation failure.

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Replicative senescence of MEFs derived from Bmi1-, M33-, and Phc2-null mice has been shown to be mediated by derepression of the central mediators of senescence signals, p19ARF and p16INK4a, which are encoded by the INK4b/ARF/INK4a region. The molecular mechanism underlying the transcriptional regulation of these genes by mammalian PcG complexes, however, has not yet been appropriately addressed except for physical interactions of Bmi1 and Phc2 gene products to ARF and INK4a genomic regions. Future Directions The significant role of the INK4b/ARF/INK4a region and its products, p15INK4b/p14(p19)ARF/p16INK4a, in suppression of tumor development is well-established. However, regulatory mechanisms controlling these genes remain to be elucidated. The entire genomic region, which covers the genomic region from p15INK4b to p16INK4a (Fig. 1), is frequently deleted in a wide spectrum of tumors as described previously. However, the precise molecular mechanism of the biallelic gene deletion has not been addressed previously. In mouse MEFs, c-Myc induces the accumulation of p19ARF/p53 at the transcriptional level and has a significant proportion of the cells undergo apoptosis, whereas Myc-induced cell cycle accelerators, e.g., cyclin E, cyclin A, and CDC25A, are upregulated. In the face of Myc overexpression, there was a strong selective advantage for cells that sustained p53 mutations or the INK4b/ARF/INK4a deletion, and once such variants emerged, these soon predominated and were able to continuously proliferate. These results suggest that genes encoded by the INK4b/ARF/INK4a region are deleted by oncogene-derived stress-induced unknown molecular mechanisms and the safe guard machineries-broken cells continuously proliferate by oncogene-derived stimulations. The precise mechanisms of the locus deletion should be addressed to understand the immortalization and malignant transformation of normal cells. A particularly important recent finding with regard to the INK4b/ARF/INK4a regulation was a coordination of transcription at the locus and DNA replication. The coordination between silencing of the locus and DNA replication was reported. In detail, a DNA replication origin in close proximity to the locus appears to transcriptionally repress p15INK4b/ARF/ p16INK4a expression in a CDC6-dependent manner. Obviously, these findings suggest a novel molecular connection between DNA replication and p15INK4b/ ARF/p16INK4a transcription. Further analysis of the cooperation between this CDC6-dependent regulation and other known regulatory mechanisms of this locus, both of genetic and epigenetic regulations, seems to be worthwhile.

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Innate Immune System

References 1. Ruas M, Peters G (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378: F115–F177 2. Lowe SW, Sherr CJ (2003) Tumor suppression by Ink4aARF: progress and puzzles. Curr Opin Genet Dev 13:77–83 3. Sherr CJ (2001) The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2:731–737 4. Sparmann A, van Lohuizen M (2006) Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 6:846–856 5. Gil J, Peters G (2006) Regulation of the INK4b-ARFINK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7:667–677

Innate Immune System Definition Comprises the cells and mechanisms that defend the host from infection by other organisms or tumor growth, in an immediate and non-specific manner. The recognition of tumor cells by ▶natural killer cells is part of an innate immune response and does not depend upon the adaptive immune system; it does not confer long-lasting or protective immunity to the host. ▶Toll-Like Receptors ▶Adaptive Immunity

system. Innate immune cells recognize common molecular patterns on infectious agents, cells infected with viruses, and transformed cells. Cells of the innate immune response include ▶macrophages, ▶dendritic cells, mast cells, neutrophils, eosinophils, natural killer (NK) cells, natural killer T (NKT) cells and γδ cells, and the cytokines they secrete on activation, as well as antimicrobicides that are made by specialized cells and tissues. ▶Immunoediting ▶Bacillus Calmette-Guérin ▶DNA Vaccination

Innexins Definition Are the invertebrate gap junction proteins. ▶Pannexins ▶Gap-Junctions

iNOS Definition

Inducible ▶nitric oxide synthase.

Innate Immunity

▶Nitric Oxide ▶Nitric Oxide Synthase

Definition

Used together with the term ▶adaptive immunity to describe the two functional parts of the immune system. The original strict separation of the two parts of the immune system is currently reconsidered because a deep and intimate interconnection and cross-talk between the humoral and cellular parts of innate and adaptive immunity does exist. Nevertheless, innate immunity describes the immediate (within hours to a few days) reaction of the immune system in response to a given challenge. Typically involves, but is not limited to, (antimicrobial) phagocytes and substances, natural killer cells, certain cytokines and the complement

Inosine Definition A nucleoside; the breakdown product of adenosine through the enzyme adenosine deaminase (ADA). ▶Adenosine and Tumor Microenvironment

Inositol Lipids

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Among different inositol lipids, the importance in ▶transmembrane signaling and regulation of cell functions are best documented for PtdIns (4,5)P2 and PtdIns (3,4,5)P3. There are several ways in which these low abundance inositol lipids (less that 1% of membrane phospholipids) could provide a signaling link or fulfill other roles in different cellular processes.

trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG) molecules. The reaction is catalyzed by phosphoinositide-specific ▶phospholipase C (PI-PLC) (Fig. 1). There are several ▶isoforms of this enzymes (PLCβ, PLCγ, PLCδ, and PLCε) linked to and activated by different cellular receptors. For example, PLCγ is regulated through tyrosine kinase receptors such as receptors for ▶epidermal growth factor, ▶fibroblast growth factor, and ▶platelet-derived growth factor, while PLCε could be a novel target for ▶RAS proteins. The second messengers generated from PtdIns(4,5)P2 interact with specific intracellular targets and, in turn, cause their activation. Ins(1,4,5)P3 binds to specific receptors in the endoplasmic reticulum causing a release of calcium from this intracellular store into the cytoplasm. Membrane resident diacylglycerol (DAG) is required for activation of several isoforms of ▶protein kinase C (PKC). These second messengers act as a common component in different signaling pathways, contributing to diverse cellular responses. Specificity of the pathways is provided at the level of a receptor and downstream components (e.g., calcium-regulated proteins and PKC substrates) present in a specific cell type or state.

Hydrolysis of PtdIns(4,5)P2 to Generate Second Messenger Molecules Hydrolysis of PtdIns(4,5)P2 occurs in response to a large number of extracellular signals and generates two ▶second messenger molecules, inositol (1,4,5)

Binding of PtdIns(4,5)P2 to Specific Proteins In addition to its role as a precursor of Ins(1,4,5)P3 and DAG, PtdIns(4,5)P2 has emerged as a highly versatile signaling molecule in its own right. These other functions are mediated through direct binding of PtdIns(4,5)P2

Inositol Lipids M ATILDA K ATAN CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, London, UK

Definition

Are a class of ▶phospholipids where inositol is the polar headgroup. The simplest inositol phospholipid is ▶phosphatidylinositol (ptdlus). The inositol moiety can be phosphorylated at several different positions giving rise to a number of other molecular species.

Characteristics

Inositol Lipids. Figure 1 Structure of phosphatidylinositol (4,5) bisphosphate (PtdIns (4,5)P2) shows a typical phospholipid containing an inositol ring as a headgroup. The positions on the inositol ring are designated 1–6 and two phosphate groups are present at positions 4 and 5. Phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3) is generated in a phosphorylation reaction; the third phosphate group is added at position 3 of the inositol ring. Hydrolysis of PtdIns (4,5)P2 at the C-bond separates the hydrophobic part that contains two lipid chains, from watersoluble inositol that contains phosphates at the positions 1, 4, and 5.

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to specific protein targets, and include fundamental processes in membrane trafficking and plasma membranecytoskeleton linkages. Many proteins that regulate actin cytoskeleton (e.g., gelsolin and profilin) and proteins involved in ▶endocytosis (e.g., dynamin and AP-2 adaptor) bind PtdIns(4,5)P2. The binding involves different positively charged protein surfaces that in some proteins are present within the modular ▶pleckstrin homology domain (PH-domain). The result of the binding could be a direct change in the protein function or a regulated membrane targeting. For example, the PH domain of phospholipase Cδ1 associates with membranes of many cell types, but after PLC stimulation and the reduction in PtdIns (4,5)P2 concentrations, it translocates to the cytoplasm. Concentration of PtdIns(4,5)P2 is not only regulated at the level of hydrolysis by PLC but also through regulation of several types of inositol lipid kinases and phosphatases. Generation of PtdIns(3,4,5)P3 and Other 3-Phosphorylated Inositol Lipids and Their Binding to Specific Intracellular Targets Inositol lipids phosphorylated at the 3-position of the inositol ring (PtdIns(3)P, PtdIns(3,4)P2, PtdIns (3,5)P2, and PtdIns(3,4,5)P3) are generated by ▶phosphoinositide 3-kinase (▶PI3-K). PI3-Ks are grouped into three classes on the bases of their structure and according to the inositol lipid they preferentially utilize as a substrate. For example, the class I PI3-Ks are receptor-regulated ▶signal-transducer proteins that preferentially phosphorylate PtdIns(4,5)P2 in vivo and generate PtdIns(3,4,5)P3 (Fig. 1). Several target proteins for PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been described and they include protein kinases such as PKB/Akt, PDK1, and Btk. In the case of PKB/Akt, the direct binding to the PH domain (with high affinity and specificity toward 3-phosphorylated inositol lipids compared with more abundant PtdIns(4,5)P2) results in both membrane targeting and conformational changes that lead to phosphorylation and activation of this protein kinase. Activated kinases, in turn, phosphorylate and regulate downstream targets and thus propagate the signal. The 3-phosphorylated inositol lipids also participate in diverse cellular functions including cell survival, proliferation, migration, and vesicle budding. In addition to regulation of PI3-K, the levels of PtdIns (3,4,5)P3 are also controlled by a 3-phosphatase. Clinical Relevance There is considerable experimental evidence that the key enzymes involved in the control of inositol lipids, ▶PIPLC and ▶PI3-K, play an important role in processes critical for tumor development and spreading, including cell proliferation, survival, and ▶migration. However, oncogenic or constitutively active mutants of either

PI-PLC or PI3-K have not yet been isolated from human tumors. In the case of PI3-K, an oncogenic form (v-p3k) of the class I PI3-K has been isolated from a chicken ▶retrovirus that causes ▶hemangiosarcomas. Another oncogenic form of class I PI3-K (mutation in the regulatory subunit) has been isolated from transformed murine lymphoid cells. Nonetheless, the importance of the control of inositol lipid levels in human cancers have been emphasized by the findings that the tumor suppressor protein ▶PTEN is a 3-phosphatase that dephosphorylates PtdIns(3,4,5)P3. The PTEN gene is deleted or mutated in a wide variety of human cancers. Many human tumors have also been found to express increased levels of PI-PLC or PI3-K. The role of a signaling protein in generation and spreading of a malignant tumor is not limited to its function as an oncogene or a tumor suppressor gene. For example, it has been documented that the activation of PLCγ is a rate limiting step in breast and prostate tumors that overexpress growth factor receptors. This type of tumor is associated with a poor prognosis. PLCγ seems to be required for cell migration but not for proliferation, and the motility and invasiveness of cancer cells are strongly inhibited either after treatment with a chemical PLC inhibitor (U73 122) or in the presence of molecular inhibitors. ▶Polyphosphoinositides

References 1. Katan M (1998) Families of phosphoinositide-specific phospholipase C: structure and function. Biochim Biophys Acta 1436:5–17 2. Katan M, Allen VL (1999) Modular PH and C2 domains in membrane attachment and other functions. FEBS Lett 452:36–40 3. Czech MP (2000) PIP2 and PIP3: complex roles at the cell surface. Cell 100:603–606 4. Rameh LE, Cantly LC (1999) The role of phosphoinositide 3-kinase lipid products in cell function. J Biol Chem 274:8347–8350 5. Wells A (2000) Tumor invasion: role of growth factorinduced cell motility. Adv Cancer Res 78:31–101

Inositol Polyphosphates Definition IPs; Inositol derivatives with various numbers of inorganic phosphate groups attached at each of the carbon atoms. ▶Pentakisphosphate

Insulin Receptor

Inositol Tetrakisphosphate

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Insulator

Definition

Definition

Inositol Polyphosphate (IP) containing four phosphate groups within the inositol ring.

Is a DNA sequence that acts as a barrier to the influence of neighboring genes.

▶Pentakisphosphate

▶CCCTC-Binding Factor

Inositol 1,4,5-trisphosphate Definition Natural compound containing three phosphate groups at the 1, 4 and 5 positions within the inositol ring. Its production is a common step seen in cell signaling. ▶Pentakisphosphate

Insulin Definition Is a natural hormone produced in the pancreas by the beta cells of the ▶islets of Langerhans; controls the level of the sugar glucose in the blood. Insulin permits cells to use glucose for energy. Cells cannot utilize glucose without insulin. People who are Type 1 ▶Diabetes mellitus must use manufactured insulin, usually in an injectable form, to replace the natural insulin that is no longer produced by their body (for instance as the result of beta-cell degeneration). People with Type 2 sometimes need to use insulin when their cells become too resistant to the insulin that they produce naturally and when oral medications are no longer working.

Inositoltrisphosphate Definition

IP3; is generated by ▶phospholipase C from phosphatidylinositol and is instrumental in Ca2+ mobilization required in signal transduction. ▶Lipid Mediators

Insulin Receptor A NTONIO B RUNETTI Department of Experimental and Clinical Medicine “G. Salvatore”, University of Catanzaro “Magna Græcia”, Catanzaro, Italy

Definition

Insertional Mutagenesis Definition Is the alteration of a gene by integration of a foreign, often exogenous, DNA sequence. For instance, a virus DNA can integrate into a gene or in the vicinity of a gene. ▶Retroviral Insertional Mutagenesis

IR; Is a phylogenetically ancient ▶receptor tyrosine kinase protein embedded in the ▶plasma membrane of virtually all cells. When the peptide hormone ▶insulin binds to the IR, the receptor becomes activated and induces a cascade of intracellular events that will lead to several metabolic and growth promoting effects.

Characteristics The IR belongs to the tyrosine kinase growth factor receptor family and functions as an enzyme that transfers phosphate groups from ▶ATP to tyrosine

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residues on intracellular target proteins. The IR consists of two identical extracellular alpha subunits (130 kDa) that house insulin binding domains, and two transmembrane beta subunits (95 kDa) that contain ligandactivated tyrosine kinase activity in their intracellular domains (Fig. 1). When insulin binds to the alpha subunits, the receptor is first activated by tyrosine autophosphorylation, and then the IR tyrosine kinase phosphorylates various intracellular effector molecules (e.g. ▶IRS proteins and ▶Shc) which in turn alters their activity, thereby generating a biological response. The IR exists as two splice variant isoforms: the IR-B isoform that is responsible for signaling metabolic responses involved mainly in the regulation of glucose uptake and metabolism by increasing glucose transporter (▶Glut4) molecules on the plasma membrane of the insulin-responsive tissues muscle, liver, and fat, and the IR-A isoform that is capable of binding ▶IGF-2 with high affinity and signals predominantly mitogenic responses. As a consequence of these cellular activities, abnormalities of IR expression and/ or function can facilitate the development of several metabolic and neoplastic disorders in humans as well as in animal models. Regulation Gene expression in eukaryotic cells is controlled by nuclear regulatory proteins (trans-acting factors) that modulate the transcription of genes by binding to specific cis-acting transcriptional elements in the promoter of target genes. The IR gene promoter extends over 1,800 bases 5′ upstream from the IR gene ATG codon, contains a series of GGGCGG repeats that are putative binding sites for the mammalian transcription factor Sp1, and has neither a ▶TATA box nor a CAAT box, reflecting the common features for the promoters of constitutively expressed genes (so-called housekeeping genes). Like other housekeeping promoters, the IR gene promoter confers a basal level of transcriptional activity common to all cells, whereas significantly higher transcriptional activity is induced in the muscle, liver, and fat, at which levels the IR has been shown to be under the regulation of hormones, metabolites, and differentiation. Promoters of genes that are activated in a tissue specific manner are often regulated by a combination of tissue specific and ubiquitous transcription factors, where the ubiquitous element facilitates or enhances the action of one or more tissue-specific transcription factors. The molecular mechanisms regulating IR gene expression are being elucidated and evidence has been provided showing that the architectural transcription factor ▶HMGA1 is required for proper transcription of the IR gene in cells expressing IRs. HMGA1 acts on the IR gene promoter as an element necessary for the formation of a transcriptionally active multiprotein–DNA complex involving, in addition to

the HMGA1 protein, the ubiquitously expressed transcription factor Sp1 and the CCAAT-enhancer binding protein beta (C/EBP-beta). By potentiating the recruitment and binding of Sp1 and C/EBP-beta to the IR promoter, HMGA1 greatly enhances the transcriptional activities of these factors in the context of the IR gene. Conversely, repression of HMGA1 function in cells and tissues adversely affects transactivation of the IR gene promoter by Sp1 and C/EBP-beta, and considerably reduces IR protein expression. Clinical Relevance The IR is of major importance in certain states of ▶insulin resistance in humans, in which abnormalities of the receptor may lead to defective transmembrane signaling. In this respect, dysfunctional IR signaling is implicated in certain common dysmetabolic disorders, including ▶obesity, type 1 and type 2 ▶diabetes, the dysmetabolic syndrome X, and the polycystic ovary syndrome (PCOS). Also, clinical syndromes due to mutations in the IR gene have been identified in patients with genetic forms of severe insulin resistance (i.e. leprechaunism, type A insulin resistance, and the Rabson–Mendenhall syndrome). Many of these patients have point mutations in the coding sequence of the IR gene, leading to reduced or absent IR expression in target tissues. Recently, defects in IR gene regulation have been reported in individuals with insulin resistance and type 2 diabetes, in which the generation of IR mRNA was considerably impaired, although the IR genes were normal. In these individuals, cellsurface IRs were decreased and the expression of HMGA1 was markedly reduced. Even though it is an open question whether IR plays a critical role in aging and longevity in mammals, disturbance of the neuronal IR seems to be of pathogenetic relevance in human Alzheimer’s disease and depressive disorders, suggesting a neurotrophic role of IR in the brain. According to recent studies, IR in the brain begins to disappear early in Alzheimer’s and continue to decline as the disease progresses. It has been shown that stimulation in the brain of a receptor that mediates insulin responses can halt or diminish the neurodegeneration of Alzheimer’s disease. Also, a high prevalence of insulin resistance has been reported in patients with depression, and an increased risk of cognitive decline has been found in women with insulin-resistant PCOS. A relation between IR and cancer has been established following the observation that overexpression of functional IRs can occur in human ▶breast cancer and other epithelial tumors including ovarian and colon cancer, in which the IR may exert its oncogenic potential via abnormal stimulation of multiple cellular signaling cascades, enhancing growth factor-dependent proliferation and/or by directly affecting cell metabolism.

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Insulin Receptor. Figure 1 In the absence of insulin, most of the IRs in the plasma membrane are in a non-tyrosine phosphorylated inactive state, and only a very small proportion of receptors are lightly phosphorylated and subjected to constitutive ▶endocytosis and recycling. Upon binding of insulin, the IR undergoes autophosphorylation which enables the receptor to have a kinase activity and phosphorylates various cytoplasmic substrates, such as IRSs. From this point, signaling proceeds via a variety of signaling pathways (i.e. PI3K signaling pathway, Ras and MAP kinase cascade) that are responsible for the metabolic, growth-promoting and mitogenic effects of insulin.

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An explanation for increased IR expression in epithelial tumors has been recently provided for several human breast cancers, in which overexpression of the transcription factor ▶AP2-alpha accounts for increased IR expression in neoplastic breast tissue. In these cases, it has been demonstrated that transactivation of the IR gene by AP-2 alpha needs direct physical association of AP2 with HMGA1 and Sp1, which represents a fundamental prerequisite for AP-2 alpha to activate IR gene transcription in neoplastic breast tissue. Epidemiological and clinical evidence points to a link between insulin resistant syndromes such as obesity and type 2 diabetes and cancer of the colon, liver, pancreas, breast and endometrium. The mechanistic link between insulin resistance and cancer is unknown, but constitutive activation of the tyrosine kinase activity of IR and related downstream signaling pathways by chronic sustained hyperinsulinemia (a hallmark of insulin resistance) in these clinical syndromes appears to have a role in the neoplastic transformation process. Thus, the IR plays a major role in the pathophysiology of a wide range of metabolic, neurodegenerative and proliferative disorders in humans. Selective modulation of IR expression and/or function may represent a useful therapeutic strategy for these diseases. Also, measures to decrease insulin resistance in insulin resistant patients may offer a general approach to prevention of cancer in these predisposed individuals.

References 1. Goldfine ID (1987) The insulin receptor: molecular biology and transmembrane signalling. Endocr Rev 8:235–255 2. White MF, Khan CR (1994) The insulin signaling system. J Biol Chem 261:1–4 3. Yarden Y, Ullrich A (1998) Growth factor receptor tyrosine kinases. Annu Rev Biochem 57:443–478 4. Foti D, Chiefari E, Fedele M et al. (2005) Lack of the architectural factor HMGA1 causes insulin resistance and diabetes in humans and mice. Nat Med 11:765–773 5. Paonessa F, Foti D, Costa Vet al. (2006) Activator protein2 overexpression accounts for increased insulin receptor expression in human breast cancer. Cancer Res 66:5085–5093

Src-homology 2 (SH2) domain containing proteins to bind and transmit signals downstream. ▶Receptor Cross-Talk ▶Insulin Receptor ▶SH2/SH3 Domains

Insulin Resistance Definition Occurs when the body does not respond properly to ▶insulin, a hormone made by the pancreas. It is characterized by the diminished ability of cells to respond to the action of insulin in transporting glucose from the bloodstream into muscle and other tissues. ▶Adiponectin ▶Diabetes

Insulin-like Growth Factor Binding Proteins Definition IGFBPs are a family of secreted proteins that bind the ▶insulin-like growth factors, IGF-I and IGF-II, with affinities comparable to their respective receptors. To date, six insulin-like growth-factor-binding proteins (IGFBP-1 to IGFBP-6) have been identified. ▶Kallikreins ▶Diabetes

Insulin-like Growth Factors Insulin Receptor Substrate Proteins

RUSLAN N OVOSYADLYY, D EREK L E R OITH Division of Endocrinology, Diabetes and Bone Diseases, Departmant of Medicine, Mount Sinai School of Medicine, New York, NY, USA

Definition

IRS proteins; Large intracellular ▶docking proteins through which the ▶insulin receptor and IGF-1Recepror propagate their signals. Receptor tyrosine phosphorylation of IRS proteins yields sites for

Definition The insulin-like growth factors (IGF-I and IGF-II) are structurally related molecules that play essential roles in the regulation of cell survival, growth, proliferation,

Insulin-like Growth Factors

differentiation and metabolism. The IGF family is comprised of (i) ligands (IGF-I, IGF-II and insulin), (ii) six well characterized high affinity binding proteins (IGFBP-1 through -6), (iii) IGFBP proteases and (iv) cell surface receptors that mediate the biological functions of IGFs. These transmembrane receptors include the IGF-I receptor (IGF-IR), IGF-II/mannose 6-phosphate receptor (IGF-II/M6PR), ▶insulin receptor (IR) and insulin receptor-related receptor (IRR). In many tumor cells the IGF-IR is often upregulated and/or hyperactivated. Furthermore, increased circulating IGF-I levels are considered a significant risk factor for the development of various types of cancers. Although the oncogenic role of IGFs (i.e. their ability to initiate ▶carcinogenesis) is still under the debate, numerous lines of evidence suggest that these powerful growth factors enhance tumor growth and ▶progression.

Characteristics IGFs The insulin-like growth factors (IGF-I and IGF-II) are ubiquitously expressed growth factors that are structurally related to insulin. However, in contrast to insulin and other peptide hormones, they are not stored within cells of a specific tissue but are produced by numerous cell types in the body and circulate in approximately 1,000-fold higher concentrations than most other known peptide hormones. These properties suggest more universal functions of the IGFs in the body compared to the more specific metabolic role of insulin. The IGFs are critical in a broad range of functions during pre- and postnatal life. In adult tissues, IGFs are important trophic factors that support normal differentiated functions of various tissues and prevent programmed cell death (▶apoptosis). IGF-I is known as a major regulator of postnatal growth. Most of the circulating IGF-I is produced by the liver, although other tissues are capable of synthesizing this peptide locally. Thus, IGF-I has characteristics of both a circulating hormone and a tissue growth factor. In contrast to IGF-I, IGF-II plays a fundamental role in embryonic and fetal growth, whereas due to ▶imprinting of the Igf 2 gene, its role in postnatal period of life is less important, especially in rodents.

IGF Receptors Most of the actions of both IGF-I and IGF-II are mediated via the IGF-IR, which is expressed by virtually all cell types except adult hepatocytes. The IGF-IR and IR belong to the large family of ▶receptor tyrosine kinases. The two receptors are structurally related and are composed of two α-subunits localized entirely extracellularly and two β-subunits spanning the membrane

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and localized primarily intracellularly. Both subunits are linked together by disulfide bonds. They assemble a α2β2-configuration with ligand binding primarily mediated by the α-subunits, which form a binding pocket. Binding of the ligand to the α-subunit leads to conformational changes resulting in stimulation of the β-subunit intrinsic tyrosine kinase activity with subsequent multisite phosphorylation of the β-subunit. The current concept is that insulin and the IGFs act as bivalent ligands, both IGF-IR and IR are capable of binding insulin and IGF-I or -II, though each receptor binds its own ligand with a 100–1,000-fold higher affinity than the heterologous peptide. In cells expressing both receptor genes, hybrid insulin/IGF-I receptors can form. The hybrid receptors have ligand specificity profiles more comparable to the IGF-IR than to the IR since they bind IGF-I with an affinity similar to the IGF-IR, but insulin with a much lower affinity. Moreover, the IR is also responsible for some of the mitogenic actions of IGF-II. IGF-II is an agonist of the A isoform of the IR. This splice variant of the IR is expressed at high levels in fetal and neoplastic tissues. IRR and hybrid IR/IRR have not yet been extensively studied, and their ability to bind all the different insulin-like peptides as well as their biological significance remains unclear. The IGF-II/M6PR is structurally distinct from the IGF-IR and is actually identical to the cationindependent mannose 6-phosphate receptor, which lacks tyrosine kinase activity and is not considered to have any role in IGF ▶signal transduction. The IGF-II/ M6PR functions as a scavenger receptor and is involved in uptake and degradation of IGF-II. The IGF-II/M6PR is strongly expressed during tissue differentiation and organogenesis, and high levels of the IGF-II/ M6PR were found in fetal tissue, which decline in late gestation and in the early postnatal period due to genomic imprinting. IGFBPs Unlike insulin, the IGFs are present in the circulation and throughout the extracellular compartments almost entirely bound to a family of multifunctional, structurally related, high affinity IGF-binding proteins (IGFBPs), which can modulate biological effects of the IGFs. To date, six IGFBPs with high affinity have been cloned and sequenced. All share structural homology with each other and specifically bind the IGFs. They differ in molecular mass, binding affinities for the IGFs, posttranslational modifications such as phosphorylation and glycosylation as well as susceptibility to proteolysis. The IGFBPs act as carrier proteins in plasma, control the efflux of the IGFs from the vascular space, and prolong half-lives of the IGFs. They regulate metabolic clearance and tissue- and cellspecific localization of the IGFs thereby modulating

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their biological actions in a negative or a positive manner. Finally, some IGFBPs also have intrinsic bioactivities that are independent of the IGFs (Fig. 1). Mechanisms The biological effects of IGFs are mainly mediated by the IGF-IR and two principal signaling pathways including ▶mitogen-activated protein kinase (MAPK) pathway that plays a pivotal role in cell growth and proliferation, and ▶phosphatidylinositol-triphosphate kinase (PI3K) pathway, which is mainly involved in mediating the metabolic, antiapoptotic and other more differentiated effects of IGF-I. Upon ligand binding and receptor autophosphorylation, the IGF-IR recruits and phosphorylates several adaptor proteins including ▶Shc and members of the insulin receptor substrate (IRS) family of proteins. They bring together and coordinate the activity of other signaling intermediates, finally resulting in activation of the ▶MAPK and PI3K pathways. Typically, the MAPK pathway is initiated by the recruitment of growth factor receptor bound 2 protein (Grb2) that via the guanine nucleotide exchange factor Son of Sevenless (SOS) stimulate the activity of the GTPases ▶Ras and Rac, which, in turn, through the sequential phosphorylation of certain kinases finally lead to activation of terminal MAP kinases ERK1/2, ▶JNK and p38 kinase. Although JNK and p38 kinase are primarily activated by environmental stressors, several lines of evidence suggest that they can also be activated in response to IGFs. The activated MAP kinases phosphorylate several important cytoplasmic substrates and also translocate to the nucleus where they phosphorylate transcription factors leading to immediate-early gene induction followed by progression of the cell cycle. Alternatively, signal transduction through the PI3K pathway results in the activation of protein kinase B also known as ▶Akt, which is known to block apoptosis by phosphorylating numerous cellular substrates such as Bad, GSK3, Foxo, Mdm2, CREB, IKK, caspase-9, p21 and p27. The PI3K pathway also activates p70S6 kinase involved in regulation of ribosome biogenesis as well as some isoforms of ▶protein kinase C, which are capable of potentiating signal transduction through the MAPK pathway. Thus, IGFs induce cell proliferation both by enhancing cell cycle progression and by inhibiting apoptosis. Furthermore, evidence for both direct and indirect interaction between the IGFIR and other growth regulatory signals has been demonstrated, thereby expanding the traditional view of highly specific IGF-IR/IGF interactions and rendering the IGF-IR central in cellular response. For instance, in many cell types IGF-IR and ▶epidermal growth factor receptor family members physically interact and transphosphorylate each other. ▶Estrogen receptor activation augments the IGF-I response in estrogen

receptor-positive MCF-7 mammary carcinoma cells at multiple levels. Moreover, estrogens enhance the tyrosine phosphorylation of the IGF-IR and IRS-1 and eventually increase expression of cyclins and reduce the level of cdk inhibitors (Fig. 2). Altered Expression of the IGF System Components in Tumor Cells The expression of the components of the IGF system is often altered in malignant cells. In certain tumors, the ▶imprinting of the Igf 2 gene is lost, and this results in increased IGF-II gene expression. In general, IGF-II is more commonly expressed by tumors than IGF-I, although increased IGF-I expression has been found in numerous tumors as well. The IGF-IR is often upregulated or hyperactivated in tumor cells. Expression of the IGF-IR is regulated by tumor suppressors and growth factors in a negative and positive manner, respectively. Tumor suppressors such as ▶Wilms Tumor-1 (WT1) and ▶p53 bind to the Igf1r gene promoter and inhibit receptor gene expression. Mutations of these genes occur in various tumors and paradoxically enhance the activity of the Igf1r gene promoter. This explains the upregulation of the IGF-IR gene expression in Wilms tumor (a pediatric kidney tumor) and ▶colon cancer, which is often accompanied by p53 mutations. Both WT1 and p53 also inhibit IGF-II gene expression. By analogy with the IGF-I receptor, IGF-II gene expression is increased when WT1 and p53 genes are mutated. Thus, the autocrine IGF-IR/IGF-II loop is turned on under these circumstances. Basic ▶fibroblast growth factor and ▶platelet-derived growth factor are capable of enhancing the IGF-IR gene expression. Since tumors often express these and other growth factors, this could also upregulate the IGF-IR gene expression. Certain ▶oncogenes can also regulate the IGF-IR at posttranslational level. For instance, ▶Src augments the phosphorylation state of the IGF-IR, thereby increasing its kinase activity. Neoplastic growth can be also enhanced by injections of recombinant human IGF-I into mice. In this case, the latency period is shortened and tumor growth is accelerated. This response is particularly enhanced in tumors with higher levels of IGF-IR expression. In contrast to the IGF-IR, the IGF-II/M6PR expression is often decreased or lost in tumor cells. The IGF-II/M6PR possesses properties of a ▶tumor suppressor gene. Tumor cell growth is inhibited when the IGF-II/M6PR expression is restored and is increased when its expression is reduced. In addition to the IGFs and their receptors, the IGFBP expression is also altered in tumor cells. Various IGFBPs are expressed by numerous tumors and often in different combinations. For example, estrogen receptor-positive breast cancer cells release IGFBP-2, whereas estrogen receptor-negative breast tumors


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Insulin-like Growth Factors. Figure 1 The IGF family is comprised of (i) ligands (IGF-I, IGF-II and insulin), (ii) IGF-binding proteins (IGFBPs), (iii) IGFBP proteases and (iv) cell surface receptors. IGFBPs act as carrier proteins in plasma, control the efflux of the IGFs from the vascular space, prolong half-lives of the IGFs, regulate their metabolic clearance, provide tissue- and cell-specific localization of the IGFs, modulate biological actions of the IGFs, and some also have intrinsic bioactivities that are IGF-independent. The IGFs can be released from the IGF/IGFBP complexes by the action of IGFBP proteases. The insulin receptor (IR), insulin-like growth factor I receptor (IGF-IR) and insulin receptor-related receptor (IRR) are heterotetrameric complexes composed of extracellular α-subunits that bind the ligands, and β-subunits that anchor the receptor in the membrane and contain tyrosine kinase activity in their cytoplasmic domains. Hybrids consist of a hemireceptor from both IR and IGF-IR. The IGF-II/M6PR is not structurally related to the IGF-IR and IR or the IRR, having a short cytoplasmic tail and no tyrosine kinase activity. IR is responsible for metabolic effects, whereas IGF-IR and hybrid IR/IGF-IR for cell survival, growth, proliferation and differentiation. The insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/M6PR) functions as scavenger receptor and is responsible for uptake and degradation of the IGFs. This receptor is not considered to have any role in IGF signaling.

release IGFBP-1 and -3. The IGFBP production can be altered by growth factors, steroid hormones, cytokines, and these changes in IGFBP levels may alter biological effects of the IGFs on tumor growth and progression. Interestingly, wild type p53 induces the expression of IGFBP-3 that seems to be critical for the inhibitory function of p53 on cell growth. Furthermore, enhanced IGFBP proteolysis is thought to contribute to carcinogenesis. Numerous IGFBP proteases produced by tumor cells mediate release of the IGFs from the IGFBP/IGF complexes that eventually leads to

increased IGF-IR stimulation. For instance, prostate cancer cells secrete ▶prostate-specific antigen (PSA), which exerts IGFBP-3 proteolytic activity thereby enhancing the local bioavailability of free IGFs. Syndrome of Hypoglycemia An emerging clinical syndrome is tumor-induced hypoglycemia. This phenomenon is often seen in terminally-ill, poorly nourished patients. In addition, it can be also observed in patients with large mesenchymal tumors in the abdomen or thorax that

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Insulin-like Growth Factors. Figure 2 Signal transduction cascades initiated by the IGF-IR. Activation of the IGF-IR kinase results in receptor autophosphorylation and tyrosine phosphorylation of several docking proteins such as Shc and insulin receptor substrate (IRS) proteins. Once activated, IRS molecules recruit Src homology 2 (SH2)-domain containing molecules such as Grb2 and the p85 subunit of phosphatidylinositol-3′-kinase (PI3-K). Grb2 via SOS stimulates the activity of the GTPases Ras and Rac, which through the phosphorylation of numerous kinases finally lead to activation of terminal MAP kinases ERK, JNK and p38 kinase. Activated MAP kinases are translocated to the nucleus where they activate a variety of transcription factors. Alternatively, the binding of the p85 and p110 subunit of PI3-K to IRS proteins generates phospholipids that participate in the activation of 3-phosphoinositide-dependent kinase (PDK) 1 and 2. In turn, they phosphorylate several targets involved in the regulation of different biological processes including glucose transport, protein synthesis, glycogen synthesis, cell proliferation and cell survival.

secrete large quantities of IGF-II. In these patients, IGF-II is not fully processed, and therefore it is poorly bound to the circulating IGFBPs. This allows IGF-II to interact more readily with insulin receptors thereby causing hypoglycemia. Clinically, tumor-induced hypoglycemia can be diagnosed in cancer patients that have normal or elevated circulating IGF-II levels, whereas their insulin, growth hormone (GH), IGF-I and IGFBP-3 levels are suppressed. These patients usually have a poor prognosis. Surgical excision and ▶chemotherapy or ▶radiation therapy-induced reduction of tumor size is ▶palliative, and GH and corticosteroid therapy provides symptomatic relief. Clinical Aspects An increasing body of evidence suggests that the IGF system is a promising target for adjuvant anticancer therapy. If chemotherapy inadequately ablates tumor

cells, then blockade of the proliferative and antiapoptotic effects of the IGFs may be helpful. The IGF system could potentially be targeted at various levels. Reduction of circulating IGF-I levels can be achieved by GHRH antagonists or somatostatins as well as GH receptor antagonists. Application of inactive IGF molecules, small-molecule competitive binding antagonists or soluble IGF-I receptors may inhibit binding of endogenous IGFs to the receptor thereby abrogating their tumor-promoting effects. The IGF-IR represents another attractive therapeutic target. Approaches that are currently being tested in preclinical and clinical studies include the use of blocking ▶monoclonal antibodies directed against the extracellular portion of the IGF-IR and ▶small molecule drugs that inhibit the tyrosine kinase activity of the IGF-IR. Application of ▶RNA interference and ▶antisense therapy to reduce the IGF-IR expression, as well as overexpression of altered or

Insulinoma

truncated IGF-IR that acts in a ▶dominant-negative manner, represent additional approaches that have been effective in laboratory studies.

References 1. Khandwala HM, McCutcheon IE, Flyvbjerg A, et al. (2000) The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocrine Rev 21:215–244 2. LeRoith D, Werner H, Beitner-Johnson D, et al. (1995) Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocrine Rev 16:143–163 3. Pollak MN, Schernhammer ES, Hankinson SE (2004) Insulin-like growth factors and neoplasia. Nat Rev Cancer 4:505–518 4. Rubin R, Baserga R (1995) Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest 73:311–331 5. Samani AA, Yakar S, LeRoith D, et al. (2007) The role of the IGF system in cancer growth and metastasis; overview and recent insights. Endocrine Rev 28:20–47

Insulinoma B OAZ H IRSHBERG Cardiovascular and Metabolic Diseases, Pfizer Inc, Groton, CT, USA

Synonyms

β-Cell tumor of the islets

Definition

Insulinomas are functioning ▶insulin producing tumors of the pancreatic ▶islets of Langerhans, resulting in hypoglycemia.

Characteristics

Insulinomas arise from the β-Cell of the pancreatic islets. The nonregulated secretion of insulin from these tumor cells into the blood stream results in fasting hypoglycemia. Insulinomas are relatively rare, with approximately four cases per million person-years. However, they are the most common tumor of the pancreatic islets. Insulinomas may appear at any age, but the majority appears in the fifth decade. Insulinomas are evenly distributed along the pancreas. Insulinomas, associated with ▶multiple endocrine neoplasia type 1 (MEN1), tend to appear one decade earlier. Insulinomas are usually solitary and their localization is even along the pancreas, exceptions are those associated with MEN1, where multiple tumors are the rule.

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Diagnosis The presence of ▶Whipple Triad (hypoglycemia and neuroglycopenic symptoms that are corrected by the administration of carbohydrate) is the hallmark of the diagnosis of insulinoma. Fasting, therefore, is the major maneuver used in the diagnosis of insulinoma and has two important purposes. The first goal is to document hypoglycemia and its relationship to the patient’s symptoms, and the second is to demonstrate inappropriate insulin concentrations in the face of hypoglycemia. The prolonged (48–72 h) fast is the gold standard for the diagnosis of hypoglycemia. This study should be conducted under supervised conditions (i.e., while hospitalized). Diagnosis of insulinoma has been centered on the 72-h fast that was introduced long before to measure insulin or insulin-related components. Now the insulin and proinsulin measurements are widely available, all of the necessary information from a fast can be derived in the first 48 h. Thus, the 48-h fast has become the new standard. The diagnosis is based on detectable insulin levels (≥6 μU/ml), detectable C-peptide, and elevated proinsulin levels, when the patient has symptoms of hypoglycemia and glucose levels tÞ

Kaplan–Meier Product Limit Estimator ▶Kaplan-Meier Survival Analysis

¼ Pðsurviving past time tÞ and is called the survival function. Note that although t measures the time-until-failure, it also represents the disease-free survival time. Fig. 1 illustrates a typical Kaplan–Meier survival function estimate.

Kaplan-Meier Survival Analysis

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Kaplan-Meier Survival Analysis. Figure 1 Kaplan–Meier survival curve.

Censoring If all individuals in the study fail, we can precisely describe the survival distribution, SðtÞ ¼ 1 FðtÞ Suppose we have decided to follow patients in a clinical trial for 5 years and that our outcome is recurrence at a specific tumor site. For those who are diagnosed with recurring tumor during the study period, exact failure times are recorded. Those who complete the 5-year follow-up but do not have a recurrence are said to be right censored. By focusing on survival instead of failure, we are able to conclude that the survival time was at least 5 years for the censored individuals without knowing the exact time of failure beyond that 5-year study period. In other words, we may not know the exact time of failure for individuals that survived longer than 5 years, but we do know they lived without recurrence for at least 5 years. In addition to ▶censoring by the limits of the study design, individuals may also be censored due to factors beyond the investigators control, such as death from other causes. Although the analysis itself does not distinguish between these types of censoring, one caveat in generalizing results is that intermittent censoring must be non-informative so that the reason for censoring should not be related to the either the time of failure or the treatment choice. In other words, this type of censoring should be random. Parametric, Semi-Parametric and Nonparametric Survival Three major divisions are made in the types of survival analyses; ▶parametric, semi-parametric and

▶nonparametric. If survival times are assumed to follow a specific mathematical distribution, the parameters of that distribution can be estimated from the sample. Perhaps the simplest parametric assumption in survival analysis is one of a constant hazard rate, λ, leading to the failure distribution, FðtÞ ¼ 1 elt , and the complementary survival distribution, SðtÞ ¼ elt that we recognize as exponential decay. Metabolism studies, for example, may exhibit exponential decay. ^l ¼ r=T The parameter, λ, is estimated from the data asP where r is the total number of events and T ¼ t is the total of all survival times. Other useful parametric survival distributions are the Weibull, lognormal and gamma distributions. Another common approach introduced; by D. R. Cox in 1972 and called ▶Cox proportional hazards modeling has become one of the most common methods of survival analysis and is based on semi-parametric estimation where the background hazard rate is not estimated. Cox P assumed the survival distribution, expð bxÞ where S ðtÞ is a baseline hazard SðtÞ ¼ ½S ðtÞ 0

0

rate that is typically treated as a nuisance, x is some covariate of interest and β is the slope parameter associated with that covariate. In this type of analysis, we are usually interested in comparing covariate parameters so that S(t) itself is not estimated fully. Finally, nonparametric methods including actuarial and life table methods are useful for summarizing survival distributions. Perhaps one of the nonparametric approaches to survival analysis that is most often used in the medical literature is that introduced by E. L Kaplan and P. Meier in 1958. Useful in visualizing

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Kaplan-Meier Survival Analysis

survival data, the Kaplan–Meier curve is plotted as a starting point in most survival analyses. Unlike the parametric and semi-parametric approaches to quantifying a survival distribution, the nonparametric Kaplan– Meier method requires few assumptions other than independence of failure time observations. Kaplan–Meier Product Limit Estimator Many of the survival analysis methods, such as Cox proportional hazards models and parametric models, require iterative solutions and specialized software. The Kaplan–Meier Product Limit Estimator, on the other hand, is easily calculated. The r event times are ordered from smallest to largest so that t1 < t2 < t3 < < tr . For example, assume the relapse times (in months) for ten cancer patients are recorded as (2, 3, 5, 7, 9, 11, 12, 12, 12, 12). If there is no censoring, the Kaplan–Meier estimator at any event time, j, is simply the proportion with event times greater than tj. For example, at t = 7 months four have relapsed and six have not so that the probability of surviving more than seven months is ^ Sð7Þ ¼ 6=10 . In the case of single right censoring at the end of the study, the calculations are similar. If the relapse times are (2, 3, 5, 7, 9, 11, 12+, 12+, 12+, 12+) ^ where “+” indicates censoring, Sð7Þ ¼ 6=10 is the proportion surviving longer than 7 months as before. In addition to the plot of these data in Fig. 1, we might choose to report summary statistics in the form of quantiles, medians or means. For these data, the median ^ is 9 months since Sð9Þ ¼ 0:5 . The mean is related to the area under the survival curve and, for data sets in which the largest observation is censored, the mean is based on the truncated failure times so that the estimate of the mean is too low. In this case, the mean is 8.5 months. The probability of surviving can be found using simple counting rules and the multiplication rule for joint conditional probabilities. At each distinct event time, there are nj individuals at risk of whom dj relapse and nj dj do not relapse. Assume the event times are recorded as follows: (2, 3, 5, 7, 9, 11, 12+, 12+, 12+, 12+). At 7 months, we find nj ¼ 7 individuals still at risk of relapse of whom dj ¼ 1 relapses and nj dj ¼ 6 do not. Applying the multiplication rule to the conditional probabilities for each distinct event time, the probability of surviving past 7 months is 9 8 7 6 6 ^ Sð7Þ ¼ ¼ 10 9 8 7 10 as we found previously. If any censored times are less than any event times, we must alter the formula slightly. In essence, the Kaplan–Meier product limit estimator is still an application of counting rules and conditional probabilities but the numbers at risk at some event times are altered due to censoring. Suppose we find that one

observation is censored prior to seven months and the event times are recorded as (2, 3, 5+, 7, 9, 11, 12+, 12+, 12+, 12+). In terms of an event, all we know of the individual who was censored at five months is that they at least survived longer than the previous event time of 3 months. The product of the conditional probabilities using only event times is 9 8 6 ^ ¼ 0:6857 Sð7Þ ¼ 10 9 7 and is slightly different from the estimate when no censoring prior to seven months was observed. The censored observation provides additional information for the first two periods by increasing the overall sample size. The median survival time for these data is 11 months, illustrating the effect of censoring on the estimates. The mean is 9.1 months but, again, may be too low due to the censoring of all observations at 12 months. In general, the Kaplan–Meier estimator is defined as Y nj dj ^ ¼ SðtÞ nj tj 40 years of age with an even sex distribution. Interestingly, >60% of GISTs are associated with exon 11 mutations in Kit/ SCF-R, but none with mutations in exon 17, including of D816. Kit exon 11 GOF mutations occur mainly in malignant GISTs, which tend to be larger, with necrosis, hemorrhage, intra-abdominal spread and liver metastases and frequent recurrences. Hence, the exon 11 mutations portend poor prognosis with a 3 year survival 65% for exon 11 mutation negative tumors, and it has been reported that Kit exon 11 mutations are an independent prognostic factor for GIST survival. The Kit/SCF-R (CD117) expression is diagnostic for GI stromal tumors versus ▶leiomyomasa and gastric Schwannomas. Most cases examined are also CD34-positive, and ultrastructurally cells look like ▶interstitial cells of Cajal (ICC), which is why it has been proposed that the ICC is the cell of origin for most GISTs. However, tumors phenotypically identical to GISTs (CD117+, most CD34+) occur as primary tumors in the omentum and mesentery as well, which indicates that GISTs might not all originate

Kit/Stem Cell Factor Receptor in Oncogenesis

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Kit/Stem Cell Factor Receptor in Oncogenesis. Figure 2 Schematic representation of mutations in Kit/SCF-R, associated with human malignancies and dysplasias. Amino acid residues are denoted with single letters, numbers indicate the residue number in the human Kit protein sequence. Point-mutated amino acid residues are shown in outline, amino acid deletions with a dash and amino acid additions are underlined. The D52N mutation has been associated with myelodysplastic syndromes that include myelofibrosis and chronic myeloid leukemia. The AY duplication, the juxtamembrane mutations and the K642E mutations have mainly been associated with gastro-intestinal stromal tumors, but also with mast cell leukemias. The D816V and E839K mutations have been connected with mast cell leukemias. The D816H mutation has been connected with seminomas and dysgerminomas. Mutations, found in the hydrophilic region between the N- and C-terminal region of the kinase domain result -due to alternative promoter usage- in truncated versions of Kit. These isoforms have been identified in various cell lines which derived from colon carcinomas and hematopoietic malignancies.

from ICCs, but rather from a multi-potential precursor cell. A characteristic of GISTs is mitochondrionrich ICCs, and some GISTs are gastro-intestinal autonomic nerve tumor (GANT)-like stromal tumors. An immunohistochemical and histological re-evaluation

of archived paraffin-embedded esophageal tumor samples disclosed that 25% of these were indeed Kit-positive GISTs with exon 11 mutations rather than leiomyomas or leiomyosarcomas, which was the original classification. This is important, since

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esophageal GISTs are malignant, while leiomyomas, which are Kit-negative, are benign. Analysis of eight GISTs devoid of exon 11 mutations in Kit/SCF-R revealed exon 9 mutations in 6 and an exon 13 mutation (K642E) in 2, the latter causing constitutive tyrosine autophosphorylation of Kit/SCF-R. Finally, in colon carcinoma cell lines certain intron 15 alternative promoter splice variants, causing either a 78 bp deletion or a truncated Kit/SCFR with 25 unique amino acids fused to the C-terminal 257 amino acids, have been reported sporadically. The functional consequences are unknown. However, a potential autocrine loop of Kit/ SCF-R and SCF might exist in colon cancer, since colonic mucosal cells usually express SCF, but areKit/ SCF-R-negative. In conclusion, a majority of GISTs harbor GOF mutations in exon 11 of Kit/SCFR and there is overwhelming evidence that these are involved in the oncogenesis. Kit expression and exon 11 mutations are both of significant diagnostic and prognostic value for GISTs, the prognosis being significantly more severe if both are present.

Acute and Chronic Myeloid Leukemias A vast number of publications have attempted to address the putative role of Kit/SCF-R in myeloid leukemias. While it is still unclear whether Kit/SCF-R is causally involved in these diseases, it does have an important diagnostic and prognostic value. While only 1–3% of normal mononuclear marrow blasts express Kit/SCF-R, a big European multi-center study concluded that 67% of all ▶acute myeloid leukemia (AML) cases, 30% of all biphenotypic acute leukemias and all undifferentiated acute leukemias express Kit/SCF-R. Kit/SCF-R is expressed mainly in M4 and M5 AML subclasses, but the highest expression levels are found on blasts in M1 and M2 subclass AML cases. A high proportion of megakaryocytic and erythroid leukemic cells are also positive for Kit expression, as is most blasts in chronic myeloid leukemia (CML). In general, Kit/SCF-R expression is useful in the differential diagnosis between AML (mostly positive) and acute lymphocytic leukemia (ALL; all negative). Negative expression for Kit/SCFR in AML also identifies with some certainty two M5b subgroups. AML blasts express between 600 and 29,000 Kit molecules/cell, but there is no correlation between expression level and prognosis. Despite conflicting reports, it seems to be the consensus from the literature that there is no correlation between Kit expression and prognosis for AML in general, but that expression of Kit/SCF-R in the M1 subclass indicates a worse prognosis. This might be due to a strong correlation between expression of a non-P-glycoprotein multidrug resistance protein and Kit/SCF-R. Mutations in exon 8 of c-kit have been identified in

a high proportion of AML cases, and all had the inv. 16 re-arrangement. Conversely, 20% of inv. 16 AMLs had c-kit exon 8 mutations. All exon 8 mutations involved either deletion or replacement of the codon for D419. The functional consequences of these mutations for the kinase activity of Kit/SCF-R are unknown at present. However, retroviral transduction of murine hematopoietic precursors with D816V-Kit and transplantation of these cells into syngeneic hosts resulted in myeloid leukemias in a significant proportion of cases, showing that GOF mutations in Kit is sufficient for leukemic progression in mice. It has been suggested that constitutive association of ▶BCR-ABL1 with activated Kit/SCF-R is responsible for the basophilia and myeloid growth in the chronic phase of ▶CML. However, the ability of SCF to stimulate blast growth has also been utilized to mobilize peripheral CD34+/CD38– and other committed progenitors in patients about to undergo bone marrow transplantation for subsequent autologous transplantation. Addition of SCF together with GCSF and with standard chemotherapy is superior to GCSF and chemotherapy for this purpose.

Germ Cell Tumors All carcinoma-in-situ (▶CIS) testis, 90% of seminomas and dysgerminomas, but only 5% of nonseminomas express Kit/SCF-R. Isochrome 12p is a marker of ▶germ cell tumors, and ▶loss of heterozygosity on the long arm of chromosome 12 implicates SCF as a tumor suppressor. Furthermore, intersex gonads (45X/46XY and other cases with additional Y chromosome material) have delayed Kit expression and increased testicular cancer risk. This could indicate an anti-oncogenic role of Kit/SCF-R and its ligand for germ cell tumor development under some circumstances. However, other results might indicate that SCF and Kit/SCF-R can drive tumor progression. Hence, ectopic expression of Kit and SCF has been found in cervical and ovarian carcinomas and ovarian teratomas. Furthermore, there is an association between mediastinal germ cell tumors (MGCT) and hematological malignancies (e.g. acute leukemia and malignant histiocytosis), and often Kit-positive areas are found in these mediastinal tumors. This and other results have made it clear that Kit/SCF-R expression is a diagnostic aid for extragonadal seminomas. All classical seminomas are Kit-positive, aneuploid and positive for placental alkaline phosphatase, while 40% of spermatocytic seminomas are Kit-positive, and all are diploid or polyploid and negative for placental alkaline phosphatase. This indicates that some spermatocytic seminomas might originate from primordial germ cells. In line with this, experimental testicular teratomas can be generated by transplanting E12 male genital ridges to


testes of adult mice. Importantly, it was recently found that tumors of seminoma/dysgerminoma type had a D816H mutation in Kit/SCF-R causing its constitutive activation. In conclusion, Kit/SCF-R expression is of diagnostic help for seminomas/dysgerminomas, and GOF mutations in Kit/SCF-R might be oncogenic and involved in the generation of such tumors. Malignant Melanoma Normal melanocytes depend on ▶bFGF, ▶HGF and SCF in vitro. ▶Melanoma cells become independent of these growth factors, in part through autocrine bFGF stimulation. Interestingly, Kit mRNA and protein are down-regulated in human and murine ▶melanoma cell lines. This correlates with in vivo findings: While Kit/ SCF-R is expressed in normal melanocytes, benign and dysplastic naevi and nontumorigenic melanomas, expression is lost in dysplastic naevi, tumorigenic primary melanomas and metastases. In addition, transfection of Kit/SCF-R into highly metastatic melanoma cell lines, induced slowed growth rate and fewer lung metastases in nude mice. The transcription factor AP-2 controls expression of c-kit and the gene for MCAM/MUC18 positively and negatively, respectively. AP-2 is downregulated in melanomas and this is thought to be the reason for loss of Kit expression, allowing the malignant cells to escape SCF-induced apoptosis. Conversely, enforced AP-2 expression suppresses tumorigenicity and metastatic potential, possibly through c-kit transactivation and subsequent SCF-induced apoptosis. It has been proposed that AP-2 loss is a crucial event in malignant melanoma development. Other Neoplastic/Malignant Lesions Kit/SCF-R and its ligand have been found co-expressed in cells from ▶small cell lung cancer and ▶neuroblastoma, and it was reported that it might be involved in malignant progression in these cases. In ▶neurofibromatosis 1 (NF-1) there is infiltration with Kit-positive mast cells in the neurofibroma tissue, which is composed mainly of Schwann cells with an increased SCF mRNA expression compared to normal skin. The mast cells produce collagen VIII, which might contribute to the fibrosis in this disease. There is an abnormally high expression level of Kit in NF-1derived Schwann cell lines and decreased neurofibromin expression (Ras-GTPase). The proliferation is Kitdependent. In myelodysplastic lesions, Kit mutations might be involved in the pathogenesis. A recurrent D52N-Kit/SCFR mutation has been reported in these cases. Finally, down-regulation of Kit/SCF-R has been reported in breast cancer and in ▶thyroid carcinoma, despite expression by normal mammary duct epithelial cells and thyroid cells. It has been proposed that the Kit/ SCF-R downregulation enables de-differentiation of the cells in these tumor types.

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Gene Transfer, Immunotherapy, Vaccination SCF might be useful in expansion of peripheral blood leukocytes and of hematopoietic progenitors in culture before retroviral transduction or re-introduction in conjuction with autologous bone marrow transplantation. Phase II and III trials are currently being conducted on advanced stages of breast cancer and certain leukemias for this purpose. SCF might also be useful in conjuction with immuno-therapy. For instance, following high-dose ▶cyclophosphamide and IL-3, ▶dendritic cells can be mobilized and expanded ex vivo from CD34+ cells in the presence of GM-CSF, TNFα, Flt3 ligand and SCF. Dendritic cells are competent antigen-presenting cells for CD8+ cytotoxic T cells, so they can be used to stimulate the host immune defense against undesirable antigens, including tumor antigens. Ongoing phase II trials are examining the use of such expanded dendritic cells for immunotherapy or vaccination. Finally, in the past five years several small molecule protein kinase inhibitors have been approved as drugs for the treatment of specific types of cancer. Among these are two that act as inhibitors of Kit tyrosine kinase activity. ▶Imatinib (STI571/GleevecTM), an Abl, PDGFR and Kit inhibitor, was originally approved by the FDA for the treatment of chronic myelogenous leukemia (▶CML) in 2001. Shortly thereafter, imatinib was shown to be efficacious for treatment of gastrointestinal sarcomas (GIST), which are caused by mutant forms of Kit and PDGFRalpha, and was approved by the FDA for treatment of GIST in 2002. In 2006, a second tyrosine kinase inhibitor (TKI), ▶sunitinib (SU11248/ SutentTM), was approved for the treatment of imatinib-resistant GIST. Several additional TKIs have either approved or are in clinical trials for the treatment of cancer and other diseases caused by mutant Kit and other activated tyrosine kinases, and the number of approved TKI drugs is likely to grow significantly in the next ten years.

References 1. Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596 2. Hubbard SR, Till JH (2000) Protein tyrosine kinase structure and function. Annu Rev Biochem 69:373–389 3. Blume-Jensen P, Hunter T (2001) Receptor tyrosine kinase-initiated signal transduction: mechanisms and specifity. In: Bertino JR (ed) Encyclopedia of cancer, 2nd edn. (Academic Press). 2:213–234 4. Besmer P et al. (1993) The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev Suppl 125–137 5. Galli SJ, Zsebo KM, Geissler EN (1994) The kit ligand, stem cell factor. Adv Immunol 55:1–96

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Klatskin Tumors

Klatskin Tumors TANIA M. W ELZEL , K ATHERINE A. M C G LYNN Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Synonyms (Peri-) Hilar Cholangiocarcinoma; Extrahepatic; cholangiocarcinoma; Cholangiocellular carcinoma; Bile duct carcinoma

Definition Klatskin tumors are anatomically defined as extrahepatic ▶cholangiocarcinomas that occur at the confluence of the left and right hepatic duct (Fig. 1a). Morphologically, most Klatskin tumors (hilar cholangiocarcinomas) exhibit a nodular-sclerosing growth pattern. Few are papillary tumors and mass-forming hilar lesions are rare. Histologically, the vast majority of these tumors are adenocarcinomas.

Characteristics History and Classification Hilar cholangiocarcinomas were first described by Klatskin and were further classified by Bismuth and Corlette (Fig. 1b): Bismuth type I tumors involve the common hepatic duct below the confluence of the right and left hepatic ducts; Bismuth type II tumors involve the confluence of the left and right hepatic duct; Bismuth type III tumors involve the common hepatic duct and the right (type IIIa) or left hepatic duct (type IIIb). Bismuth type IV tumors involve either the confluence of the hepatic ducts and the right and left hepatic duct, or are multicentric. Epidemiology Over the last decades, the incidence of intrahepatic cholangiocarcinoma increased in the United States, as it did worldwide. In contrast, the incidence of extrahepatic cholangiocarcinoma remained constant (Fig. 2). A recent study showed, that extrahepatic cholangiocarcinomas (including Klatskin tumors and distal extrahepatic lesions) represent 50% of all cholangiocarcinomas in the Surveillance Epidemiology and End Results (SEER) cancer registries of the United States. A frequently cited study reported that 67% of cholangiocarcinomas were hilar tumors. However, the study was a retrospective examination of cholangiocarcinoma patients who underwent surgical exploration at a tertiary medical center. Due to the study design, the patients were unlikely to be representative of all cholangiocarcinoma patients in the United

Klatskin Tumors. Figure 1 (a) Anatomical location of Klatskin tumors. (b) Bismuth Corlette classification of Klatskin tumors (hilar cholangiocarcinoma).

States. In addition, the International Classification of Diseases for Oncology (ICD-O) assigned Klatskin tumors a histology code rather than a topography code, and the histology code was cross-referenced to the topography code for intrahepatic rather than extrahepatic tumors. This misclassification has introduced error into the reporting of cholangiocarcinoma rates in the U.S. SEER cancer registries, making it impossible to define Klatskin tumor incidence on a populationbased level.

Klatskin Tumors

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Klatskin Tumors. Figure 2 Age-adjusted incidence rates for intrahepatic CC (ICC), extrahepatic CC (including Klatskin’s tumors under 8162/3; ECC) diagnosed in SEER9 registries, 1973–2002. The dashed line represents the incidence of tumors reported in SEER to histology code 8162 (Klatskin). It must be noted that these tumors are unlikely to comprise all Klatskin tumors, since according to the ICD-O coding (1992–to date) could also be coded as ECC using other histology codes.

Etiology and Risk Factors There is recent evidence that the molecular pathogenesis of Klatskin tumors/extrahepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma may differ. Conditions that are associated with biliary inflammation increase the risk for cholangiocarcinogenesis. Well defined risk factors for Klatskin tumors and other extrahepatic cholangiocarcinomas include primary sclerosing cholangitis, infection with liver flukes (Opisthorchis viverrini, Clonorchis sinensis), choledochal cysts, ▶Caroli Syndrome, biliary stones, smoking and ▶Thorotrast exposure. Hepatitis C virus has been reported to be associated with intrahepatic cholangiocarcinoma and, in some reports, with hilar cholangiocarcinoma. Many patients do not exhibit any of the known risk factors, highlighting the need for further studies on etiopathogenesis of these tumors. Biology Diagnosis Clinical Symptoms. Patients present with obstructive jaundice (icterus, dark urine, pale stools, pruritus) and weight loss, and frequently signs of bacterial cholangitis. Abdominal pain and ascites may be present in patients with advanced disease. Biochemical Investigations. Parameters indicating obstructive cholestasis such as increased alkaline phosphatase, gamma glutamyltranspeptidase, serum bilirubin, and sometimes transaminase concentrations, may be found in patients with Klatskin tumors. ▶Tumor markers such as ▶CA 19-9 and ▶CEA, may

be elevated. These tumor markers, however, are not specific for Klatskin tumors/cholangiocarcinoma and CA19-9 may also be elevated in conditions with benign cholestatic disease. Imaging Methods. Accurate diagnosis is key to delineate benign from malignant biliary strictures and to determine the resectability of Klatskin tumors. Ultrasound is the initial diagnostic test to evaluate hilar cholangiocarcinoma and may show intrahepatic duct dilatation and caliber changes of bile ducts, but may fail to show liver infiltration of smaller tumors. On ▶computed tomography (CT), the primary obstructing hilar lesion may be invisible. On contrast enhanced CT, (infiltrative) lesions may show as thickening of the duct. Further signs include dilatation of the intrahepatic bile ducts or the atrophy-hypertrophy complex. Used to assess resectability, the accuracy of CT ranges from 60 to 75%. On ▶magnetic resonance imaging, Klatskin tumors appear hypointense on T1and hyperintense on T2-weighted images. Compared to the liver parenchyma, most Klatskin tumors are hypovascular. Gadolinium chelates and ferumoxide contrast agents can be used to assess liver invasion because of a good liver-tumor contrast. Magnetic resonance cholangiopancreatography (MRCP) can be used to visualize the obstructing lesion and intrahepatic bile duct dilatation and, in a small study, was shown to have an accuracy of 84% to assess the level of bile duct involvement. Endoscopic retrograde cholangiopancreatography (ERCP) can be used to endoscopically determine the

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location and extent of biliary tract obstruction. ERCP also allows palliative stent implantation for biliary drainage, and to obtain tumor cytology or biopsy. However, according to the specific growth pattern of Klatskin tumors, the sensitivity of brush cytology is 30%, and the combination of brush cytology/biopsy only ranges up to 70%.

Treatment To date, the only curative approach to hilar cholangiocarcinomas is surgery with resection of the bile ducts and hepatojejunostomy, and/or partial hepatectomy to achieve tumor free margins. However, at the time of diagnosis, only few/a small proportion of patients have resectable disease. Resection is contraindicated for Bismuth Type IV tumors or in the presence of any of the following: vascular encasement, occlusion or invasion of the main portal vein or hepatic artery, distant or lymph node metastases, liver invasion, or invasion of extrahepatic organs. Surgery for hilar cholangiocarcinoma with curative intent is associated with 5-year survival rates of 40%. Recently, there has been some evidence that liver transplantation, together with neoadjuvant chemoradiation may improve survival in selected patients with unresectable hilar cholangiocarcinoma. ▶Palliative approaches include the endoscopic placement of biliary stents or percutaneous biliary drainage to improve the symptoms of cholestasis. ▶Photodynamic therapy with hematoprophyrin based photosensitizers, in addition to bilateral endoprosthesis insertion, has been shown to prolong survival by several months and improve quality of life in several randomized controlled trials. So far, ▶radiation, as well as systemic ▶chemotherapy has not led to a significant improvement of survival rates, however, several new protocols are currently under investigation.

4. Nakeeb A, Pitt HA, Sohn TA et al. (1996) Cholangiocarcinoma: a spectrum of intrahepatic, perihilar, and distal tumors. Ann Surg 224:463–473 5. Slattery JM, Sahani DV (2006) What is the current state-of the-art imaging for detection and staging of cholangiocarcinoma? Oncologist 11:913–922

KLK3 ▶PSA

KLRK1 ▶NKG2D Receptor and Cancer

Knobloch Syndrome Definition Is an autosomal recessive disease that is characterized by ocular defects leading to retinal detachment, macular degeneration and blindness.

Knock-Down Survival The prognosis of hilar cholangiocarcinoma is poor, with 5 year survival rates ranging between 5 and 10%.

Definition

▶Knockdown

References 1. Klatskin G (1965) Adenocarcinoma of the hepatic duct at its bifurcation within the porta hepatis. An unusual tumor with distinctive clinical and pathological features. Am J Med 38:241–256 2. Bismuth H, Corlette MG (1975) Intrahepatic cholangioenteric anastomosis in carcinoma of the hilus of the liver. Surg Gynecol Obstet 140:170–176 3. Welzel TM, McGlynn KA, Hsing AW et al. (2006) Impact of classification of hilar cholangiocarcinomas (Klatskin tumors) on the incidence of intra- and extrahepatic cholangiocarcinoma in the United States. J Natl Cancer Inst 98(12):873–875

Knock-in Definition A genetically engineered mouse that has a different version of a gene inserted into its genome. ▶Mouse Models

Koch’s Postulates

Knock-Out

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Knudson Hypothesis

Definition

Definition

Gene invalidation resulting in the absence of expression of the corresponding protein. A commonly used model is the ▶Knock-out mouse.

Originally described as a model where in retinoblastoma two genetic “hits” result in cancer; subsequently, the two “hits” were identified as occurring as inactivating mutations in the two alleles of a gene today known as the ▶retinoblastoma gene (RB1). The two mutations can occur somatically in an individual. More often, one mutation inactivates one allele in a germ cell, the developing embryo then has one inactivated and one normal allele. Because of the ▶recessive nature of the RB1 gene, there will be no phenotype in the child, it does have however an increased risk. In most cases, the remaining normal allele will mutate in the retina during early childhood resulting in cells with both RB1 alleles inactivated. As the consequence, the child will develop retinoblastoma. This two-hit hypothesis has been generalized to entertain the idea that most cancers develop following this model. Accordingly, numerous ▶loss-of-heterozygosity studies have been done on virtually every tumor entity to identify genomic regions deleted on one tumor cell chromosome with co-existing gene mutation on the other chromosome. Only in few models the two-hit model could be convincingly verified, and today the Knudson hypothesis more or less appears to be restricted to few cancer types. Instead, there are convincing data now to support the idea that cellular effects supporting the process of malignant cellular conversion can result from the inactivation of one of the alleles. The basic concept here is ▶haploinsufficiency.

▶Gene Knockout ▶Mouse Models

Knock-out Mice ▶Knock-out Mouse

Knock-Out Mouse Definition Knock-out mouse is a genetically engineered mouse carrying one or more of its genes made inoperable through a ▶gene knockout (usually an inactivating mutation). Knockout is a technical approach to functionally characterize a gene that has been sequenced but has an unknown or incompletely known function. Because genes between mouse and humans are evolutionary conserved and, a priori, are assumed to have similar functions, the phenotype that a knock-out gene has in the mouse may be used to extrapolate its function in humans. For many genes, however, disease-associations in humans did not identify a similar phenotype in a knock-out mouse. Different strains of the mouse also may develop different phenotypes upon knock-out of a particular gene.

Koch’s Postulates R OY J. D UHE´ Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA

Knockdown

Synonyms Henle–Koch postulates

Definition

Definition

Gene knockdown refers to the experimental reduction of expression of a gene. This is commonly achieved by introducing small interfering RNAs (▶SiRNAs) or other anti-sense oligonucleotides into cells.

Characteristics

▶Antisense Nucleic Acid

A set of criteria used to identify the specific pathogen that causes an infectious disease.

Koch’s postulates are attributed to Robert Koch, who received the 1905 Nobel Prize in Medicine or

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Koch’s Postulates

Physiology “for his investigations and discoveries in relation to tuberculosis.” Jakob Henle was a professor at the University of Göttingen when Koch enrolled as a student there in 1862, and Henle was one of the early proponents of the idea that contagious diseases were caused by microorganisms. In the early days of bacteriology there were numerous heated arguments over the identity of pathogenic agents. The presence of commensal microorganisms alongside pathogenic microorganisms often resulted in the misidentification of the real disease-causing organism. At that time there was also a school of thought which held that there were no true bacterial species, but rather that a bacterium could adopt nearly limitless morphologies and physiologies, which would allow it to cause tuberculosis, anthrax, food spoilage, or other problems depending on the particular form adopted by the bacterium. It was against this backdrop that Koch proposed the following three criteria (as translated by Thomas M. Rivers) for identifying a microbiological cause of disease. 1. “The parasite occurs in every case of the disease in question, and under circumstances which can account for the pathological changes and clinical course of the disease.” 2. “It occurs in no other disease as a fortuitous and nonpathogenic parasite.” 3. “It, after being fully isolated from the body and repeatedly grown in pure culture, can induce the disease anew.” Koch famously applied these principles as he isolated Bacillus anthracis and Mycobacterium tuberculosis and identified them as the causes of anthrax and tuberculosis, respectively. By establishing a theoretical framework for determining cause and effect, Koch’s postulates had a profound effect on infectious disease research. Within a few decades, the identities of dozens of new pathogens were revealed. Yet these precepts had obvious limitations arising from the simple fact that not all pathogens can be propagated in pure form as autonomous living organisms. Viruses were the first such examples to be considered by the scientific community, and it was soon apparent that Koch’s postulates had to be revised to accommodate the scientific discoveries of the twentieth century. Over the years since Koch’s postulates were first postulated, many investigators have articulated a revised set of criteria to permit the systematic evaluation of viruses, prions, serum cholesterol, tobacco smoke, chromosomal translocations (▶chromosomal translocation) and other factors as the underlying cause of a disease, contagious or otherwise. In 1976, Alfred S. Evans proposed the following ten criteria as a “unified concept” for disease causality, which could be applied to either chronic or acute diseases.

1. Prevalence of the disease should be significantly higher in those exposed to the putative cause than in case controls not so exposed. 2. Exposure to the putative cause should be present more commonly in those with the disease than in controls without the disease when all risk factors are held constant. 3. Incidence of the disease should be significantly higher in those exposed to the putative cause than in those not so exposed as shown in prospective studies. 4. Temporally, the disease should follow exposure to the putative agent with a distribution of incubation periods on a bell shaped curve. 5. A spectrum of host responses should follow exposure to the putative agent along a logical biologic spectrum from mild to severe. 6. A measurable host response following exposure to the putative cause should regularly appear in those lacking this before exposure (i.e. antibody, cancer cells) or should increase in magnitude if present before exposure; this pattern should not occur in persons so exposed. 7. Experimental reproduction of the disease should occur in higher incidence in animals or man appropriately exposed to the putative cause than in those not so exposed; this exposure may be deliberate in volunteers, experimentally induced in the laboratory, or demonstrated in a controlled regulation of natural exposure. 8. Elimination or modification of the putative cause or of the vector carrying it should decrease the incidence of the disease (control of polluted water or smoke or removal of the specific agent). 9. Prevention or modification of the host’s response on exposure to the putative cause should decrease or eliminate the disease (immunization, drug to lower cholesterol, specific lymphocyte transfer factor in cancer). 10. The whole thing should make biologic and epidemiologic sense. The correct identification of the cause of a cancer is essential, because the cause can either be avoided, thereby preventing cancer, or the cause can be targeted through drug design, thereby curing the cancer. It is also clear that if one invests preventive or drug design efforts into the wrong cause, then one will have accomplished naught. Obvious limitations of Koch’s postulates arise when one attempts to apply them to noncontagious diseases, when suitable animal models for reproducing the disease do not exist, or when there is an inordinately long latent period between initial exposure to the causal agent and the disease’s manifestation. These circumstances often apply to the development of cancer. The ability of a pathogenic agent to cause cancer is also dependent upon the presence or absence of various tumor

KSP Mitotic Spindle Motor Protein

suppressors, which causes some individuals to be more susceptible to developing cancer than others. For these and other technical reasons, it is often unwise to dogmatically invoke Koch’s postulates in an effort to disprove a particular agent as a cause of disease. An infamous controversy arose in the late 1980s and early 1990s when Koch’s postulates were invoked in an attempt to disprove that HIV-1 caused AIDS, although that pathogenic link is now universally accepted. Koch’s postulates are of great historical significance because they marked the application of logic and reason to the field of pathology. As many scholars have noted, it is unwise to insist upon the application of the original postulates when we now know that there are pathogenic mechanisms that do not conform to the lifestyles of protozoa, fungi, or bacteria. It is the rigorous application of logic and reason to the elucidation of the cause of disease which remains as the lasting legacy of Koch’s postulates.

References 1. Rivers TM (1937) Viruses and Koch’s postulates. J Bacteriol 33:1–12 2. Evans AS (1976) Causation and disease: The Henle–Koch postulates revisited. Yale J Biol Med 49:175–195

Kostmann Syndrome Definition Synonym: Severe Congenital neutropenia; Is an inherited disorder of the bone marrow. Children born with this condition lack ▶neutrophils (a type of white blood cell that is important in fighting infection, also called granulocytes). These children suffer frequent infections from bacteria which in the past led to death in three-quarters of cases before 3 years of age. Children with hostmann syndrome have an increased risk of developing ▶acute myeloid leukemia (CML) or ▶myelodysplasia. ▶myelodyplastic syndromes

Kringle Domain Definition

Is a triple-disulfide-loop structure spanning 80 amino acids and playing a role in protein-protein

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interactions. This domain was first found in bovine prothrombin. ▶Macrophage-stimulating Protein ▶Scatter Factor

KSA ▶EpCAM

KSP ▶KSP Mitotic Spindle Motor Protein

KSP Mitotic Spindle Motor Protein W EIKANG TAO Department of Cancer Research, Merck Research Laboratories, West Point, PA, USA

Synonyms Eg5; Kinesin spindle protein; KSP; Kinesin-5; Kinesinlike protein KIF11

Definition KSP mitotic spindle motor protein is a microtubuleassociated molecular motor belonging to the ▶kinesin superfamily, which plays an essential role in the separation of centrosomes, the assembly of bipolar spindles, and the faithful segregation of chromosomes into daughter cells during ▶mitosis.

Characteristics

KSP is a plus-end-directed ▶microtubule motor protein, well conserved from yeast to mammals. It can generate directed mechanical force by hydrolyzing ATP and move along microtubule filaments from the minus end (oriented towards the nucleus) to the

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plus end (oriented towards the cell periphery). The KSP protein contains a highly conserved N-terminal motor domain encompassing the head and neck motifs, a coiled-coil stalk domain, and a tail region containing a characteristic BimC box close to the C-terminus. Based on the N-terminal motor domain sequence, the vertebrate KSP belongs to the BimC subfamily of ▶kinesins, a large family of ▶microtubule-based motor proteins. More recently, KSP was classified to the kinesin-5 subgroup according to a new nomenclature system. The globular motor domain of KSP is responsible for catalyzing hydrolysis of ATP, interacting with microtubules and nucleotides, and driving the movement along microtubules empowered by ATP hydrolysis, whereas the stalk domain mediates protein–protein interactions to form homotetramers. Active KSP is a homotetramer in which two polypeptides first dimerize to form a parallel coiled-coil and then two dimers form an antiparallel tetramer containing four motor domains with a dimeric motor unit at each end (Fig. 1). As a tetramer, KSP is able to crosslink and translocate along two adjacent microtubules with each dimeric motor unit interacting with a single microtubule fiber. Fluorescence studies suggest that when binding to two antiparallel microtubules, KSP can slide them apart, thereby pushing spindle poles apart. If binding to two parallel microtubules, KSP can mediate bundling of parallel microtubules, contributing to the assembly and integrity of mitotic spindles (Fig. 2). Like other ▶kinesins of the BimC subfamily, human KSP is required early in prometaphase for the separation of duplicated centrosomes and the formation of bipolar mitotic spindles. Inhibition of KSP with neutralizing antibodies, small molecule inhibitors, or small interfering RNAs (siRNAs) causes formation of the characteristic monopolar mitotic spindle (also termed monoaster), a radial array of microtubules surrounded by a ring of chromosomes, and cell cycle arrest at prometaphase. Further studies indicate that suppression of KSP activates the ▶spindle assembly checkpoint, thereby preventing

the onset of anaphase and causing mitotic arrest. Prolonged mitotic arrest induces apoptosis. Inhibition of KSP does not affect postmitotic cells, indicating that KSP is only required for cell proliferation and that it functions exclusively in mitosis. Consistent with its exclusive role in cell division, KSP is preferentially expressed in proliferating tissues, such as thymus, bone marrow ,and intestine epithelium, with minimal or no expression in nonproliferating tissues such as the central nervous system. In addition, it displays much more abundant expression in actively proliferating tumor tissues than in normal adjacent tissues. Regulation The ATPase activity of KSP is essential for its function and it is stimulated by the interaction with microtubules. The control of ATPase activity by interactions with microtubules is thought to prevent futile hydrolysis of ATP. It has been shown that there is an evolutionarily conserved p34cdc2 phosphorylation site (Thr-927 in human KSP) located in the BimC box of KSP, and that p34cdc2-mediated phosphorylation of this site controls the association of KSP with the microtubule spindles. Moreover, KSP can be phosphorylated by ▶Aurora kinase, a mitotic kinase, but the functional importance of this phosphorylation remains to be determined. Relevance to Cancer The mitotic spindle is a protein complex consisting of multiple components including microtubules and motor proteins and it is a pharmaceutically validated target for cancer therapeutics. ▶Vinca alkaloids and ▶taxanes that interfere with the dynamics of microtubules by binding to β-tubulin perturb mitosis and have been used in the clinic for decades for the treatment of many human malignancies. However, since microtubules have essential functions beyond mitosis in nonproliferating cells, including intracellular transport

KSP Mitotic Spindle Motor Protein. Figure 1 Schematic representation of the structures of KSP dimmer and tetramer.


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K KSP Mitotic Spindle Motor Protein. Figure 2 Schematic illustration of KSP functions.

and organelle positioning, drugs that target microtubules cause microtubule-dependent side effects, such as peripheral neuropathy. In addition, an increasing number of tumors have acquired resistance to these antimicrotubule drugs via various mechanisms including acquired mutations on β-tubulin, altered expression of tubulin isoforms, changed microtubule dynamics as well as elevated expression of membrane drug efflux pumps like ▶P-glycoproteins (P-gp). Thus, agents that are able to selectively disrupt the mitotic spindle via a novel mechanism of action and without P-gp liability are greatly desired for cancer therapy. There are several important traits that make KSP an attractive target for cancer treatment: (i) KSP is a crucial component of the mitotic spindle and is essential for spindle bipolarity and proper segregation of chromosomes during mitosis. (ii) KSP functions exclusively in dividing cells and there is no evidence that inhibition of KSP affects nonproliferating cells. (iii) KSP is overexpressed in tumor tissues relative to adjacent normal tissues. (iv) Since KSP is an ATPase and the ATPase activity is required for its function, KSP is a druggable target. Indeed, a number of small molecule inhibitors of KSP ATPase with distinct chemical structures have been generated and currently at different stages of development. Some specific inhibitors of KSP have exhibited broad antiproliferative activity in tumor cell lines and significant antitumor efficacy in various murine tumor models. A few KSP inhibitors

have entered clinical trials. Among them ispinesib demonstrated some activity in taxane-treated patients with metastatic breast cancer in a phase II study. As expected, the clinical trial data indicate that KSP inhibitors could not exhibit neurotoxicity because of their lack of effect on microtubule dynamics. Although KSP inhibitors hold great promise for cancer treatment, their clinical antitumor activity remains to be determined. The clinical development of these novel antimitotic agents holds many challenges. A critical issue is how to identify the responders in the clinic, or what ▶biomarkers could predict clinical response of tumors to KSP inhibitors. Preclinical studies suggest that tumor cells with a competent spindle assembly checkpoint are more sensitive to KSP inhibitor-mediated cell death than those with a deficient spindle checkpoint. This awaits confirmation in the clinic. Additionally, a better understanding of the mechanism that mediates the lethality of these agents or the link between KSP inhibition and induction of cell death is required to guide the selection of dosing schedules and the identification of determinants of drug sensitivity. Finally, since antimicrotubule drugs and KSP inhibitors may kill cancer cells through a partially overlapping pathway, elucidation of the mechanisms that cause resistance to microtubule inhibitors in the clinical setting would tell whether KSP inhibitors should be used for the treatment of antimicrotubule agent-resistant tumors.

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Ku70

References 1. Wood KW, Cornwell WD, Jackson JR (2001) Past and future of the mitotic spindle as an oncology target. Curr Opin Pharmacol 1:370–377 2. Bergnes G, Brejc K, Belmont (2005) Mitotic kinesins: prospects for antimitotic drug discovery. Curr Topics Med Chem 5:127–145 3. Miki H, Okada Y, Hirokawa N (2005) Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol 15:467–476 4. Tao W, South V, Zhang Y et al. (2005) Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell 8:49–59 5. Valentine MT, Fordyce PM, Block SM (2006) Eg5 steps it up! Cell Div 1:31

Kupffer Cells P ETER T HOMAS Departments of Surgery and Biomedical Sciences, Creighton University, Omaha, NE, USA

Synonyms Hepatic (Liver) fixed macrophages

Definition

Kupffer cells are ▶macrophages that are fixed within the sinusoids of the liver. They are the body’s most abundant source of fixed macrophages.

Characteristics

Ku70 Definition The protein of 70-kDa molecular weight is a DNAhelicase involved in the DNA double-strand break repair system (▶Double-strand break DNA repair), more precisely the nonhomologous end joining. ▶Ku Antigen, 70-kDa Subunit ▶Lupus Autoantigen p70 ▶Thyroid-Lupus Autoantigen (TLAA) ▶Thyroid Autoantigen, 70-kDa (G22P1) ▶Securin

Kulchitsky Cell Carcinoma ▶Extrapulmonary Small Cell Cancer

Kunitz Trypsin Inhibitor Definition KTI; Antinutritional/allergen protein found in soybean that KTI acts as a inhibitor of pancreatin and chymotrypsin. ▶Lunasin

Kupffer cells were first described in 1876 and are named for, the German pathologist Karl Wilhelm von Kupffer who called them the star cells (sternzellen) of the liver. Kupffer cells are tissue macrophages that are located in the hepatic sinusoidal blood flow attached to the endothelial cell lining. They represent 10% of all liver cells and are the body’s largest population (approximately 80%) of fixed macrophages. They are the first macrophage population to contact bacteria, bacterial debris and ▶endotoxins arising in the gastrointestinal tract. Kupffer cells are both highly endocytic and phagocytic and a major function is to remove particulate matter from the circulation. They are also an important component of innate immunity which is the most rapid response to dangerous stimuli and can modulate immune responses by antigen presentation and suppression of T-cell activation. Thus, the main role of Kupffer cells is to protect the body by removing potentially harmful substances from the blood. In addition Kupffer cells function to remove and destroy old red blood cells. Kupffer cells have also been implicated in cancer cell surveillance (see below). Kuppfer cells originate in the bone marrow. These bone marrow monocytes enter the circulation and become implanted in the liver where they differentiate into fixed tissue macrophages. Kupffer cells are considered terminally differentiated and the cells no longer divide (Fig. 1). Kupffer Cells and Liver Disease Kupffer cells are involved in the development of alcoholic liver disease. Kupffer cells become activated when they are exposed to the bacterial cell wall component endotoxin (Lipopolysaccharide, LPS). This results in the secretion of signaling molecules including ▶cytokines and ▶reactive oxygen species (ROS), which can be damaging to liver cells especially those that have been affected by alcohol ingestion and contain a large amount

Kupffer Cells

Kupffer Cells. Figure 1 Section of mouse liver showing stellate shaped Kupffer cells (arrows). The cells are stained by the immunoperoxidase method for the presence of a breast cancer associated antigen, the gross cystic disease fluid protein (GCDFP-15), 15 min after intravenous injection of the purified protein.

of fat (alcoholic steatosis). Endotoxin is produced by gram negative bacteria in the large intestine and ingestion of alcohol alters the permeability of the intestine (leaky gut) to endotoxin causing an increased level in the portal blood entering the liver. To activate Kupffer cells and other macrophages endotoxins combine with a serum protein, lipopolysaccharide binding protein (LBP). This complex reacts with a cell surface receptor, ▶CD14 which in turn interacts with the Toll like receptor ▶TLR4 and the intracellular signaling protein ▶MyD88. This causes a cascade of intra-cellular signals that activate transcription factors such as ▶NFκB and result in the synthesis and secretion of cytokines and ROS. Relationship of Kupffer Cells to Primary and Secondary Liver Cancers The liver is a favored site for the secondary spread of many cancers including those of the large intestine and breast. The role of Kupffer cells in the development of cancers in the liver is controversial. There is no doubt that they play an important part in both the development of primary liver cancer (▶hepatocellular carcinoma or hepatoma) and in the formation of ▶metastasis from a distant site including cancers of the lungs, breast, colon and other gastrointestinal cancers. Some experimental studies have shown that elimination of Kupffer cells from the liver results in enhanced metastatic disease. Other studies have implicated Kupffer cells as agents that interact with the tumor cells entering the liver and their pro-inflammatory responses can induce tumor cell implantation and growth allowing increased metastatic disease. In primary liver cancer recent evidence has shown a role for Kupffer cell activation in carcinogenesis and progression of the

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cancer. Some liver carcinogens are able to activate Kupffer cells and induce cytokines and other factors that promote hepatocyte growth. These factors can also effect growth of pre-neoplastic cells and lead to the development of hepatoma. Evidence has accumulated that the pro-inflammatory cytokine IL-6 plays an important role in promoting hepatoma cell survival and proliferation. This tumor cell related increase in IL-6 is mediated through the MyD88/NFκB signaling cascade in Kupffer cells. In the case of colorectal cancer metastasis to the liver cytokines produced by Kupffer cells play a part in both tumor cell implantation and their subsequent survival and growth. During the development of colorectal cancer metastasis to the liver, Kupffer cells respond to the glycoprotein carcinoembryonic antigen (▶CEA). The cells bind CEA via a pentapeptide that reacts with a surface protein that is identical to the RNA binding protein hnRNP M4. This results in Kupffer cell activation and a cytokine cascade that includes IL-1, IL-6, IL-10 and TNF-α. The high local concentration of cytokines produced within the hepatic sinusoids has multiple effects that influence cancer cell implantation, survival and proliferation. The initial effect of the Kupffer cell produced cytokines seems to be on the sinusoidal endothelial cells. They become activated and produce adhesion molecules such as ▶E-selectin and ▶ICAM-1 on their cell surfaces. This allows the cancer cells to attach to the endothelial cell layer and arrest in the liver sinusoids. When this happens the tumor cells can break through the endothelial cell layer and invade the parenchyma Cytokines, in particular IL-10 are also protective against the effects of ischemia/reperfusion (IR) injury caused by blockage of the blood supply by tumor cell infiltrates into the small sinusoids. IR causes oxidative stress and cell death. Secretion of anti-inflammatory cytokines such as IL-10 counteracts these effects and aids the initial survival of the cancer cells following implantation in the sinusoids. These studies were largely carried out using human colorectal cancer cells in culture or in metastasis models in immuno-suppressed mice and imply that Kupffer cells are important participants in metastasis development. However, other experiments using syngeneic animal tumors have shown a role for Kupffer cells in preventing liver metastasis. Experiments where Kupffer cells have been depleted prior to injection of tumor cells have often resulted in extensive tumor growth in the liver. Similarly when Kupffer cells have been activated prior to tumor cell injection reduced tumor growth in the liver is seen. Kupffer Cell Cancers Cancer originating in Kupffer cells is very rare and few cases have been reported in the literature. They are described as Kupffer cell ▶sarcomas or hemangioendotheliosarcomas of the liver. The tumors contain

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Kuzbanian

phagocytic giant cells and numerous other cells with morphology similar to Kupffer cells. These hemorrhagic tumors tend to be quickly fatal with abdominal discomfort, ascites and jaundice as symptoms. Kupffer cell cancers will metastasize to distant sites including the pancreas and lung. Similar tumors to those found in humans can be induced in rats by exposure to low doses of carcinogens during liver regeneration.

Kuzbanian Definition

▶ADAM10

References 1. Von Kupffer C, Uber Sternzellen der Leber (1876) Arch Mikr Anat 12:353–358 2. Bilzer M, Roggel F, Gerbes AL (2006) Role of Kupffer cells in host defense and liver disease. Liver Inter 26:1175–1186 3. Burston J (1958) Kupffer Cell sarcoma. Cancer 11:798–802

Kv10.1 ▶Ether à-go-go Potassium Channels

L

L-PAM Definition

▶Melphalan

Lactate Definition A 3-carbon carboxylic acid also known as 2-hydroxypropanoic acid. ▶Warburg Effect

L&H Definition Lymphocytic and/or histiocytic. ▶Hodgkin Disease, Clinical Oncology

L&H Cells Definition Lymphocytic and Histiocytic Cells.

Lactate Dehydrogenase Definition LDH is a ubiquitously expressed enzyme. Serum levels of LDH can be used to monitor treatment of testicular cancer, ▶Ewing sarcoma, non Hodgkin lymphoma and some types of leukemia. Elevated levels of LDH can also be caused by a number of non-cancerous conditions, including heart failure, hypothyroidism, anemia, as well as lung and liver diseases. ▶Serum Biomarkers ▶Testis Cancer ▶Hodgkin Disease

Label-Free Analysis Definition A biochemical analysis employing detection method without the need of label such as fluorescence or radioactive tag. ▶Surface Plasmon Resonance

Labile Factor Definition

▶Factor V.

Lactoferricin Antiangiogenesis Inhibitor DAVID W. H OSKIN Departments of Pathology, and Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada

Definition Lactoferricin is a cationic peptide that is generated by the acid-pepsin hydrolysis of mammalian ▶lactoferrin present in the secretory granules of neutrophils (polymorphonuclear leukocytes), as well as in exocrine

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Lactoferricin Antiangiogenesis Inhibitor

secretions, including milk, tears, and saliva. Interestingly, substantial quantities of lactoferricin are generated in the stomach following the ingestion of lactoferrin-containing milk. Lactoferricin possesses potent antimicrobial, antiviral, immunomodulatory, and antitumor activities. In terms of anticancer activity, the best studied of the lactoferricins is bovine lactoferricin, which consists of amino acid residues 17–41 from the NH2-terminal region of bovine lactoferrin (Fig. 1). A disulfide bond that forms between cysteine residues located at each end of the peptide creates a looped or hairpin structure. In an aqueous environment, bovine lactoferricin assumes an ▶amphipathic, twisted β-sheet configuration with clear positively charged and hydrophobic faces. The relatively high proportion of asymmetrically clustered basic amino acid residues (arginine and lysine) and the hydrophobic tryptophan residues that in part comprise bovine lactoferricin are believed to be important for the peptide’s biological activity.

Characteristics The in vitro and in vivo anticancer activity of bovine lactoferricin has been attributed to the selective cytotoxic effect (either by membrane lysis or ▶apoptosis induction) exerted by this cationic peptide on a broad range of cancer cell types, including leukemias, lymphomas, fibrosarcomas, and various carcinomas. However, recent studies indicate that bovine lactoferricin is also able to inhibit ▶angiogenesis. A similar, but less potent, antiangiogenic activity has also been reported for bovine lactoferrin. Although ▶neovascularization is normally tightly regulated by the opposing effects of proangiogenic and antiangiogenic factors, this

process becomes dysregulated during tumor growth. The action of proangiogenic factors, in combination with basement membrane degradation by proteolytic enzymes, results in the proliferation, migration, and differentiation of tumor-associated endothelial cells. Neovascularization is an essential step in ▶tumorigenesis since the new blood vessels provide oxygen and nutrients to rapidly dividing cancer cells and remove metabolic wastes from the tumor ▶microenvironment. This allows solid tumors to grow in size, as well as promoting the development of metastatic disease. Diffusible, tumor-associated growth factors that stimulate angiogenesis include ▶vascular endothelial growth factor165, ▶platelet-derived growth factor, ▶heparin-binding epidermal growth factor-like growth factor, and basic ▶fibroblast growth factor (also known as fibroblast growth factor 2). Bovine lactoferricin is a potent in vivo inhibitor of vascular endothelial growth factor165- and basic fibroblast growth factor-induced angiogenesis in the mouse ▶Matrigel-plug assay. This finding is consistent with the observation that administration of bovine lactoferricin by subcutaneous injection reduces the number of tumor-associated blood vessels in B16-BL6 melanoma-bearing mice. In addition, bovine lactoferricin exhibits a dose-dependent inhibitory effect on the in vitro proliferation of human umbilical vein endothelial cells in response to vascular endothelial growth factor165 or basic fibroblast growth factor, as well as inhibiting the in vitro migration of human umbilical vein endothelial cells across transwell membranes in response to vascular endothelial growth factor165 or basic fibroblast growth factor. Bovine lactoferricin therefore inhibits endothelial cell proliferation and

Lactoferricin Antiangiogenesis Inhibitor. Figure 1 Primary structure of bovine lactoferricin. The amino acid sequence of bovine lactoferricin is indicated by single-letter code (see the accompanying key). Basic and hydrophobic amino acid residues are important for peptide function and are colored red and blue, respectively. The disulfide bond, indicated by the solid bar between cysteine residues at opposite ends of the peptide, forms a loop consisting of 18 amino acids. Numbers indicate the sequence position in bovine lactoferrin.

Lactoferricin Antiangiogenesis Inhibitor

migration, which are essential steps in neovascularization. Importantly, bovine lactoferricin does not affect the viability of human umbilical vein endothelial cells, suggesting that the antiangiogenic activity of bovine lactoferricin is independent of its membranelytic and apoptosis-inducing activity. Vascular endothelial growth factor165 and basic fibroblast growth factor must first interact with ▶heparan sulfate proteoglycans on the surface of endothelial cells in order for these proangiogenic factors to bind to and trigger signal transduction through their respective cellsurface receptors. Other heparan sulfate proteoglycandependent proangiogenic factors that have been linked to tumor progression include platelet-derived growth factor and ▶heparin-binding epidermal growth factorlike growth factor. Positively charged bovine lactoferricin mediates its inhibitory effect on basic fibroblast growth factor- and vascular endothelial growth factor165-induced angiogenesis by interacting with negatively-charged heparin-like structures on the surface of human umbilical vein endothelial cells, thereby competing with basic fibroblast growth factor and vascular endothelial growth

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factor165 for the heparan sulfate proteoglycans that are required for these proangiogenic factors to bind to and signal through their specific cell-surface receptors (Fig. 2). Although not yet formally proven, it is likely that bovine lactoferricin will have a similar inhibitory effect on angiogenesis induced by other heparin-binding tumorassociated proangiogenic factors. However, ▶electrostatic interactions alone do not govern the binding of bovine lactoferricin to the heparan sulfate proteoglycans that are involved in cell-surface receptor signaling caused by heparin-binding proangiogenic factors. Thus, a scrambled form of bovine lactoferricin that retains the net positive charge of the native peptide is unable to inhibit the binding of vascular endothelial growth factor165 or basic fibroblast growth factor to human umbilical vein endothelial cells. The primary and secondary structure that is conferred on bovine lactoferricin by its amino acid sequence is therefore an important factor in the selectivity of bovine lactoferricin for heparin-like structures that are involved in basic fibroblast growth factor and vascular endothelial growth factor165 interactions with their respective receptors on human endothelial cells.

L

Lactoferricin Antiangiogenesis Inhibitor. Figure 2 Model of bovine lactoferricin-mediated blockade of heparin-binding growth factor-induced angiogenesis. (a) Heparin-binding proangiogenic growth factors such as vascular endothelial growth factor165 (VEGF) must interact with heparin-like binding sites on heparan sulfate proteoglycans in order to bind to and signal through receptors on the surface of endothelial cells. (b) Bovine lactoferricin (Lfcin) complexes with heparin-like binding sites on cell-surface heparan sulfate proteoglycans, thereby competing with heparan sulfate proteoglycan-dependent proangiogenic growth factors for heparin-like binding sites and preventing proangiogenic growth factor receptor signaling from taking place.

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Lactoferrin

Clinical Perspectives Starving a solid tumor of its blood supply by preventing or interfering with tumor-induced neovascularization has generated considerable interest as an alternative to conventional chemotherapy for the prevention of tumor growth and metastasis. Indeed, several different inhibitors of angiogenesis are currently being used in the treatment of human cancers. However, the results obtained to date in clinical practice have been less than was hoped for on the basis of preclinical studies, most likely because the current generation of antiangiogenic agents only target a single proangiogenic growth factor receptor, whereas multiple proangiogenic factors are typically associated with tumor-induced angiogenesis. In this regard, the ability of bovine lactoferricin to inhibit neovascularization in response to multiple heparin-binding growth factors may allow bovine lactoferricin to be more effective than current antibody-based antiangiogenic agents for the blockade of tumor-induced angiogenesis. However, the susceptibility of bovine lactoferricin to enzymatic degradation and inactivation through interactions with anionic serum components remains a major obstacle to any future use of this host defense peptide as an antiangiogenic agent. One possible solution is to synthesize an all-▶D amino acid analogue of bovine lactoferricin since cationic peptides that consist of all-D amino acids exhibit dramatically increased stability in serum. Alternatively, tumor-targeted ▶liposomes might be used to encapsulate and deliver bovine lactoferricin directly to tumor sites while retaining the peptide’s ability to mediate antiangiogenic activity. Although preclinical studies have revealed that bovine lactoferricin is a potent inhibitor of angiogenesis, additional research will be needed in order to realize the potential of bovine lactoferricin as a novel antiangiogenic agent for the treatment of human cancers.

5. Yoo Y-C, Watanabe S, Watanabe R et al. (1997) Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. Jpn J Cancer Res 88:184–190

Lactoferrin Definition Is an 80-kDa iron-binding glycoprotein belonging to the transferrin family. Lactoferrin has important multifunctional roles in host defense. ▶Lactoferricin Antiangiogeneis Inhibitor

LAIR1 Definition Leukocyte-associated immunoglobulin-like receptor 1.

LAK ▶Activated Natural Killer Cells

LAMB References 1. Gifford JL, Hunter HN, Vogel HJ (2005) Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci 62:2588–2598 2. Mader JS, Hoskin DW (2006) Cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment. Expert Opin Investig Drugs 15:933–946 3. Mader JS, Smyth D, Marshall JS et al. (2006) Bovine lactoferricin inhibits basic fibroblast growth factor- and vascular endothelial growth factor165-induced angiogenesis by competing for heparin-like binding sites on endothelial cells. Am J Pathol 169:1753–1766 4. Vogel HJ, Schibli DJ, Jing W et al. (2002) Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. Biochem. Cell Biol 80:49–63

▶Carney Complex

Lamellar Phase Definition The most common lipid secondary structure, also called the lipid bilayer, which defines most phospholipid membranes found. ▶Membrane-Lipid Therapy

Laminin Signaling

Lamellipodia

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Laminin Signaling

Definition

R EUVEN R EICH

Areas at the edge of adherent cells that extend away from the cell body by the pushing of internal actin filaments as they polymerize. Flattened sheet-like structures composed of a crosslinked F-actin meshwork that project from the cell membrane. Often associated with the leading edge of migrating cells.

Department of Pharmacology, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel 2 Reuven Reich is affiliated with the David R. Bloom Center of Pharmacy at the Hebrew University

▶Cortactin ▶Migration

Laminin Definition LM are large, heterotrimeric, cruciform matrix glycoproteins composed of highly homologous α, β, and γ subunits. Five α (α1–5), four β (β1–4), and three γ (γ1–3) chains variably assemble to create 14 known isoforms that convey a variety of important biological functions. Specific LM isoform expression and posttranslational processing can directly influence cellular response to growth factors, intracellular signaling, cell proliferation, susceptibility to apoptosis and migratory capacity. Changes in LM isoform expression in vessel walls have been shown to foster angiogenesis as well as leakage in vessels, rendering them attractive to tumor cells and susceptible to metastatic invasion. Laminins are expressed in both normal and malignant tissue, but different specific isoforms predominate in each case. ▶Aging and Cancer ▶Laminin Signaling ▶Tissue Inhibitors Of Metalloproteinases

Laminin-Receptor Definition

Are proteins that bind ▶laminin and transduce a certain signal into the cell bearing the receptor. ▶Laminin Signaling

1

Definition

▶Laminins are a family of glycoproteins with an apparent molecular weight between 400 and 900 kDa. They are heterotrimers of three subunits, α, β, and γ held together by disulphide bonds to form triple helical coiled coil in a shape of a cross. Five α chains, three β chains, and three γ chains have been identified and by combination they assemble to form over 14 laminin isoforms that have different tissue distributions and functions. Laminins are essential for basement membrane assembly, promote cell attachment and ▶angiogenesis, induce neurite outgrowth, affect gene expression and are involved in cell proliferation, migration, and differentiation. Biochemical dissection related some of the laminin functions to specific parts of the glycoproteins. It appears that different parts in the molecules have different effects on cells. Some of these parts are cryptic and interact with cells only after proteolytic cleavage of the laminin molecules. In vitro, most structural and functional studies have been performed on laminin-1 (α1β1γ1).

Characteristics Laminin Signaling Laminins activate various ▶signal transduction pathways. It was shown that ▶chemotaxis induced by laminin-1 is pertussis toxin sensitive, indicating on the involvement of a pertussis toxin-sensitive G protein in the process, while ▶haptotaxis seems to involve a different mechanism. It was shown that human osteoclast-like cells selectively recognize laminin isoforms. The cells adhered to laminin-2 but not to laminin-1, and a sharp increase in intracellular Ca2+ was detected upon addition of soluble laminin-2 to the cells. Another study showed that laminin-1 induced a rapid and transient mRNA expression of c-fos and c-Jun in PC12 cells, and stimulated the DNA binding activity of the complex of these proteins to the ▶AP-1 site. In tumor cells, addition of laminin-1 resulted in a time- and dose-dependent activation of phospholipase D (PLD) followed by generation of ▶phosphatidic acid that is involved in signal transduction events leading to the induction of ▶MMP-2 and enhanced invasiveness of metastatic tumor cells. This effect of laminin-1 was not seen in normal cells in vitro. Laminins’ signaling has

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been shown to involve kinase/phosphatase cascades since bound laminin-1 and laminin-5 induce protein ▶dephosphorylation in neural cells during process formation. A recent study performed in our laboratory showed that mitogen activated protein kinases (▶MAPK) are involved in laminin signaling. Addition of exogenous soluble laminin-1 resulted in a significant decrease in the ▶phosphorylation (activation) of ERK, JNK, and p38 after 30 min of incubation. This laminin-induced dephosphorylation of all MAPK was dose-dependent and transient. Another study demonstrated that incubation of macrophages with a peptide from the laminin-α1 chain, but not intact laminin-1, triggered protein kinase C-dependent activation of ERK1/2, leading to upregulation of proteinase expression. Several recent studies using laminin-5 have shown activation of ERK1/2 via focal ▶adhesion kinase (FAK), while other studies have shown activation of Rac1 via phosphoinositide 3 kinase (PI3K). Although some functions may be common to all laminin variants, other may be unique and isoform-specific, depending on the tissue or organ in which they are expressed. In addition, various signal transduction pathways may be activated by different ▶laminin receptors. Laminin Receptors The biological effects of laminins are mediated by numerous laminin receptors that are divided into two major groups: ▶integrin and nonintegrin receptors (Table 1). Integrins Integrins are a large family of cell-membrane receptors for extracellular matrix proteins (Table 1). Integrins are heterodimeric combination of various α-subunits with various β-subunits. The ligand specificity for different integrins can be altered depending on the type of divalent cation present, the surrounding lipid environment, and various cell-specific factors. Inside

the cell, the short cytoplasmic domains of integrins associate with various cytoskeletal proteins that mediate integrin signal transduction. At least eight integrins bind laminin; some of them bind additional extracellular matrix components as well. Integrins recognize mainly laminin-α chains and hence determine cell adhesion and response to laminin isoforms. Two possible integrin-related signal transduction pathways have been identified. First is direct signaling, where binding to integrins by extracellular proteins triggers intracellular signaling events. The second is integrin modulation of mitogen-initiated signaling; in this case, integrin-mediated cell anchorage influences signaling pathways activated by growth factors. In general, integrin direct signaling activates FAK, small GTPases of the ▶Rho family, and MAPK, resulting in accumulation of highly ▶phosphorylated proteins and cytoskeletal molecules at the adhesion sites. Binding to integrins is followed by receptor clustering that initiates activation and autophosphorylation of FAK. Tyrosinephosphorylated FAK can recruit Src family kinases to focal contact sites. This sets up additional tyrosine phosphorylation of proteins such as cytoskeletal proteins and adaptor proteins such as Grb2. Small GTPases of the Rho family (Rho, Rac, and Cdc42) are involved in ▶integrin signal transduction and affect cytoskeleton arrangement. Rho contributes to cell adhesion to extracellular matrix. Rac and Cdc42, via PI3K, mediate the increase in cell motility and invasiveness induced by the integrin. Some integrins activate MAPK cascades. For example, laminin binding to the integrin α6β4 results in activation of an associated kinase and consequently tyrosine phosphorylation of the β4-subunit cytoplasmic domain, followed by association of the adaptor protein Shc with tyrosine-phosphorylated β4 integrin subunit. Shc is then phosphorylated on tyrosine residues, presumably by an integrin-associated kinase, and combines with the adaptor protein Grb2 which exists in a complex with the ras GTP exchange factor

Laminin Signaling. Table 1 Laminin receptors and their additional ligands Receptor Integrins α1β1 Integrins α2β1 Integrins α3β1 Integrins α6β1 Integrins α6β4 Integrins α7β1 67 kDa laminin receptor Dystroglycan Heparan sulfate

Ligands Collagen (I,II,IV), laminin (1, 2) Collagen (I,II,IV), laminin (1, 2), chondroadherin ▶Fibronectin, collagen (I), laminin (2, 5, 8, 10, 11), nidogen, epiligrin, perlecan Laminin (1, 2, 5, 8, 10, 11) Laminin (1, 2, 5, 10) Laminin (1, 2, 8, 10) Laminina Laminin (1, 2), agrin, perlecan Laminin (1, 2), collagen XVIII

Laminin-4 receptor interactions presumed to be similar to those of laminin-2. a Most studies on laminin-1.

Laminin Signaling

SOS. This leads to Ras activation followed by activation of a kinase cascade consisting of Raf, MEK (MAPK/ ERK kinase), and ERK, resulting in increased cell motility and proliferation. In addition, integrin α6β4 activates the JNK cascade, via Rac1, resulting in jun protein expression. Jun associates with fos, whose expression is induced by ERK cascade, to form the AP-1 transcription factor. In human hepatocellular carcinoma cells, laminin-binding integrin α6β1 stimulation resulted in FAK tyrosine phosphorylation, leading to FAK–GRB2 association and ERK cascade activation, which promotes tumor cell migration. Interestingly, aggregation of integrin receptors, even in the absence of ligand occupancy, is sufficient to induce a prompt transmembrane accumulation of at least 20 signal transduction molecules, including Src, Rho, Rac1, Ras, ERK1/2, and JNK. Nonintegrin Receptors The 67kDa laminin receptor is a nonintegrin receptor. A highly conserved 37 kDa protein is the precursor of the receptor, but the exact manner by which it configures its mature form is not clear. The amino acid sequence of the 37 kDa precursor is extremely well conserved through evolution, however, it corresponds to that of additional proteins, suggesting a multifunctional protein. The cDNA of the 37 kDa precursor is virtually identical to a cDNA encoding the ribosomal protein p40. In addition, the 37 kDa precursor acts as a receptor for cellular prion protein and is involved in the life cycle of prions. It has also been found that the 37 kDa precursor is identical to the oncofetal antigen protein that is expressed by tumors. The 67 kDa laminin receptor mediates cell attachment to laminin. Colocalization of the 67 kDa laminin receptor with the cytoskeleton constituents α-actinin and vinculin, and the focal adhesion plaque was found. The receptor is involved in several physiological processes such as implantation [56], invasive phenotype of trophoblastic tissue, angiogenesis, T-cell biology, and shear stress-dependent endothelial nitric oxide synthase expression. Increased expression of the 67 kDa laminin receptor correlates with cell proliferation, migration, and ▶invasion capacity. Clinical data suggest a correlation between 67 kDa laminin receptor expression in tumor cells and tumor progression. Expression of the receptor has been shown to be upregulated in neoplastic cells compared to their normal counterparts and directly correlates with an enhanced invasive and metastatic potential in numerous malignancies. Malignant ▶mesothelioma is one of the most aggressive human cancers, however, no tumor is less susceptible to distant ▶metastasis and still associated with such high mortality rates. In a recent study, we wound frequent expression of 67 kDa mRNA but very rare expression of the protein in clinical malignant

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mesothelioma samples in contrast to metastatic breast or lung carcinomas. These findings suggest that the differences between malignant mesothelioma and carcinomas regarding expression of the 67 kDa laminin receptor may account at least in part for the reduced ability of MM to metastasize to distant organs, due to lack of the signaling mediated by the receptor. By stable transfection of A375SM melanoma cells, we established lines expressing reduced or elevated 67 kDa laminin receptor. We found that stable antisensetransfected cells that expressed reduced 67 kDa laminin receptor demonstrated significantly less aggressive tumor phenotype, as reflected by their reduced invasiveness through Matrigel, diminished attachment to laminin, and decreased MMP-2 expression and activity. Further, the basal phosphorylation extent (activity) of ERK, JNK, and p38 was significantly higher in cell lines expressing reduced 67 kDa laminin receptor, compared to parental cells. The increase in MAPK phosphorylation in cells expressing reduced 67 kDa laminin receptor was accompanied by a significant reduction in MKP-1 mRNA level and a significant increase in PAC-1 mRNA level. It seems that the 67 kDa laminin receptor induces downregulation of MKP-1 expression that may contribute to the reduced activity (dephosphorylation) of MAPK induced by the receptor, which is followed by an upregulation of PAC-1 expression, possibly as a negative feedback. 67 kDa Laminin Receptor and Integrins There are studies that indicate an association between the 67 kDa laminin receptor and the α6 integrin subunit that is a part of the laminin-binding integrins α6β4 and α6β1. Biochemical analyses indicate on coimmunoprecipitation of the 67 kDa laminin receptor with the α6 integrin subunit. Specific reduction of the α6 integrin subunit by an antisense was accompanied by a proportional decrease in the cell surface expression of the 67 kDa laminin receptor. Other studies targeting the 67 kDa laminin receptor, showed a significant reduction in one of the α6 integrin subunit isoforms. Analysis of α6 integrin subunit and of the 67 kDa laminin receptor in ▶ovarian carcinoma samples showed no statistical correlation between the two. ▶Dystroglycan Dystroglycan consists of two subunits, which are translated from a single mRNA as a propeptide that is proteolytically cleaved into two noncovalently associated proteins. Dystroglycan was originally isolated from skeletal muscle as an integral membrane component of the dystrophin–glycoprotein complex (DGC). The exact function of the entire DGC is not completely determined but evidence indicates that it confers structural stability to the sarcolemma during contraction. In fact, mutations in components of this complex

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lead to various types of muscle disorder such as Duchenne muscular dystrophy and limb-girdle muscular dystrophies. Dystroglycan is also expressed in many other cell types and it plays important roles outside skeletal muscle. It has been implicated in early mouse development, structure and function of the central nervous system, myelination and nodal architecture of peripheral nerves, epithelial morphogenesis, cell adhesion, synaptogenesis, and signaling. In addition, several extra- and intracellular proteins are less tightly associated with the DGC, such as nitric oxide synthase [nNOS], dystrobrevin, and laminin-2. Molecules that bind to the cytoplasmic tail of β-dystroglycan include the signaling molecule Grb2, components of the ERK– MAP kinase cascade including MEK and ERK, and rapsyn. Binding of laminin-2 to dystroglycan induces phosphorylation of Grb2 followed by Sos binding. This phosphorylation initiates activation of Rac1 pathway that is further followed by MAPK activation.

References 1. Aumailley M, Smyth N (1998) The role of laminins in basement membrane function. J Anat 193:1–21 2. Barresi R, Campbell KP (2006) Dystroglycan: from biosynthesis to pathogenesis of human disease. J Cell Sci 119:199–207 3. Ekblom P, Lonai P, Talts JF (2003) Expression and biological role of laminin-1. Matrix Biol 22:35–47 4. Givant-Horwitz V, Davidson B, Reich R (2005) Laminininduced signaling in tumor cells. Cancer Lett 233:1–10 5. Schneider H, Muhle C, Pacho F (2006) Biological function of laminin-5 and pathogenic impact of its deficiency. Eur J Cell Biol doi:10.1016/j.ejcb.2006.07.004

Langerhans Cell Definition

Langerhans’ cells are immature ▶dendritic cells found in skin containing Birbeck granules and expressing CD1a. They are the most efficient antigen processing and presenting cells of dendritic cell family. ▶Langerhans Cell Histiocytosis ▶Birbeck Granules

Langerhans Cell Histiocytosis A KIRA M ORIMOTO Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan

Synonyms Histiocytosis X

Definition

▶Langerhans Cell Histiocytosis (LCH), previously referred to as Histiocytosis X, is a rare clonal disorder of Langerhans cell proliferation, involving the skin, bone and other organs. The disease family consists of the syndromes originally described as ▶eosinophilic granuloma, ▶Hand–Schüller–Christian disease, and ▶Letterer–Siwe disease. Modern classification of LCH consists of single-system versus multisystem and unifocal versus multifocal.

Langerhans, Islets of Characteristics Definition Are groups of specialized cells in the pancreas that produce and secrete hormones. Named after the German pathologist Paul Langerhans (1847–1888), who discovered them in 1869, these cells are arranged in groups that Langerhans likened to little islands in the pancreas. There are five types of cells in an islet: alpha cells that produce ▶glucagon, which raises the level of glucose (sugar) in the blood; beta cells that produce ▶insulin; delta cells that produce ▶somatostatin which inhibits the release of numerous other hormones in the body; and PP cells and D1 cells, about which little is known. Degeneration of the insulin-producing beta cells is the main cause of type I (insulin-dependent) diabetes mellitus.

Epidemiology Most patients diagnosed with LCH are children with a peak percentage of diagnoses occurring between 1 and 3 years of age. The incidence of LCH has been estimated to be five cases per million per year in children. It appears to be more common in boys than in girls (1.2–2:1). The incidence of LCH in adults is thought to be one-half of that in children. Development of the disease is usually sporadic; however, the fact that about 1% of patients have relatives with LCH and monozygotic twin pairs are concordant for LCH suggests a genetic predisposition. A higher frequency of malignant disorders has been reported in patients with LCH than in the normal population. Acute lymphoblastic leukemia is the most common malignancy preceding or co-occurring with LCH.

Langerhans Cell Histiocytosis

Etiology Whether LCH is a neoplasm or is reactive in nature has been a controversial issue. Pathological Langerhans cells (LCH cells) are monoclonal, and sometimes show chromosomal deletion or gain, suggesting a neoplastic etiology. While LCH lesions often regress spontaneously, there is no evidence that LCH cells are immortalized, supporting the possibility of a reactive nature. Although infection may play a role in the development or reactivation of LCH, no well-accepted environmental risk factors are associated with the disease, except for cigarette smoking in adult pulmonary LCH. It has been demonstrated that 77% of adult patients with LCH pulmonary lesions are smokers.

Pathophysiology LCH lesions not only contain LCH cells but also various inflammatory participants, including T lymphocytes, macrophages, plasma cells, eosinophils, osteoclast-like multinucleated giant cells and neutrophils. These cells stimulate each other to produce abundant cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-2, IL-4, IL-5, IL-7, IL-10, transforming growth factor (TGF)-ß, ▶RANKL and ▶osteoprotegerin. This cytokine storm plays a role in the proliferation of LCH cells and of other infiltrating cells, and is responsible for the various clinical features of LCH. LCH cells in the bone, lymph node and some skin lesions contain immature ▶dendritic cells. These cells do not express CD83 or CD86, but do express intracellular the major histocompatibility complex (MHC) class II antigen. They also express the immature dendritic cell marker ▶CCR6 and produce the ligand for CCR6 (▶CCL20/MIP-3α) as well as ▶CCL5/ RANTES and ▶CXCL11/I-TAC. These ligands may play a role in recruiting eosinophils and CD4 positive T cells into the lesions, respectively. LCH cells show a greater proliferative capacity and a lower antigen presenting capability, suggesting maturation is arrested at an activated state. It is hypothesized that IL-10 as well as TGF-ß could be key factors in the inhibition of maturation of these cells.

Clinical Manifestations LCH affects a number of different organs, so clinical signs and symptoms may be extremely variable. Patients can present with either single-system or multisystem involvement. Single-system presentations most often occur in the bone with single-site or multifocal involvement, but can also occur in the skin. Most commonly, the initial manifestations include the occurrence of soft tissue mass, bone pain, skin rash and fever. Laboratory findings include normochromic and normocytic

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anemia and an elevated erythrocyte sedimentation rate. Elevations in IgM are also common. The bone is involved in about 80% of patients with LCH. The skull is most often affected followed by the extremities, ribs, spine, and mandible and maxilla. Osteolytic “punched out” lesions with sharp margins are typically seen on X-ray. Bone lesions may be asymptomatic or accompanied with pain and soft tissue swelling, which may cause compression of adjacent structures such as the optic nerve or the spinal cord. The clinical course of LCH when localized solely to the bone is generally benign and it sometimes resolves spontaneously over a period of months to years. However, it may result in permanent sequelae including the collapse of vertebral bodies, orthopedic deformities and growth impairment. Skin involvement is seen in approximately half of patients. Patients present with lesions that are seborrhea-like eruptions on the scalp or an erytematous rash on the trunk, abdomen and inguinal areas. Ulcerative lesions in the genital or inguinal region may also be present. There may be bleeding into the lesions, even in the absence of thrombocytopenia. Lymph nodes in the cervical, axillary and inguinal areas are most commonly affected. Rarely, the nodes can become massive and cause upper airway obstruction. Earinvolvement usually appears as an aural discharge caused by external otitis, which is often associated with the destruction of mastoid. Ossicle or vestibular damage of the middle ear may cause a loss of hearing. Hepatosplenomegaly occurs in 20% of patients with the infiltration of histiocytes into hepatic sinuses. Various degrees of liver dysfunction may appear, including hyperbilirubinemia, hypoproteinemia, hypoalbuminemia, elevation of γ-GTP, alkaline phosphatase and/or transaminases, ascites and edema. Histological examination of the liver shows portal infiltrates which can cause bile duct destruction and periportal fibrosis (sclerosing cholangitis) leading to biliary cirrhosis with portal hypertension and ultimately secondary hypersplenism. Pulmonary involvement in children is usually part of multisystem disease, but in adults the lung involvement may be solitary and frequently regresses after the cessation of smoking. The lung pathology is associated with tachypnea, dyspnea, cyanosis, cough, pleural effusion and recurring pneumothorax. High resolution computed tomography may reveal reticular or micronodular opacities as well as large nodules and honeycombing. Typical histological findings are alveolar destruction and diffuse interstitial infiltration of histiocytes. Pulmonary fibrosis develops in 10% of patients and can lead to respiratory failure. Hematopoietic involvement is seen in disseminated LCH and defined by anemia (hemoglobin < 10 g/dl in the absence of iron deficiency), leukopenia (leukocytes < 4.0 × 109/l) or thrombocytopenia (platelets < 100 × 109/l)

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with or without bone marrow involvement. In severe cases, serious anemia and thrombocytopenia may develop, often associated with a secondary hemophagocytic syndrome. Oral mucosa infiltration may appear as ulcerations or swelling of gingiva resulting in the loss of teeth. Infiltration of the small bowel may occur and cause malabsorption of nutrients. Diarrhea with blood and/or mucus suggests involvement of the colon. Occasionally the pancreas is also involved. In the central nervous system (CNS), infiltration and dysfunction of the pituitary gland and/or adjacent hypothalamus occurs in about 20% of cases in those with multisystem involvement. The most frequent manifestation is diabetes insipidus (DI), which may precede, co-occur, or follow other symptoms and signs of the disease. DI occurs more often among patients with skull involvement (known as “CNS risk lesions”). Infiltration of the anterior pituitary is less common and may cause growth retardation and panhypopituitarism. These pathologies usually develop in those with DI after a disease course of 10 years. Magnetic resonance imaging (MRI) findings of LCH involvement of the pituitary gland and the hypothalamus are demonstrated by the loss of the physiologic high intensity signal of the posterior pituitary lobe on T1-weighted images. There can also be thickening of the pituitary stalk or a hypothalamic mass. Progressive, degenerative CNS disease may develop over the years after onset of disease, often when the disease is considered quiescent. CNS involvement causes ataxia, tremor, dysarthria, dysphagia and hyperreflexia. Changes in personal behavior, judgment and cognitive function may also develop. In this case, MRI studies using T2-weighted or FLAIR images may reveal bilateral symmetric lesions in the cerebellar white matter and basal ganglia. Histologically, the neurodegenerative LCH is characterized by the presence of CD8 positive T lymphocyte infiltration, microglia activation, gliosis, neuronal and axonal destruction with secondary demyelination. There may be a lack of ▶CD1a positive LCH cells, as is seen in autoimmune encephalitis. Currently there is no established therapy for LCH-CNS disease. Pathology and Diagnosis A pathological examination is indispensable in the diagnosis of LCH. With hematoxylin-eosin staining, LCH cells have a distinctive homogeneously stained pink cytoplasm. The nuclei appear twisted with a longitudinal groove and a small nucleolus, often with a “coffee bean” appearance. Immuno-histochemical staining of ▶S-100 protein and ▶langerin (CD207) is helpful for detection of LCH cells. In active lesions of the disease, LCH lesions show granulomas caused by the aggregation of LCH cells as well as a number of

various inflammatory cells. A definitive diagnosis can be made by either positive staining for CD1a or electron microscopic demonstration of ▶Birbeck granules in the granulomatous lesional cells. In the later stages of LCH, macrophages are more predominant than LCH cells in the lesions, and xanthomatous and fibrotic changes are characteristic. It is not uncommon that lesions at different stages of disease may be mixed in the same organ simultaneously. Prognosis The clinical course of LCH varies quite widely depending on the extent of organ involvement. Multisystem disease can be separated into two categories based on whether or not “risk organs” are involved. Risk organs are defined as the liver/spleen, lung or the hematopoietic system. LCH may resolve spontaneously in patients with localized, unifocal disease. Patients with single-system disease or without risk of organ involvement have a mortality of less than 5%. Prognosis is worse in children with multisystem and risk organ involvement who often have fatal outcomes despite intensive treatment Mortality rates of 10–50% have been reported. Infants younger than 2 years at diagnosis tend to have risk organ involvement, more often than older children; however, a recent study revealed onset age itself is not a prognostic factor. A major, positive prognostic factor appears to be a favorable response to the first 6 weeks of systemic multi-agent chemotherapy. Patients without an initial response tend to have an extremely high mortality rate with reports of 15–70%. This is in contrast to that of less than 5% in patients with a good initial response. In adults, lung disease may be a life threatening complication; it has been reported to contribute to a mortality rate of approximately 25%. Reactivation can occur unpredictably in more than half of patients, even those treated with multi-agent chemotherapy. Reactivated lesions may sometimes resolve spontaneously but there is an increased risk of permanent sequelae. Treatment Treatment of LCH should be planned according to the clinical presentation and the extent of organ involvement. In single-system LCH, the major aims of treatment are to lessen symptoms and to reduce the chance of permanent sequelae. In the case of a single bone lesion without symptoms, a wait-and-see approach or diagnostic curettage is the standard method of care. Steroids may be used for symptomatic bone lesions. Systemic chemotherapy with vinca alkaloids and corticosteroids for 6 or 12 months could be applied for patients with CNS-risk lesions or multifocal symptomatic bone disease. Radical operation of jaw lesions is discouraged as this often results in disfigurement and loss of

Laparoscopy

teeth. Radiation is rarely used because of the reported increased risk of secondary tumors. When there is skin involvement only a wait-and-see approach is considered optimal. Alternatively, patients can be provided therapies such as topical corticosteroids or thalidomide. In patients with isolated pulmonary LCH with functional impairment, systemic chemotherapy is indicated to reduce further parenchymal destruction. In multisystem LCH, the major aims of treatment are to increase survival and to reduce the incidence of late sequelae. Systemic chemotherapy with vinca alkaloids and corticosteroids for 12 months is the most commonly used regimen. In cases with risk-organ disease, more aggressive chemotherapy combined with agents such as cytarabine (Ara-C), 6-mercaptopurine and methotrexate may be considered. Etoposide (VP-16) is no longer considered a reasonable therapeutic agent because there is no reported significant efficacy and it has been shown to cause therapy-related acute myeloid leukemia (t-AML). In patients with refractory progressive disease, myeloablative therapy with a combination of high dose Ara-C and cladribine (2-CdA) is currently being tested. Immuno-suppressive therapy with cyclosporin A, anti-thymocyte globulin or anti-TNF agent, and immuno-modulation agents like thalidomide, IFN-α or bisphosphonate are also used on an investigational basis. Allogeneic hematopoietic stem cell transplantation has proved to be efficacious in some cases. Additionally, liver or heart and lung transplants have been performed successfully in patients with end-stage organ involvement. Late Sequelae Permanent sequelae are common events in many LCH patients. They are most often the result of the infiltrative nature of the disease itself which causes tissue destruction and granulomatous fibrosis or gliosis of various tissues. Seventy percent of patients with multisystem disease and 25% of single-system disease patients suffer one or more life-long sequelae, including DI (24%), orthopedic problems (20%), hearing loss (13%), neurologic problems (11%), growth hormone deficiency, loss of teeth, pulmonary fibrosis and biliary cirrhosis with portal hypertension. t-AML may develop as a consequence of LCH treatment with chemotherapy, especially following the use of topoisomerase II inhibitors, such as VP-16. The cumulative incidence of t-AML in patients treated with VP-16 for LCH has been estimated to be around 1%. In addition, secondary solid tumors, particularly sarcomas and brain tumors may develop in irradiated areas.

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2. McClain KL (2005) Drug therapy for the treatment of Langerhans cell histiocytosis. Expert Opin Pharmacother 6(14):2435–2441 3. Savasan S (2006) An enigmatic disease: childhood Langerhans cell histiocytosis in 2005. Int J Dermatol 45 (3):182–188 4. Morimoto A, Ikushima S, Kinugawa N et al. (2006) Improved outcome in the treatment of pediatric multifocal Langerhans cell histiocytosis: results from the Japan Langerhans cell Histiocytosis Study Group-96 protocol study. Cancer 107(3):613–619 5. Donadieu J, Egeler RM, Pritchard J (2005) Langerhans cell histiocytosis: a clinical update. In: Weitzman S, Egeler RM (eds) Histiocytic disorders of children and adults; basic science, clinical features and therapy. Cambridge University Press, Cambridge, pp 95–129

Langerin Definition Is a cell surface receptor that induces the formation of the ▶Birbeck granule. ▶Langerhans Cell Histiocytosis

LAP Definition Latency-Associated Peptide. ▶Transforming Growth Factor Beta

Laparoscopy Definition

References

Examination of the abdominal and pelvic structures within the peritoneum using an illuminated tubular instrument, which is passed through the abdominal wall via a small incision. It can be used for diagnosis and for certain operations.

1. Henter JI, Tondini C, Pritchard J (2004) Histiocyte disorders. Crit Rev Oncol Hematol 50(2):157–174

▶Endometriosis

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Lapatinib

Lapatinib Definition

Is an anti-cancer drug, a ▶tyrosine kinase inhibitor of human ▶epidermal growth factor receptor type 2 (HER2, (synonym ▶HER2/neu, also epidermal growth factor receptor (EGFR). Lapatinib is active in combination with capecitabine in women with HER2positive metastatic breast cancer that has progressed after ▶trastuzumab-based therapy.

is just in the lung or has spread to other places), tumor size, the type of lung cancer, whether there are symptoms, and the patient’s general health. Patients unsuitable for surgery may be offered curative intent radiotherapy. ▶Adjuvant therapy may be given to more advanced resected cases. For late stage cases, chemotherapy with or without palliative radiotherapy are the conventional options, although the long term survival rates are very low.

Large Granular Lymphocyte Large Cell Calcifying Sertoli Cell Tumor

▶Activated Natural Killer Cells

Definition

LCCST; Is a rare type of ▶testis cancer.

Large Tumor Suppressor Gene ▶Lats in Growth Regulation and Tumorigenesis

Large Cell Medulloblastoma Definition Variant of medulloblastoma accounting ~5% of cases. Characterized by more abundant cytoplasm than seen in classic medulloblastoma and large areas of necrosis. ▶Medulloblastoma

Laryngeal Carcinoma C HARLOTTE J IN Departments of Clinical Genetics, University Hospital, Lund, Sweden

Definition

Large-Cell Undifferentiated Carcinoma Definition

About 10–15% of ▶lung cancer are this type. It can start in any part of the lung. It tends to grow and spread quickly. Non-small cell lung cancer is a common disease. It is usually treated by surgery (taking out the cancer in an operation) or radiation therapy (using high-dose x-rays to kill cancer cells). However, chemotherapy may be used in some patients. The prognosis (chance of recovery) and choice of treatment depend on the stage of the cancer (whether it

The vast majority of malignant neoplasms of the larynx arises from the surface epithelium and therefore classified as keratinizing or nonkeratinizing ▶squamous cell carcinomas (SCC). The other rare malignant forms include verrucous carcinoma, adenocarcinoma, fibrosarcoma, and chondrosarcoma. Histopathologically, laryngeal SCC can further be classified into: well differentiated (more than 75% keratinization), moderately differentiated (25–75% keratinization), poorly differentiated (1 cm of healthy tissue around the tumor, in all directions, when the tissue is a muscle or adipose tissue, while when the tissue is periostium, vessel sheath, epineurium, or muscular fascia (that act as barriers) healthy tissue >1 mm may be considered sufficient. Radiotherapy. Radiotherapy plays a well-defined role in local control in ▶STSs. In particular, in adult STSs irradiation is recommended not only after incomplete resection, but also after wide excision, especially in case of large tumors. Certainly, the indication for radiotherapy has to be stricter in children and young adolescents with NRSTSs than in adults, given the higher risk of severe late effects of radiotherapy (in particular, the risk of retardation or arrest of irradiated bone growth, the risk of functional impairment and that of second postirradiation tumor): the patient’s age must always be borne in mind when planning radiotherapy in order to keep its sequelae to a minimum. In patients who received initial wide resection, in fact, the indication for radiotherapy is still debated due to the problem of sequelae, though reported

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Non-Rhabdomyosarcoma Soft Tissue Sarcomas

Non-Rhabdomyosarcoma Soft Tissue Sarcomas. Figure 3 Overall survival of NRSTSs according to histotypes, tumor grade, surgical margins (IRS group), tumor size. (Ferrari et al (2005) J Clin Oncol 23:4021–4030).

series showed a favorable trend for the addition of radiotherapy and therefore seemed to suggest the use of postoperative irradiation in those cases with tumor larger than 5 cm. The indication for postsurgical radiotherapy is clearer, instead, after initial marginal resection (the so-called IRS group II patients): when suspected microscopical residual tumor is left in situ (and when a re-excision is considered unfeasible), radiotherapy should be given, since the risk of local recurrence appears very high. The local treatment strategy is more complicated in patients whose tumors are judged unresectable at diagnosis, and thus receive initial chemotherapy (IRS group III patients). For these patients, delayed surgery is the treatment of choice, but surgery and radiotherapy should be discussed in a multidisciplinary setting and combined in order to define (and customize) the best local treatment for each patient, considering again that radiotherapy should be administered not only considering the need to maximize the chances of local control, but also containing the radiation-related sequelae and preserve function: for instance, irradiation after delayed surgery could be avoided in younger patients, while it might be recommended in the case of large tumors; postoperative radiotherapy may be easier to plan and carries a lower risk of complications, but preoperative irradiation can improve the chances of achieving free margins at the

secondary resection, may reduce the risk of intraoperative contamination, and smaller radiotherapy fields and lower doses can often be used. Chemotherapy. While conservative surgical resection unquestionably remains the mainstay of treatment for NRSTSs, and the effectiveness of radiotherapy is widely appreciated, oncologists are still wondering about systemic chemotherapy and NRSTSs, as adult ▶STSs, continue to be considered scarcely chemosensitive (unlike the case of ▶RMS, which is a highly chemosensitive tumor). However, it is generally agreed that the outcome is reasonably good in patients with resected, small, noninvasive NRSTSs (survival rate up to 90%), whereas even after initial gross resection, the prognosis for patients with high-grade and large invasive tumors would be unsatisfactory if the treatment were limited to surgery alone (with or with out radiotherapy). The risk of developing distant metastases, particularly to the lung, is higher for high malignant NRSTSs, and the role of different chemotherapy regimens has been variously investigated. Several findings emerging from adult trials have suggested that the combination of fulldose ifosfamide and ▶doxorubicin (▶adriamycin) was the regimen achieving the highest response rate in STSs. Of course, chemotherapy has to be recommended in frontline treatment in patients with metastatic and advanced disease at diagnosis, and also in all those

Non-Rhabdomyosarcoma Soft Tissue Sarcomas

cases where the surgeon is unsure of being able to achieve a complete resection at the first attempt. Neoadjuvant chemotherapy might convert such cases into conservative complete resections, as well as treating any micrometastases promptly, since these patients have a high risk of distant dissemination whatever the local control measures adopted. Overall, the response rate (complete response plus partial response >50%) of NRSTSs to frontline chemotherapy is considered around 40%, though more recent findings would suggest better percentage of response when minor responses were also included (and some cases that had initially been considered unresectable reportedly underwent complete delayed surgery after minor tumor shrinkage induced by chemotherapy). Even more doubts surround the role of ▶adjuvant chemotherapy, though recent evidence seems to suggest that it may have a more significant beneficial impact in selected cases than is generally believed. Despite the relatively good prognosis for patients who received initial resection, a particular group of high-risk patients can be in fact earmarked: the combination of two variables – high tumor grade plus large tumor size – gives rise to a very high risk of metastases, irrespective of the results of initial surgery, suggesting that systemic chemotherapy should, in principle, be used to improve survival. In patients with these characteristics, in fact, the metastases-free survival is around 30–40%, but it seems clearly better in those patients who received adjuvant chemotherapy, as compared with those who have not been given. Of course, the debate on whether or not to provide adjuvant chemotherapy for STS remains wide open. The role of adjuvant chemotherapy in preventing distant recurrences after initial surgery has long been a point of controversy in the field of clinical studies on adult STS. Most randomized trials performed by North American and European groups showed neither statistically significant benefit for patients given adjuvant chemotherapy, but some of the negative results recorded in those studies need to be reconsider since these trials did not use the combination of drugs currently recognized as the most effective in STS (ifosfamide, in particular, was not included in most of these studies), nor had they selected patients most likely to respond to chemotherapy (tumors of diverse histology, grade, and size were grouped together). A completely separate discussion should be dedicated to the role of chemotherapy in synovial sarcoma. Over the years, different strategies have been developed for pediatric and adult oncology protocols dealing with synovial sarcoma. High rates of response to chemotherapy were recorded in pediatric series, so synovial sarcoma came to be considered an “RMS-like” tumor and most pediatric patients were consequently included in RMS protocols, receiving adjuvant chemotherapy regardless of the risk factors (i.e., even after

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the complete excision of very small tumors), whereas adjuvant chemotherapy was generally used only in adult patients as part of trials with a no-therapy control arm and including all soft tissue sarcoma histotypes. The various reported experiences with this tumor in the field of pediatric oncology reported a 5-year overall survival rate around 80%, i.e, higher than the one usually reported in adult series, and an approximately 60% rate of response to chemotherapy in patients with measurable disease, i.e., higher than the response rate usually reported for other adult STS. Data from adult literature, instead, reported less satisfactory overall results: this finding may have to do with a different incidence of adverse prognostic factors in different age groups (i.e., large tumors were more frequent in older patients), but the different results might also be related, to some degree at least, to the different treatment strategies adopted, and particularly to the different use made of chemotherapy. A formal demonstration of the efficacy of adjuvant chemotherapy in synovial sarcoma is not available, but various data would nonetheless suggest that it does have a part to play. It may be that adjuvant chemotherapy for all synovial sarcoma patients (as pediatric oncologists did) is tantamount to overtreatment: it is probable that a subset of low-risk patients – i.e., completely resected, with tumor smaller than 5 cm – may be identified for which adjuvant chemotherapy can be omitted without jeopardizing the results. Particular Histotypes The above discussed considerations are suitable for most of the NRSTS histotypes, and in particular for the so-called “adult-type” NRSTS. However, there are some particular entities that are characterized by peculiar biology and clinical course, and deserve to be considered separately. Extra-osseous primitive peripheral neuroectodermal tumor (pPNET)/Ewing sarcoma is less frequent than the skeletal ▶Ewing sarcoma, but it is likely that there are no biological differences between Ewing sarcomas arising at different sites, so the clinical considerations should be the same: it is a high malignant tumor, with a strong propensity to give metastases, that should be treated with a multimodality strategy including, in all cases, multi-agent chemotherapy. ▶DSRCT is a very aggressive neoplasm, usually arising in the abdominal cavity, often characterized by a poor outcome despite the various intensive multimodality treatment approaches attempted over the years (i.e., aggressive surgery, radiotherapy, intensive chemotherapy including high-dose myeloablative chemotherapy (▶myeloablative megatherapy) with stem cell rescue). Soft part ▶rhabdoid tumors represent the soft tissue counterpart of the intracranial and renal entities: they are very rare and very aggressive disease, for which

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Non-small Cell Lung Cancer

improvement in genetic studies are strongly needed to cast light on their common biology in order to think to new treatment approaches. The heterogeneous assortment of NRSTS includes tumors as ▶epithelioid hemangioendothelioma, a neoplasm that sometimes arises at soft part, as a single lesion located in the extremities or cervical district, with very little propensity to metastasize or become fatal, but sometimes develop at bone, lung, and liver, often multifocal or metastatic, with indolent course but also a not negligible risk of death, for which treatment with alpha-interferon may have a significant role, probably due to an antiangiogenic effect. Finally, infantile fibrosarcoma is a peculiar tumor of infants (it is the most common soft tissue sarcoma under 1 year of age): its clinical behavior may widely vary (i.e., it can rapidly grow and also give metastatic spread, but some cases of spontaneous regression have been described). However, chemotherapy is fairly effective in this tumor, also utilizing mild alkylating/ anthracyclines-free regimens, and, consequently, the overall prognosis is very good. Future Issues Despite their heterogeneous nature, NRSTS currently have to be analyzed as a group because of their rarity and the availability of few therapeutic options (i.e., surgery remains the keystone of treatment, and few drugs are effective). However, this situation is changing and the next steps may go in the direction of histology-driven therapies: drugs other than ifosfamide–doxorubicin combination have proved fairly active against particular histotypes (i.e., ▶taxanes for ▶angiosarcoma, ▶gemcitabine for ▶leiomyosarcoma, ▶ET-743 for ▶myxoid liposarcoma). But what physicians are waiting is to really improve the understanding of the biology of these tumors, paving the way toward novel molecular therapeutic approaches. The specific chromosomal translocations occurring in ▶STS may become the targets of new molecular agents specifically designed to influence the tumor’s biology. Of course, if the near future sees STS studied no longer as a mixed bunch, but focusing separately on each histotype, this will only be feasible (especially for investigators working on pediatric NRSTS) if there is close cooperation between international groups, and between pediatric and medical oncology groups.

References 1. Okcu MF, Hicks J, Merchant TE et al. (2006) Nonrhabdomyosarcomatous soft tissue sarcomas. In: Pizzo PA, Poplack DC (eds), Principles and practice of pediatric oncology, 5th edn. Lippincott Williams & Wilkins, Philadelphia, pp 1033–1073 2. Ferrari A, Casanova M (2005) New concepts for the treatment of pediatric non-rhabdomyosarcoma soft tissue sarcomas. Expert Rev Anticancer Ther 5(2):307–318

3. Spunt SL, Poquette CA, Hurt YS et al. (1999) Prognostic factors for children and adolescents with surgically resected nonrhabdomyosarcoma soft tissue sarcoma: an analysis of 121 patients treated at St Jude Children’s Research Hospital. J Clin Oncol 17:3697–3705 4. Ferrari A, Casanova M, Meazza C et al. (2005) Adult-type soft tissue sarcomas in pediatric age: experience at the Istituto Nazionale Tumori in Milan. J Clin Oncol 23:4021–4030 5. Okcu MF, Munsell M, Treuner J et al. (2003) Synovial sarcoma of childhood and adolescence: a multicenter, multivariate analysis of outcome. J Clin Oncol 21:1602–1611

Non-small Cell Lung Cancer Definition

NSCLC; About 80–85% of all cases of ▶lung cancer are of the non-small cell type. There are three sub-types of NSCLC. The cells in these sub-types differ in size, shape, and chemical make-up.

Non-syntenic Definition Refers to genes or genetic loci that lie on different chromosomes, i.e. are not genetically linked.

Non-Viral Vector for Cancer Therapy ATSUKO F UJIHARA 1,2 , YASUFUMI K ANEDA 1 1

Department of Gene Therapy Science, Osaka University, Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, Japan 2 Department of Urology, Kyoto Prefectural University of Medicine, Kyoto, Japan

Definition Non-viral vectors can deliver therapeutic molecules into cells for the treatment of cancer. The molecules used for this purpose are usually anticancer drugs, short interfering RNA (▶siRNA), and DNA (Table 1). Nonviral vectors can also be employed for the delivery

Non-Viral Vector for Cancer Therapy

of proteins, peptides, and messenger RNA, but such agents are not so frequently used for cancer therapy. Anti-sense oligonucleotides (▶Antisense DNA therapy) were previously employed to suppress gene expression, but siRNA has recently taken their place.

Characteristics Viral vectors are generally constructed by inserting a therapeutic gene into an engineered viral genome. In contrast, construction of non-viral vectors does not require viral genome engineering and these vectors are synthesized from chemical products or native materials, after which they associate with therapeutic molecules to promote more efficient delivery. Non-viral vectors are generally less efficient at promoting gene delivery and gene expression than viral vectors (▶Viral vectormediated gene transfer), but pose less risk and can also be employed for drug delivery. The most representative non-viral vectors are ▶liposomes and ▶polymers. Physical methods, such as naked DNA injection, electroporation, and sonoporation, also allow non-viral Non-Viral Vector for Cancer Therapy. Table 1 Therapeutic molecules for cancer treatment delivered using non-viral vectors Anticancer drugs Bleomycin Cisplatin Doxorubicin Methotrexate

siRNA

DNA

Rad51 VEGF HIF-1α β-catenin

p53 B7–1 IL-12 HSV-tk

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delivery of molecules, but no carrier or vector is associated with the therapeutic molecules. Liposomes Liposomes are artificial phospholipid bilayer vesicles formed from an aqueous suspension of phospholipid molecules that can be employed for the targeted delivery of macromolecules. Cationic liposomes can bind DNA more tightly and show improved transfection efficiency. However, DNA is taken up into cells by ▶endocytosis during lipoplex-mediated transfection. The main problem with endocytosis-mediated delivery is that therapeutic molecules are often subject to degradation within endosomes or lysosomes, as shown in Fig. 1. To solve this problem, several methods have been tried. One method is to employ a neutral lipid (DOPE) that facilitates the endosomal release of DNA. Its discovery has led to the use of a mixture of cationic lipids and DOPE for lipofection. Further analysis of various lipids has revealed that a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2hydroxyethyl) ammonium bromide and cholesterol is capable of destabilizing the endosome membrane more effectively than DOPE. To further protect therapeutic molecules delivered by liposomes, DNA is now conjugated with cationic molecules. For example, protamine sulfate or adenovirus mµ protein is conjugated with DNA, after which the newly formed complexes are incorporated into or mixed with cationic liposomes. Investigations of these methods have shown that cationic liposomes containing the HLA-B7 and β-2 microglobulin genes can induce antitumor immunity in HLA-B7-negative

Non-Viral Vector for Cancer Therapy. Figure 1 Pathways by which therapeutic molecules can be introduced into cells using liposomes or fusion-liposomes. Molecules delivered in liposomes escape into the cytoplasm from the endocytotic pathway, while molecules delivered by fusion-liposomes directly reach the cytoplasm by membrane fusion.

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Non-Viral Vector for Cancer Therapy

▶melanoma patients, and a number of centers have performed clinical trials of a liposomal drug (Allovectin-7) for the treatment of metastatic melanoma. Delivery of the β-interferon gene by cationic liposomes has also been evaluated for the treatment of patients with ▶glioblastoma. In addition, several other clinical trials have tested the delivery of various anticancer agents using liposomes. Tissue targeting (▶Targeted drug delivery) is important for the efficient delivery of therapeutic molecules with minimum toxicity. Especially in cancer therapy, tumor-specific delivery is essential for treating metastases. Generally, there are two approaches to tissue targeting, which are known as ▶therapeutic active targeting and ▶therapeutic passive targeting (Fig. 2). Passive targeting is the method used for most cancer-targeting vectors, because tumor vasculature has an abnormally high permeability due to its incomplete architecture. If a vector remains stable in the circulation and is not trapped by the reticuloendothelial system, it can cross the walls of tumor vessels and accumulate in tumor tissues. To achieve this, the diameter of a vector should be less than 300 nm (preferably less than 150 nm), and the vector should be coated with polyethylene glycol. In addition, the vector should associate effectively with tumor cells. In order to promote the internalization of therapeutic DNA by tumor cells, various cell receptor ligands (such as folate and transferrin) have been used to take advantage of receptor-mediated endocytosis. Polymers Polymers are also used as DNA carriers and can be divided into two categories based on their biodegradability. Various cationic non-biodegradable polymers have

been widely investigated for enhancing the internalization of therapeutic molecules. The most common linear cationic polymers are poly (ethyleneimine) and polyL-(lysine). Other polymers include poly (N-ethyl4-vinylpyridinium bromide), poly (dimethylaminoethyl methacrylate), chitosan, and dimethylaminodextran, or branched polymers such as poly (amidoamine) ▶dendrimer and branched poly (ethyleneimine). Since DNA is a large and negatively charged molecule, it is difficult to attach it directly to the cell membrane (also negatively charged) and achieve internalization into cells. It is well known that cationic polymers can easily form complexes with negatively charged DNA via electrostatic interactions. Such complex formation condenses the DNA molecule and converts the net electrical charge to positive under appropriate conditions. This condensation and the positive charge of a DNA-cationic polymer complex facilitate attachment to cells and subsequent internalization via the normal endocytosis pathway. In addition to electrostatic interactions, hydrophilicity and hydrophobicity can be utilized to incorporate therapeutic molecules. Block copolymers with both hydrophilic and hydrophobic characteristics can be assembled to form a micelle structure that incorporates anticancer drugs or DNA. Such ▶micelles are small enough (50–100 nm in diameter) to reach tumors by extravasation from their vessels. Targeting molecules can be also bound to particles of the nanometer order in size for tissue targeting. Biodegradable polymers have been used to achieve controlled release of DNA, thus enhancing and prolonging gene expression. Controlled-release technology increases the concentration of DNA and

Non-Viral Vector for Cancer Therapy. Figure 2 Two different approaches for tumor-specific delivery. (a) Vectors that possess tumor-specific molecules such as monoclonal antibodies and ligands recognize tumor cells by molecular interactions. (b) Vectors with a small size and polyethylene glycol coating passively accumulate in tumor tissues, which are characterized by a high interstitial pressure, enhanced vascular permeability, and the lack of functional lymphatic drainage.

Nonsense Mutation

prolongs its persistence at an injection site. By using this method, the biological activity of an antitumor DNA plasmid (NK4) is enhanced. Virosomes In order to enhance the efficiency of delivery, trials of viral envelopes or other proteins have been performed. Empty viral envelopes without the viral genome that are used to incorporate drugs and non-viral vectors decorated with viral components are called ▶virosomes. The representative virosomal vectors are described below. Fusion-Liposomes To avoid degradation prior to reaching the cytoplasm, fusion-mediated delivery systems have been developed (Fig. 1). A fusigenic viral liposome with a fusigenic envelope derived from ▶HVJ (▶Sendai virus) was constructed first. Primitive ▶HVJ-liposomes are made by fusion of liposomes with UV-inactivated HVJ. Reconstituted fusion liposomes can also be created. Use of HVJ-liposomes to deliver anticancer treatment has already been investigated in animal models. A melanoma-associated antigen gene (▶Melanoma antigen) or RNA injected into skeletal muscle or the spleen successfully evokes antitumor immunity and prevents the growth of melanoma. A radiation-inducible HSV-TK gene driven by the Egr-1 promoter enhances the efficacy of radiotherapy for ▶hepatocellular carcinoma when delivered by HVJ-anionic liposomes. Using an ▶EBV replicon plasmid containing the HSV-TK gene, ▶suicide gene therapy (▶HSV-TK/Ganciclovir mediated toxicity) is more effective against melanoma in tumor-bearing mice. A similar approach to enhance the efficiency of gene transfer uses fusion peptides derived from influenza virus hemagglutinin for receptor-mediated gene delivery. Combining transferrin/poly-L-lysine/DNA complexes with the hemagglutinin peptide increases the efficiency of gene transfer into cultured cancer cells by more than 1,000-fold compared with transfer in the absence of this peptide. HVJ-Envelope Vector To simplify the vector system and to increase the efficiency of gene delivery, plasmid DNA has been incorporated into inactivated HVJ particles without liposomes by detergent treatment and centrifugation. The HVJ-envelope (inactivated Sendai virus) vector can deliver DNA, siRNA, proteins, and anticancer drugs to cells both in vitro and in vivo. Rad51 siRNA has been delivered to tumors in mice with approximately 50% efficiency by this HVJ envelope vector. After Rad51 siRNA was delivered to tumors using this envelope vector, sensitivity to ▶cisplatin was enhanced more than tenfold compared with use of cisplatin alone. The HVJ envelope vector can also enhanced transfection efficiency after conjugation with a biocompatible

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polymer or magnetic beads. The vector itself is an effective anticancer agent because it can induce T cellmediated antitumor immunity through activation of dendritic cells and inhibition of regulatory T cells.

References 1. Conwell CC, Huang L (2005) Recent advances in nonviral gene delivery. In: Huang L, Hung MC, Wagner E (eds) Non-viral vectors for gene therapy. Elsevier Academic Press, USA, pp 3–18 2. Kakizawa Y, Kataoka K (2002) Block copolymer micelles for delivery of gene and related compounds. Adv Drug Deliv Rev 54:203–222 3. Kaneda Y, Tabata Y (2006) Non-viral vectors for cancer therapy. Cancer Sci 97:348–354 4. Kaneda Y, Yamamoto S, Nakajima T (2005) Development of HVJ envelope vector and its application to gene therapy. In: Huang L, HungMC, Wagner E (eds) Non-viral vectors for gene therapy. Elsevier Academic Press, USA, pp 307–332 5. Kostarelos K, Miller AD (2005) What role can chemistry play in cationic liposome-based gene therapy research today? In: HuangL, HungMC, Wagner E (eds) Non-viral vectors for gene therapy. Elsevier Academic Press, USA, pp 71–118

Nonparametric Definition Nonparametric analysis refers to empirical estimation of a distribution without relying on an underlying population distribution typically performed by using some form of ranking methods. ▶Kaplan–Meier Survival Analysis

Nonseminomatous Germ Cell Tumor ▶Testicular Cancer

Nonsense Mutation Definition A mutation that occurs within a codon and changes it to a stop codon.

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Nonspecific Cross Reacting Antigen

Nonspecific Cross Reacting Antigen Definition

The second member of the ▶CEA gene family to be discovered. NCA contains the characteristic IgV domain at the N-terminus but has only two IgC2 like domains. It is linked to the cell membrane via a glycosyl phosphatidyl inositol moiety.

Nonsteroidal Anti-inflammatory Drugs S ATISH K. S RIVASTAVA , KOTA V. R AMANA Dept of Biochemistry and Molecular Biology, University Of Texas Medical Branch, Galveston, TX, USA

Definition NSAIDs; Are a structurally diverse group of similarly acting compounds that prevent symptoms of pain, fever, and inflammation without steroid chemistry. Characteristics NSAIDs are the most commonly prescribed drugs worldwide for the treatment of pain and ▶inflammation. They are effective as ▶antipyretic and ▶analgesic and are also effective in prevention of cardiovascular complications. In recent years, evidence from animal as well as prospective and retrospective clinical studies indicate that NSAIDs may lower the risk of cancer development and more importantly progression of cancer, especially colorectal and breast cancers. Types of NSAIDs Depending on their strength, duration of action, and elimination from the body NSAIDs have been classified as . Salycylic acids: Aspirin, Salsalate, and Diflunisal . Acetic acids: Sulindac, Indomethacin, Diclofenac, and Tolmetin . Propionic acids: Ibuprofen, Naproxen, Flurbiprofen, Oxaprozin, Ketoprofen, and Fenoprofen . Enolic acids: Meloxicam and Piroxicam . Fenamic acids: Meclofenamate and Mefenamic acid . Pyranocarboxylic acids: Etodalac . Napthylalkanones: Nabumetone . Pyrroles: Ketorolac

. ▶COX-2 inhibitors: Celecoxib, Valdecoxib (Bextra), and Rofecoxib (Vioxx). Bextra and Vioxx were withdrawn from market. Mechanism of Action Most NSAIDs act as nonselective inhibitors of the enzyme ▶cyclooxygenase – they inhibit both the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isoenzymes. Cyclooxygenases catalyze the formation of ▶prostaglandins and ▶thromboxane from ▶arachidonic acid (Fig. 1). Prostaglandins act as messenger molecules in the process of inflammation and neoplasia. Overexpression of COX-2 stimulates angiogenesis, formation of new blood vessels, in tumors. Angiogenesis is required for the transition of normal cancers to invasive cancer leading to metastasis. COX-2 can stimulate ▶angiogenesis by promoting production of ▶vascular epithelial growth factor (VEGF), ▶matrix metalloproteinases, and prostaglandins. Further, COX-2 increases production of antiapoptotic protein ▶Bcl-2 and thus causes resistance to death of cancer cells. Cancer Chemoprevention ▶Inflammation originating from infections, chronic injury, or ▶autoimmune diseases can cause immune response, which subsequently forms ▶free radicals and ▶reactive oxygen species (ROS) through a cascade of reactions. These interact with cellular DNA, proteins, and lipids and cause cellular and genomic damage. In addition, the signals initiated by ROS release ▶eicosanoids which trigger cell proliferation, cause resistance to ▶apoptosis and facilitate carcinogenesis [2]. Although NSAIDs have been used for many years to treat rheumatic diseases and inflammatory symptoms, in recent years a large number of studies suggest the important chemopreventive properties of NSAIDs against various forms of cancer such as ▶colorectal cancer, ▶breast cancer, ▶bladder cancer, ▶prostate cancer, and ▶lung cancer. Colorectal Cancer Adenomatous polyps are the initial risk for most of the colon cancer tumors. ▶Cox-2 overexpression and ▶PGE2 production has been observed in various colorectal ▶adenomatous polyps and tumors but not in normal. Treatment of patients with familial adenomatous polyps with sulindac for 9 months resulted in a 44% and 35% decrease in the number and size of colonic polyps. Similarly, ▶celecoxib resulted in ~30% decrease in the number and size of colonic polyps. In another study, aspirin treatment also significantly reduced the risk of colon polyps. Thus, these and other ongoing clinical studies suggest the significance of NSAIDs in chemoprevention of colorectal cancer.

Nonsteroidal Anti-inflammatory Drugs

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N Nonsteroidal Anti-inflammatory Drugs. Figure 1 COX-2 inhibition by NSAIDs. Cyclooxygenases catalyze arachidonic acid to prostaglandin H2, which is further catalyzed to prostaglandin E2, D2, F2, and I2 and thromboxane A2. The prostaglandins cause cell growth, angiogenesis, and metastasis, leading to cancer development.

Breast Cancer Increased levels of ▶PGE2 production and COX2 expression along with ▶aromatase have been observed in the breast cancer biopsies. Subsequently, in a number of studies NSAIDs such as ▶aspirin have been used to prevent breast cancer-related complications. One study has reported a 21% reduction in the incidence of breast cancer in women taking NSAIDs at least twice a week for a period of 5–9 years and a 28% reduction after 10 or more years of use. Further, COX2 overexpressing transgenic mice developed mammary tumors after several cycles of pregnancy and lactation while wild type animals remained tumor free. In another study, a 35-day course of ibuprofen administered to rats with carcinogen-induced mammary tumors, led to a significant reduction in tumor volume. In animal models, celecoxib was found to be effective in preventing tumors associated with breast cancer.

The combination of standard chemotherapy drugs with COX-2 inhibitors has been shown to increase the efficacy of chemoprevention to breast cancer in a number of studies. Prostate Cancer Several investigations suggest that chronic ▶inflammation leads to ▶prostate cancer. Even though overexpression of COX-2 in malignant prostate tissues is not well noticed, the use of NSAIDs such as sulindac and flubiprofen has been shown to decrease risk of prostate cancer in animal models. Bladder Cancer The expression of COX-2 has been shown to increase in bladder transient cell carcinoma. Most of the studies performed using NSAIDs in animal models of ▶bladder cancer suggest that regular use of NSAIDs

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Noonan Syndrome

could prevent the risk of bladder cancer and is more effective with combinational therapy. COX-2 inhibitors are in ongoing clinical trials in preventing bladder cancer recurrence after transurethral resection.

Nootropic Definition

Lung Cancer COX-2 overexpression has been observed in the biopsies of ▶non-small cell lung cancer. In patients with asbestosis, idiopathic fibrosing alveolitis, and in heavy tobacco users the increased incidence of lung cancer is associated with overexpression of COX-2. Clinical studies have shown that NSAIDs are effective drugs in reducing risk of lung cancer, especially in heavy smokers. Conclusions A number of clinical, epidemiological, and animal studies suggest that NSAIDs could be promising anticancer agents. Targeted inhibition of cyclooxygenases alone or in combination with standard chemotherapy could be effective in the treatment of colorectal, bladder, and breast cancers. NSAIDs could also be effective in preventing recurrence of various types of tumors. To discover novel and most potent inhibitors of COX enzymes requires extensive investigations of molecular mechanism(s) by which COX and PGE2 promote angiogenesis and tumorigenesis. An exciting area for future investigations will be to test the nonNSAIDs such as ▶aldose reductase inhibitors alone or in combination with standard chemotherapy for the prevention of colon cancer as well as other oncogenic pathways.

Improvement in functions of brain. ▶Grape Seed Extract

Normoxia Definition Is the physiological (normal) O2 partial pressure distribution in a defined tissue, allowing unrestricted function and activity of cells making up the tissue (or organ). Normoxic refers to a physiologically adequate supply of oxygen. ▶Oxygenation of Tumors

Norton-Simon Hypothesis

References

T IFFANY A. T RAINA , L ARRY N ORTON

1. Dubois RN, Abramson SB, Crofford L et al. (1998) Cyclooxygenase in biology and disease. FASEB J 12:1063–1073 2. Gius D, Spitz DR (2006) Redox signaling in cancer biology. Antioxid Redox Signal 8:1249–1252 3. Tammali R, Ramana KV, Singhal SS et al. (2006) Aldose reductase regulates growth factor-induced cyclooxygenase-2 expression and prostaglandin e2 production in human colon cancer cells. Cancer Res 66:9705–9713

Breast Cancer Medicine Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Definition The Norton-Simon hypothesis states that the rate of cancer cell death in response to treatment is directly proportional to the tumor growth rate at the time of treatment.

Characteristics

Noonan Syndrome Definition

▶Autosomal dominant disorder presenting with characteristic facies, short stature, skeletal anomalies, congenital heart defects, and predisposition to tumors. ▶Neuro-Cardio-Facial-Cutaneous Syndromes

Several experimental models have attempted to describe the fundamentals of tumor cell growth and kinetics. From these systems arose an improved understanding of tumor growth characteristics, a foundation for the key principles of chemotherapy and eventually, recognition of the importance of dose scheduling. Specifically, the Norton-Simon hypothesis of tumor kinetics was an important advance in the history of oncology. The concept of ▶dose-density is derived from its principles and ultimately led to improved survival in patients with cancer as a result of optimal dose scheduling.

Norton-Simon Hypothesis

The Log-kill Model The Norton-Simon hypothesis builds upon the ▶logkill hypothesis established by Skipper, Schabel and Wilcox. These investigators used a murine leukemia model to describe tumor growth and cell death. A unique feature of this L1210 mouse system is its rapid and exponential growth pattern. The log-kill concept of tumor cell growth suggests that tumor growth is exponential and that growth rate is a constant. In response to treatment with a dose of drug, a constant fraction of cells are killed regardless of the size of the tumor at the start of therapy. Therefore, one dose of drug leads to cytoreduction from 106 cells to 104 cells in the same way that one dose at 104 cells reduces tumor volume to 102 cells. Theoretically, enough doses of enough drugs over time could cytoreduce a tumor to 100 kDa) single pass transmembrane-spanning proteins (Fig. 1). The primary amino acid sequence of the extracellular domain of Serrate/Jagged and Delta reveals a striking similarity to the extracellular domain of Notch proteins. Their extracellular domains are composed of between 1 and 16 tandem copies of an EGF-like repeat. One common feature shared among the ligand proteins is a 45 amino acid sequence rich in cysteine. It is termed the DSL domain and it is located in the N-terminal of the EGF-like repeats. This domain is thought to play a role in ligand-receptor interactions. Unlike Notch proteins, the intracellular sequences of Serrate/Jagged and Delta are short (ca. 100–150 amino acids) and contain no sequence homology to any other proteins in the database. The function of the intracellular sequences of Serrate/Jagged and Delta is not understood. However, the integrity of these sequences are important since deletion of the intracellular domain from either Delta or Serrate/Jagged leads to a phenotype similar to that exhibited by loss of Notch signaling in Drosophila. In addition, Serrate/Jagged proteins contain a cysteinerich domain (CR) located between the EGF-like repeats and the outer face of the plasma-membrane. The presence of this domain is used to classify ligand proteins as either Serrate- or Delta-like. ▶Endocytosis plays an important role in Notch activation and inhibition. It was shown that endocytosis of DSL ligands are required for Notch activation in a neighboring cell. It has been determined that Liquid facet, Neuralized and Mindbomb are required for ubiquitination and endocytosis of DSL ligands. However, the mechanism by which endocytosis of DSL ligands activates Notch pathway in the neighboring cell has not been determined. Additionally, it has been shown that ubiquitination and endocytosis of Notch by Numb,

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Notch/Jagged Signaling. Figure 1 Structure of Notch and DSL proteins. Notch proteins are transmembranespanning receptors for DSL proteins. Notchic is thought represent an activated Notch molecule. In T-cell Leukemia, the chromosomal translocation t(7;9)(q34;q34.3) fuses the T-cell receptor β locus to the Notch1 locus at the indicated break point (BP). The result of this translocation is the constitutive production of NotchIC-like molecules. RAM, binding domain for CSL transcription factors; ANK, 7-tandem copies of ankyrin repeats; OPA, Glutamine-rich region; PEST, a domain thought to be involved in protein turnover. Putative nuclear localization sequences are indicated by small boxes and flank the ANK domain. Notch ligands are encoded by the Serrate/Jagged and Delta genes. DSL, a 45 amino acid sequence unique to DSL proteins thought to be involved in interactions with Notch. CR, a cysteine-rich domain of unknown function used to classify DSL proteins as either Serrate/Jagged or Delta. The intracellular sequences of the DSL proteins are of variable length and contain no recognizable motifs.

α-adaptin and AIP4/Itch leads to degradation and, therefore, inhibition of Notch. There are four known mammalian Notch proteins, termed Notch1–4. These proteins are related through a high degree of sequence identity and structural organization. Notch proteins are membrane-spanning receptors with molecular weights of approximately 300 kDa (Fig. 1). The extracellular domain of Notch1 is composed of approximately 1,750 amino acids, which include 36 tandem repeats of a sequence resembling ▶Epidermal Growth Factor (EGF) and three repeats of a motif designated as lin-12 repeats. The cytoplasmic domain comprises a sequence of approximately 750 amino acids with no apparent enzymatic activity, but containing seven tandem copies of an ankyrin-like repeat (CDC10/ANK), a region rich in glutamine (OPA), and a region rich in proline, glutamate, serine and threonine

(PEST). The CDC10 and OPA repeats are thought to mediate protein-protein interactions, whereas the PEST domain might target the proteins for degradation. The RAM domain is located between the ANK repeats and the interface of the plasma-membrane. This domain is the primary binding site for the transcription factor CSL (CBF1/Supressor of Hairless/Lag1) (Fig. 1). Notch proteins are synthesized as precursor proteins of approximately 300 kDa. During trafficking through the Golgi network, Notch undergoes a proteolytic processing event that produces the mature receptor. This processing step appears to be carried out by a furin-like protease and the resulting mature Notch receptor is composed of two fragments, the extracellular fragment and an intracellular fragment that remains embedded in the plasma-membrane. One of the major questions in Notch signaling is what is the mechanism of ligand

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activation of Notch? One proposed mechanism is that the association of Notch with a DSL ligand on a neighboring cell induces a proteolytic event that releases the intracellular domain of Notch from its membrane tether (Fig. 2). Ligand-induced processing involves at least two distinct cleavage events. The first cleavage event linked to ligand binding is carried out by an ▶ADAM-type ▶metalloprotease that cleaves Notch in the extracellular domain close to the plasma-membrane. This cleavage is thought to induce a conformational change that allows the intracellular domain of Notch (NotchIC) to be released from the plasma membrane following cleavage by a presenilin-dependent γ-secretase-like protease. Currently, it is not understood how ligand engagement of the receptor induces these processing events or if activation of Notch can occur through interactions with ligands present in the same cell. Moreover, there is still debate over the possibility of signaling from membrane-tethered

forms of Notch that do not require additional proteolytic processing. Then, NotchIC translocates to the nucleus to form a transcriptional activation complex with the DNAbinding factor CSL and co-activators belonging to the Mastermind-like family of proteins (Maml). Recently, two CSL-NotchIC-Mastermind ternary complex structures bound to DNA were determined for C-elegans and human orthologous proteins. These studies showed that CSL simultaneously mediates interactions with NotchIC, Mastermind and DNA. The structures revealed that the N-terminal helical region of Mastermind forms a tripartite complex with ankyrin repeats 3–7 of NotchIC and the C-terminal domain of CSL. Additionally, the C-terminal helix of Mastermind interacts with the N-terminal domain of CSL. The activation complex exhibits a rapid turn-over rate, in part as a consequence to phosphorylation events triggered by cyclinC-CDK8 and the activity of E3-ligases of the Sel-10 family.

Notch/Jagged Signaling. Figure 2 Model for Notch signaling. Notch signaling is thought to occur following the interaction of DSL proteins and Notch proteins on neighboring cells. This interaction triggers a series of proteolytic processing events (indicated by scissors). Once Notch is cleaved, it is released from its membrane tether and translocates to the nucleus. In the nucleus NotchIC displaces the HDAC/co-Repressor complex and interacts with the transcription factors CSL and Maml and thereby activates gene expression.


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The Notch transcriptional activation complex participates in the regulation of gene expression that, in part, serves to govern cellular processes such as differentiation, proliferation and apoptosis. Numerous Notch target genes have been identified, including ▶c-myc, ▶cyclin D1, Jagged, p21, IL-4 and Fringe. Notch directly upregulates c-myc, a potent oncogene, in T-cells and mammary epithelial cells. Notch has also been found to directly induce the expression of cyclin D1, a positive cell cycle regulator, as well as indirectly suppress the pro-apoptotic protein, ▶p53, via downregulation of ▶ARF by a yet unidentified mechanism. Additionally, Notch activates two families of basic helix-loop-helix proteins, HES and HEY, which homo- and heterodimerize to actively repress target genes (Fig. 2).

Notch/Jagged Signaling. Figure 3 Effects of Fringe on Notch signaling. Fringe modifies Notch by adding O-fucose glycan to the EGF repeat region which modulates Notch affinity for its ligands. Upon expression of Fringe, Delta proteins are able to initiate Notch signaling, while Jagged proteins cannot.

Bi–Directional, Jagged1-Mediated Signaling The presence of a cysteine-rich region (CR) is used to classify Notch ligands into Delta-like or Serrate-like. Delta1, 2 and 4, not Jagged2 or Delta 3, also contain a putative PDZ-ligand domain (RMEYIV). The significance of the PDZ-ligand domain and the downstream events is not well understood in the context of NotchDSL signaling. However, it has been shown that overexpression of Jagged1 can induce cellular transformation in RKE cells, which is dependent on its PDZ-ligand domain (PSD-96/DLG/Zo-1).Both the intracellular and extracellular domains are required for Jagged1-mediated transformation. To date, a number of Jagged1 target genes have been identified, including Notch3, Delta1 and Jagged1, itself, although whether they are direct targets has not been determined. Deletion of the PDZ-ligand domain of Jagged1 prevents upregulation of Delta1 and Jagged1, but not Notch3, suggesting that there are at least two signaling pathways downstream of Jagged1. Fringe proteins provide another level of specificity between the two classes of DSL proteins. Fringe modifies Notch by adding O-fucose glycan to the EGF repeats which results in a decreased affinity for Jagged proteins and a higher affinity for Delta proteins. Therefore, upon expression of Fringe, Delta proteins are able to initiate Notch signaling, while Jagged proteins cannot (Fig. 3). A possible model for bidirectional signaling in the Notch pathway may involve a PDZ mechanism mediated by both Delta and Jagged proteins. In this model, Jagged1 could signal in both directions in the absence of Fringe, whereas the Delta signal would always be bidirectional. In the case of Jagged2, signaling would only be in the Notch direction and this signal could be attenuated by Fringe. In contrast, Delta3 is insensitive to Fringe and would always allow signaling in the Notch direction. This model provides exquisite flexibility for signal specificity and accounts for the multiple distinct DSL proteins.

The major question is: what is the mechanism by which the Notch activation complex exerts the pleiotropic effects that are observed in distinct cellular contexts? Over the last several years many proteins have been identified that can physically interact with NotchIC. However, a clear picture has not emerged to describe the mechanism of Notch signaling. Deltex, an ▶E3 ubiquitin ligase, is an exclusively cytoplasmic proline-rich protein localized to endosomal vesicles. It genetically acts as an enhancer of Notch signaling and has been shown to physically associate with Notch through the ankyrin repeat domain. As previously explained, data showed that Deltex ubiquitinates Notch preventing its degradation by sequestering it to endosomal vesicles. Mastermind like-1 (Maml1) is an integral component of the Notch pathway whose function is poorly understood. Maml1 encodes a nuclear co-activator protein that binds to the ankyrin repeat domain of Notch proteins. It forms a trimeric complex with the intracellular domain of Notch and the DNA binding protein, CSL. It is thought that Mastermind functions, at least in part, by recruiting histone acetyltransferases such as p300. CSL is the collective term for the mammalian counterparts to Su(H) and Lag1. CSL proteins are transcriptional regulatory proteins that repress transcription under non-induced conditions. Repression by CSL is thought to be mediated by a histone deacetylase (HDAC) complex containing n-CoR/Smrt. Upon ligand activation of Notch it is thought that NotchIC translocates to the nucleus and interacts with Skip to displace the HDAC complex from CSL which leads to the formation of a transcriptional activation complex (Fig. 2). Additional proteins have been identified that appear to genetically inhibit Notch signaling. These proteins are Dishevelled (Dsh), Numb and Ikaros. Dsh is a protein

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with unknown biochemical function that is a component of the Wnt signal transduction system. Dsh has been shown to physically interact with the carboxyterminal portion of Notch and appears to influence Notch signaling. Numb is a cytoplasmic protein that has homology to phosphotyrosine binding (PTB) domains and may provide a link to protein tyrosine kinase signaling cascades. The molecular basis of these interactions and the mechanism of inhibition of Notch signaling are not known. Additionally, it has been shown that Notch activation of target genes is perturbed by Ikaros, a transcriptional regulator required for development of all lympoid derived cells. This inhibition is the consequence of the ability of Ikaros to bind CSL consensus sequences. Proviral insertion mutagenesis experiments performed in NotchIC transgenic mice using Moloney murine leukemia virus showed an insertion into the Ikaros locus in 40% of tumors generated. These proviral insertions result in truncated Ikaros proteins, and those lacking DNA binding domains may then function as dominant negative inhibitors to full-length Ikaros by forming dimers. These dimers prevent Ikaros-mediated inhibition of Notch transactivation of target genes. Clinical Relevance T-Cell Leukemia The human homologue of Notch1 (TAN1) was cloned from a T-cell acute lymphoblastic leukemia (T-ALL) that harbored the chromosomal translocation, t(7;9) (q34;q34.3). This translocation joins a portion of NOTCH1/TAN1 to the T-cell receptor β locus. This translocation generates aberrant Notch proteins that lack most of the extracellular domain and are not tethered to the plasma membrane. As described above, these forms of Notch are thought to be constitutively active. This translocation can be identified in approximately 10% of all T-ALL cases. Additionally, 50% of human T-ALL exhibit activating Notch1 mutations in the heterodimerization domain and/or the C-terminal PEST region. Cervical Carcinoma In situ expression studies comparing normal and neoplastic cervical epithelium, showed an aberrant Notch expression in the development of ▶cervical carcinoma. In normal cervical tissue, expression of Notch is limited to the basal layer of the stratified epithelium. However, in dysplastic tissues both Notch and its ligands’ expression is increased compared to normal tissues, indicating that inappropriate activation of Notch signaling might play a role in the generation of cervical neoplasia. Approximately 99% of cervical neoplasms are positive for ▶Human Papillomavirus (HPV) E6 and E7. HPV infection of cervical epithelial cells frequently leads to cellular transformation. HPV viral proteins, E6 and E7, transform cells, in part by targeting the tumor suppressors

p53 and Rb for degradation. It has been demonstrated that E6 and E7 upregulate Notch and presenelin expression, and that growth of CaSki cells (cervical carcinoma cells expressing HPV16) are Notch-dependent. Additionally, the Notch1 gene is an integration site for HPV16. Paradoxically, data indicate that Notch1 must be downregulated in invasive cervical cancers expressing E6 and E7, suggesting that Notch1 plays a positive role in early cervical carcinogenesis, but suppresses E6 and E7mediated transformation in later stages of tumorigenesis. B-Cell Leukemia ▶Epstein-Barr Virus (EBV) is the etiologic agent of ▶Burkitt lymphoma. The latent viral protein EBNA2 is a transcriptional activator that functions by binding to host cell CSL proteins and displacing the HDAC repressor complex in an analogous manner to the model for NotchIC. Therefore, in transformation of B-cells by EBV infection, EBNA2 may provide a function similar to NotchIC by usurping CSL proteins. Notch can induce the same phenotypic changes as EBNA2 in Burkitt lymphoma cells. Mammary Carcinoma Infection of mice with MMTV has been used as an insertional mutagen to identify genes that contribute to the generation of mammary carcinoma. Among the genes affected by such mutations is Int-3, a Notch gene family member now termed Notch4. The mutations result in aberrant expression of truncated Notch4 proteins comprising only the intracellular portion of the molecule. Additionally, in MMTV-neu/ErbB2 insertional mutagenesis studies, Notch1 insertions were found in 8% of tumors screened. Insertions were found in the genomic region between the transmembrane domain and lin-12 repeats. Furthermore, it has been demonstrated that the expression of constitutively active Notch1, Notch3 or Notch4 is sufficient to induce breast tumors in mouse models. Moreover, it was determined that loss of myc prevents the formation of MMTV-Notch1IC nonregressing mammary tumors. Additionally, it was recently shown that human breast tumor samples expressing high levels of Jagged1 have poor clinical outcome compared to low Jagged1 expressing samples. Prostate Carcinoma Jagged1 expression correlates with ▶prostate cancer progression, metastasis and recurrence. Additionally, downregulation of Jagged1 expression results in growth inhibition of prostate cell lines, PC3, Du145, LNCaP and C4-2B. Taken together, these data suggest that deregulated Jagged1 expression may contribute to prostate cancer cell growth either by changing the microenvironment and increasing Notch activation in neighboring cells, via its downstream, PDZ-dependent


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Notch/Jagged Signaling. Table 1 Evidence of Notch’s function as an oncogene and tumor suppressor in human and mouse models Evidence of the oncogenic potential of Notch Human cancers Non-small cell lung carcinoma T-ALL Ovarian serous carcinoma Cervical carcinoma Mouse models of cancer Brain cancer (choroid plexus) Breast cancer T-ALL Evidence of a tumor suppressor role for Notch Mouse models of cancer Squamous cell carcinoma Basal cell carcinoma

Genetic alteration Increased Notch3 expression via translocation (15;19)(15, 25) Notch1 activating mutations (25, 72) Notch3 gene amplification (49) Increased expression of activated Notch1 and Notch2 (75) Genetic alteration Constitutively active Notch3 expression (14) Constitutively active Notch1, Notch3 or Notch4 expression (24) Constitutively active Notch1 expression (3, 25) Genetic alteration dn MAML1 (52) Notch1 knockout (45)

signaling events in the cell in which it is expressed or bi-directionally. Feline Leukemia Virus (FeLV) Induced T-Cell Leukemia Infection of cats with replication competent Feline Leukemia Virus (FeLV) yielded T-cell leukemia that harbored recombinant FeLV that had transduced a portion of the feline Notch2 gene. The transduced gene encodes a Notch2 protein analogous to those expressed in human T-ALL. Additionally, aberrant expression and/or activation of the various Notch isoforms have also been recently implicated in contributing to non-small lung carcinoma, ovarian serous carcinoma, brain cancer, squamous cell carcinoma and basal cell carcinoma (Table 1). Cross-Talk Between Signaling Pathways Notch linked to cancer through its frequent mutation in T-ALL, can integrate with other pathways to accelerate oncogenesis. Like Wingless (Wnt) and ▶Sonic Hedgehog (Shh), the Notch signaling pathway is essential in controlling both developmental processes and tumorigenesis. For example, in human and murine medulloblastoma, Notch and Shh synergize to promote tumor proliferation and survival. Also in ▶medulloblastoma, Shh dependent tumor growth involves synergy with Notch and Wnt. Notch/Wnt crosstalk has been suggested in Melanoma, activation of Notch1 enhances primary melanoma cell growth and the potential for metastasis through β-catenin upregulation. An important positive oncogenic collaboration was also shown in breast cancer cells between Notch and Wnt. Notch as a Potential Therapeutic Target Due to the important role of Notch signaling in cancer development, it has been proposed that targeting the

Notch signaling steps including receptor/ligand binding, release of NotchIC and downstream targets could have antitumor effects. One of the approaches used to inhibit Notch signaling is to suppress the γ-secretaseproteolytic step leading to the release of active NotchIC. Inhibitors for γ-secretase have been studied for decades for their potential to block the formation of beta-amyloid peptide (Aβ) associated with Alzheimer disease. Aβ peptide is synthesized much in the same way that active NotchIC is generated, via a series of proteolytic cleavages, the final of which is cleavage of the precursor by γ-secretase. It has been demonstrated that γ-secretase inhibitors are capable of preventing Notch receptor activation and are currently being tested for their antitumor effects. Encouragingly, various γ-secretase inhibitors have been successfully used in mouse cancer models to inhibit tumorigenesis. Inhibition of tumor growth was demonstrated in xenografts of the ▶Kaposi sarcoma cell line, SLK, upon injection of the tumor with a γ-secretase inhibitor, GSI-I. Additionally, intraperitoneal injection of a γ-secretase inhibitor, DBZ, inhibited epithelial cell proliferation in intestinal adenomas from Apc–/– mice and induced goblet cell differentiation. Currently, Merck is recruiting or has completed recruitment of patients for phase I clinical trial to test safety/tolerability and efficacy of a Notch inhibitor, MK0752, in relapsed or refractory T-ALL patients and advanced breast cancers (ClinicalTrials.gov Identifier: NCT00100152 & NCT00106145, respectively).

References 1. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689 2. Grabher C, von Boehmer H, Look AT (2006) Notch1 activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia. Nat Rev Cancer 6:347–359

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3. Hurlbut GD, Kankel MW, Lake RJ et al. (2007) Crossing paths with Notch in the hyper-network. Curr Opin Cell Biol 19:166–175 4. Kovall RA (2007) Structures of CSL, Notch and Mastermind proteins: piecing together an active transcription complex. Curr Opin Struct Biol 17:117–127 5. Ascano JM, Beverly LJ, Capobianco AJ (2003) The C-terminal PDZ-ligand of JAGGED1 is essential for cellular transformation. J Biol Chem 278:8771–8779

4-NQO Definition 4-Nitroquinoline 1-oxide 4-nitroquinoline 1-oxide (4NQO) is a UV-mimetic mutagen that causes DNA bulky lesions and can induce tumors in laboratory animals. ▶Mutagen Sensitivity

NPM Definition Nucleophosmin; a ubiquitous protein that is normally involved in maturation of ribosomes and nucleocytoplasmic protein trafficking.

NR1C ▶Peroxisome Proliferator-Activated Receptor and Cancer

▶Anaplastic Large Cell Lymphoma

NR3C4 NPM–ALK

▶Androgen Receptor

Definition A protein derived from the fusion gene NPM-ALK that is characteristically expressed in ▶anaplastic large cell lymphoma. It contains a portion of the wild-type ALK and NPM proteins and, as a dimer, maintains the phosphorylation activity of ALK (ALK protein).

Nrf2 Definition

NQO1

Nuclear factor erythroid 2-related factor 2, is a master transcription regulator of cytoprotective genes. It forms hetrodimer with partners such as small Maf and binds to ▶ARE (a cis-acting DNA regulatory element) and stimulates transcription of the downstream gene. ▶Phase 2 Enzymes

Definition NAD(P)H:quinone oxidoreductase-1, synonym quinone reductase (QR) or DT-diaphorase, catalyzes the obligatory two-electron reduction and ▶detoxification of quinones, stabilizes ▶p53 and strengthens cellular antioxidant defense. ▶Benzene and Leukemia ▶Phase 2 Enzymes

NSC-638850 ▶UCN-01 Anticancer Drug

Nuclear Export Signal

NSC-684766 ▶Trabectedin

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Nuclear Atypia Definition Abnormality of the cell nucleus that may be associated with a precancerous condition.

NSC-710428

Nuclear Envelope Breakdown

▶Epothilone B Analogue

Definition

NSC-312887 ▶Fludarabine

NSC-362856 ▶Temozolomide

NSC-10514-F ▶Cladribine

NSCLC Definition

▶Non-small cell lung cancer.

NEBD; The nuclear envelope consists of two concentric lipid layers fused to one another that are punctured by nuclear pore complexes, which allow passive and regulated transport of proteins and RNA between the nucleus and cytoplasm. Underlying the inner lipid bilayer is the nuclear lamina, composed predominantly of polymerized intermediate filaments known as nuclear lamins. Nuclear envelope breakdown (NEBD) requires vesicularization of the lipid bilayers, depolymerization of the nuclear lamins to disassemble the nuclear lamina, and the falling apart of nuclear pore complexes. The best understood of these three processes, nuclear lamina disassembly, is caused by a multisite phosphorylation of the nuclear lamins. The number of kinases directly involved in depolymerization of nuclear lamins is unclear, however at least cyclin B1/CDK1 and ▶protein kinase C are likely to be involved. ▶G2/M Transition

Nuclear Export Signal Definition NES; Amino acid sequence that when present in a protein in appropriate location confers the ability to be recognized by the nuclear export machinery to permit its translocation to the cytoplasm. Several amino acid sequences can act as NES. This domain targets its protein for export from the cell nucleus to the cytoplasm through the nuclear pore complex. The NES is recognized and bound by the transporting molecule family exportins. ▶Snail Transcription Factors

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Nuclear Factor-kB K UNAL S AIGAL , F RANCISCO G. P ERNAS , C ARTER VAN WAES National Institute on Deafness and Other Communication Disorders and National Cancer Institute, NIH, Bethesda, MD, USA

Definition Nuclear Factor-kappaB (NF-kappaB, NF-κB) represents a family of transcription factor proteins that regulate expression of multiple genes important in cell survival, host responses to injury and infection, and pathogenesis of various diseases, including cancer. Originally, the transcription factor was discovered as a bacterial lipopolysaccharaide (LPS) induced nuclear transcription factor regulating expression of kappa light chains in B lymphocytes. Currently, it is considered one of the major links between immunity, inflammation, and cancer. Aberrant activation of NF-κB has been linked with a variety of malignancies including head and neck squamous cell carcinoma, ▶lung cancer, ▶esophageal cancer, ▶breast cancer, ▶prostate cancer, ▶pancreas cancer, ▶colon cancer, ▶cervical cancer, ▶melanoma, and ▶hematological malignancies, ▶leukemias and lymphomas.

Characteristics The original link between pathogens, NF-κB activation, and development of cancer was identification of REV-T, a transforming oncogene contained in avian ▶retroviruses, which leads to reticuloendothelial lymphomatosis. Like its mammalian homologues (▶REL A, REL B, cREL), it shares a C-terminal transactivation domain. In mammalian cells, the NF-κB family includes five known subunits: NF-κB1 (p105/p50), NF-κB2 (p100/p52), REL A (p65), cREL, and RELB (Fig. 1). Each subunit contains a 300 amino acid sequence in its N-terminus known as the REL homology domain. In addition, cREL, REL A, and REL B each contain a transactivation domain in their C-termini. NF-κB1 p105 and NF-κB2 p100 are initially expressed containing contiguous ankyrin repeats. Processing of NF-κB1 and NF-κB2 by the proteasome results in degradation of the ankyrin domains to produce the NF-κB p50 and p52 subunits. The NF-κB and REL subunits form homo- and heterodimers, excluding RELB, which appears to form only heterodimers. The most ubiquitous of these combinations is the p50/REL A heterodimer. In most cells in the resting state, these homo- and heterodimers remain predominantly in the cytoplasm in dormant form, complexed with inhibitor-κBs (IκBα, β, γ), which mask the nuclear localization sequence and DNA binding pocket.

Regulation Activation of inhibitor-κB kinases (IKKs) by various cell stimuli leads to phosphorylation and subsequent ubiquitinylation of IκB molecules (Fig. 1). Ankyrin/IκB degradation by the ▶proteasome results in exposure of the nuclear localization sequence and DNA binding sites of NF-κB and REL dimers, thus allowing nuclear translocation and DNA binding to 9–10 bp κB binding sequences in DNA of the promoter of target genes. Additional phosphorylations of some of the subunits or cofactors by IKK and other kinases, regulate whether bound NF-κB/ REL complexes repress or transactivate target genes. In a current paradigm, IKK and NF-κB activation is separated into the “classical” (canonical) and “alternative” (noncanonical) pathways (Fig. 1). The classical pathway is typically induced by inflammation, injury, infection, and cytokines such as tumor necrosis factor (TNF), or interleukin-1 (IL-1). It appears to be important for cell survival as well as for innate and adaptive immunity. The alternative pathway, which is activated by other TNF family members, has been implicated in regulation of survival of premature B lymphocytes and development of peripheral lymphoid tissue. In disease states such as cancer, infection, and inflammation, the canonical pathway is often aberrantly activated and NF-κB1/REL A are localized to the nucleus. Clinical Relevance Several viral genes which activate the IKK/NF-κB pathway have been identified in ▶Epstein–Barr virus, human T cell leukemia virus, ▶hepatitis B virus, ▶hepatitis C virus, and human papillomaviruses, and have been implicated in their role in pathogenesis of ▶Hodgkin disease, leukemias, ▶hepatocellular carcinoma, and ▶cervical cancer and head and neck ▶squamous cell carcinomas. Chronic bacterial-induced ▶inflammation by ▶Helicobacter pylori and colonic flora in inflammatory bowel disease has been implicated in gastric and colon carcinomas. NF-κB activation by chemical promoters and DNA damage is more widely involved in cancer development and ▶progression. Examples of this include nicotine and other carcinogens in tobacco and the betel nut (areca) ▶betel Quid, that have been linked to NF-κB activation in pathogenesis of malignancies of the lung as well as head and neck. In addition to such inducible effects, aberrant activation may result from mutation or overexpression of a variety of growth factor receptors (▶EGFR, ▶ErbB2, etc.), cytokines (▶TNF-α, IL-1), cell adhesion molecules (▶integrins), and signaling kinases (▶RAS, ▶BCR-ABL1), which can activate NF-κB via IKK, and promote molecular pathogenesis of epithelial and lymphoid malignancies. In addition to such endogenous stimuli, NF-κB may also be activated by exogenous stimuli, secondary to stress, hypoxia, chemotherapy, and radiation. Inducible activation by these factors

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N Nuclear Factor-κB. Figure 1 NF-κB activation. In the classical pathway, stimulation by IL-1, integrins, ErbB Family growth factors, and TNF-α leads to activation of an inhibitor-κB kinase (IKK) α, β, γ complex, which in turn leads to phosphorylation, and subsequent ubiquitinylation of IκB molecules. Following proteasome processing, the liberated complex NF-κB1/RELA or NF-κB1/cREL) translocates to the nucleus to promote target genes. In the alternative pathway, stimulation via ligands including CD40L and LTβ leads to activation of the intermediary NF-κB inducing kinase (NIK), an IKKα dimeric complex, and proteasome processing of P100/RELB to p52/RELB. TNF-α may also activate NIK in the alternative pathway. The liberated NF-κB2/RELB complex translocates to the nucleus to promote target genes.

can promote therapeutic resistance to chemotherapeutic agents, immune toxins (i.e., TNF), and radiation therapy. NF-κB promotes expression of a diversity of genes involved in proliferation, cell survival, invasion, ▶inflammation, and ▶angiogenesis that have been implicated in cancer. These include genes crucial to cell proliferation and survival such as ▶cyclin D (key in progression of cell cycle from G0 to G1 and S phase), and ▶BCL-2 and ▶IAP gene families (inhibit mitochondriamediated ▶apoptosis). Expression of NF-κB modulated angiogenesis factors such as GRO1, IL-8, and ▶VEGF allow tumors to obtain sufficient neovasculaization for tumorigenesis. NF-κB-mediated alterations in expression or interaction of cell adhesion molecules (integrins, CAMs) and ▶matrix metalloproteinases (MMPs) with matrix or other cells have been implicated cell migration, invasion, and metastasis.

With all of the above mechanisms by which NF-κB plays a role in cancer progression, inhibiting the transcription factor has become an important candidate for therapeutic investigation. As a result of its central role in inflammation, the first drugs to be used for cancer therapy and prevention and found to inhibit NF-κB were corticosteroids and ▶nonsteroidal anti-inflammatory drugs (NSAIDs). Corticosteroids in combination with cytotoxic chemotherapy are now a mainstay of therapy in certain leukemias, lymphomas, and myelomas. NSAIDS, several of which inhibit IKK as well as arachidonic acid synthesis, have been shown to reduce development of inflammatory and heritable colon carcinomas in experimental models and clinical studies. Natural compounds such as ▶curcumin, which comes from tumeric (a common Indian spice), have been shown to effectively inhibit NF-κB activation, and have been implicated in the

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reduced incidence of colon cancer in India. Standard ▶chemotherapy agents, such as ▶cisplatin, have been shown to have cytotoxic activity as a result of inhibition of NF-κB activation, in addition to their DNA damaging capability. Recently, agents more specifically targeting the proteasome, IKKs, and other upstream kinases have been investigated. The 26S ▶proteasome inhibitor ▶bortezomib (VELCADE, Millennium pharmaceuticals) when used alone or in combination with other therapies has been shown to have preclinical and phase I/II clinical activity in ▶multiple myeloma, mantle cell lymphomas, ▶Waldenstrom macroglobulinemia, ▶lung cancer, and head and neck ▶squamous cell carcinoma. It is currently approved for treatment of patients with therapy-resistant multiple myeloma. Because of remaining concern regarding the broader effects of proteasome or other NF-κB inhibitors on its physiologic roles in immunity, inflammation, and cellular homeostasis, the role of these agents remains investigational. Aberrant and inducible activation of NF-κB via multiple signaling pathways has been observed in multiple human malignancies. While the specific role it plays in the progression of each of these diseases is yet to be fully elucidated, its activation in most forms of cancer points to its overall importance in cancer. As more is learned about the molecular regulation of NF-κB and its target oncogenes, novel targeted therapies may allow improvement in current treatment outcomes.

References 1. Baldwin AS (2001) Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest 107:241–246 2. Gilmore TD (2006) NF-κB: from basic research to human disease. Oncogene 25:6679–6899 3. Hiscott J, Kwon H, Genin P (2001) Hostile takeovers: viral appropriation of the NF-kappaB pathway. J Clin Invest 107:143–151 4. Karin M (2006) Nuclear factor-κB in cancer development and progression. Nature 441:431–436 5. Van Waes C (2007) NF-κB in pathogenesis, prevention and therapy of cancer. Clin Cancer Res 13:1076–1082

Nuclear Hormone Receptors Definition Are ligand-activated transcription factors that regulate gene expression by interacting with specific DNA sequences upstream of their target genes. Upon ligand binding, these cytosolic proteins translocate to the nucleus to bind as dimers to response elements in the promoter regions of target genes and stimulate or

suppress gene transcription (e.g. retinoids bind to retinoic acid receptors and retinoid X receptors, which bind as dimers to ▶retinoic acid response elements and retinoid X response elements in the promoters of retinoid-responsive genes). ▶Carotenoids

Nuclear Imaging Definition The use of photon-emitting radionuclides to visualize tumor boundaries.

Nuclear Localization Signal Definition

NLS; Is an amino acid sequence which acts like a “tag” on the exposed surface of a protein. This sequence is used to confine the protein to the cell nucleus through the nuclear pore complex and to direct a newly synthesized protein into the nucleus via its recognition by cytosolic nuclear transport receptors.

Nuclear Magnetic Resonance Definition NMR. ▶Magnetic Resonance Imaging

Nuclear Magnetic Resonance Spectroscopy Definition A method that uses the nuclear isotopes of atoms to determine the tertiary structure of a molecule, generally in aqueous solution. ▶Structural Biology

Nucleolin

Nuclear Pore Complex

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Nucleolin

Definition

ATSUKO M ASUMI

NPC; A large multiprotein complex, embedded in the double membrane nuclear envelope, which provides the sole channel for nucleocytoplasmic trafficking of ions, small molecules, proteins, RNAs, and ribonucleoprotein particles in eukaryotic cells through both a passive diffusion and an energy-dependent signal-mediated active transport.

Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Musashimurayama-shi, Gakuen 4-7-1, Tokyo, Japan

▶Major Vault Protein

Nuclear Receptor Definition Nuclear receptors are ligand-inducible transcription factors, such as receptors for ⇒ thyroid hormones and ⇒ steroid hormones, retinoids (⇒ ▶retinoic acid) and ⇒ ▶vitamin D. They typically form homo- or heterodimers that bind to specific DNA binding elements. ▶Estrogen Receptor ▶Orphan Nuclear Receptors and Cancer ▶Parathyroid Hormone-Related Protein ▶Thyroid Hormone Receptors ▶Steroid Hormone Receptors

Nuclear Receptor Coactivator 3 ▶Amplified in Breast Cancer 1

Nuclear Translocation Definition Transport of proteins through nuclear pores (usually by a specific transport system) into the nucleus, enables transcription factor proteins to get access to the nucleus.

Definition Is a ubiquitous, nonhistone nucleolar phosphoprotein of eukaryotic cells and is present in abundance at the sense fibrillar and granular regions of the nucleolus. Nucleolin is also able to localize in the nucleus, in the cytoplasm, and at the cell surface. Nucleolin is an RNA-binding protein and multifunctionally involved in many cellular processes, including ribosome biogenesis, the processing of ribosomal RNA (rRNA), mRNA stability, transcriptional regulation, and cell proliferation, and it is also a downstream target of several signal transduction pathways.

Characteristics Regulation of Nucleolin Function as a Phosphoprotein The multiple activities of nucleolin are regulated by covalent modifications, most notably phosphorylation, methylation, proteolysis, and ADP-ribosylation. Nucleolin function is coupled to growth control by its phosphorylation. Active rRNA transcription is positively correlated with highly phosphorylated nucleolin. In growing cells, casein kinase II (CKII) phosphorylates nucleolin. Phosphorylation by CKII enhances nucleolin (~110 kDa molecular size) as a substrates for protease to produce smaller fragments 30 and 72 kDa proteins, which trigger rDNA transcription by RNA polymerase I. Both CKII activity and nucleolin phosphorylation are enhanced after stimulation with mitogens, in regenerating rat liver after partial hepatectomy and in tumor cells. In addition, nucleolin phosphorylation by CKII and rRNA synthesis is dependent on hormones, such as dexamethasone and androgen, and growth factors. Major phosphorylation sites of nucleolin are serine and threonine. Serine phosphorylation is mediated by CKII and related to nucleolar function in the control of rDNA transcription, and threonine phosphorylation is linked to cdc2 kinase during mitosis through condensation of nucleolar chromatin, leading to increased rDNA transcription by RNA polymerase I in the G1 to S phase of cell cycle. Thus, sequential CKII and cdc2 phosphorylation modulates nucleolin function in regulating nucleolar structure and activities between interphase and the mitotic phase during cell growth. Nucleolin Function of Cell Cycle and Ribosomal Biogenesis Nucleolin levels are highest in tumors or other rapidly dividing cells. Nucleolin is necessary for cell

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Nucleolin

proliferation and nucleogenesis and is downregulated during differentiation. In nondividing cells, nucleolin level is low and is preferentially associated with chromatin. In G1 phase, nucleolin decondenses the chromatin by replacing histones. Hormones such as ▶androgen and growth factors regulate the expression and phosphorylation of nucleolin by CKII, resulting in increased DNA transcription by RNA polymerase I in the G1 to S phase of cell cycle. Nucleolin binds to the 5′ end of the external transcribed spacer region of preRNA and participates in the preRNA processing along with fibrillarin and small nucleolar RNA. The 48S RNA is rapidly cleaved to yield the mature 18S, 28S, and 5.8S rRNA species during the transcription of rDNA by RNA polymerase I. During ▶mitosis, nucleolin is phosphorylated by cdc2 kinase and condensates the chromatin. The nucleolus may be involved in functions other than ribosome biogenesis. The functions of nucleolin are the result of assemblies of nucleolin with other factors to form large complexes which induce or regulate cell growth and cancer cells. The Role for Nucleolin in Cancer Role of Nucleolin in B-cell Lymphomas Nucleolin is a subunit of the ▶transcription factor LR1 (switch region binding protein), which activates expression of the c-myc gene and the EBNA-1 (▶Epstein-Barr virus nuclear antigen 1) gene, which promote cells to malignancy. LR-1 is a B-cell-specific DNA-binding protein that contains two polypeptides of 106 kDa and 45 kDa. The 106-kDa component of LR-1 is nucleolin. Although nucleolin is ubiquitous, LR1–DNA binding activity is ▶B cell specific. LR-1 is present only in activated B cells, not resting B cells. LR-1 regulates the promoter of Epstein-Barr virus, which produces EBNA-1 protein. Nucleolin that is component of LR-1 may contribute to cell-type specific transformation by Epstein-Barr virus. Nucleolin’s Interaction with Cellular Protein, p53, and Rb Nucleolin is directly involved in two major cellular tumor suppressors, ▶Rb and ▶p53. The p53 gene is commonly mutated in human cancer cells. The p53 protein has a critical role in cellular responses to DNA damage and other stresses by inhibiting proliferation or inducing G1 arrest, resulting in program cell death. Heat shock induces nucleolin to relocalize from nucleus to nucleoplasm. This nucleolin relocalization is dependent on p53 and cellular stress, including formation of p53–nucelolin complex. Nucleolin relocalization and complex formation are required the p53 C-terminal regulatory domain. Nucleolin–p53 interaction indicates the p53-dependent mechanism in which cell stress mobilizes nucleolin to cause the transient replication inhibition and DNA repair. In addition, ribosomal protein L26 (RPL26), nucleolin, and p53 protein itself bind to the 5′ UTR of p53 mRNA. p53 translation

and induction after DNA damage is controlled by these factors. Increased levels of RPL26 enhance DNAdamage-induced p53 translation and its cellular function, such as G1 arrest and ▶apoptosis. In contrast, nucleolin suppresses the translation and induction of p53 after DNA damage. Nucleolin has an opposite effect of p53 and RPL26. High risk ▶human papillomaviruses (HPVs) play a central etiologic role in the development of ▶cervical cancer, and in this process the deregulated expression of high-risk HPV oncogenes is a critical event. Cervical cancer represents the second most common malignancy in women, with an annual incidence of ~500,000 new cases, and nucleolin is involved in HPV18-induced cervical carcinogenesis. Nucleolin controls the chromatin structure of the HPV18 enhancer in vivo and is directly linked to HPV18-induced cervical cancer formation. Nucleolin binds to the HPV18 enhancer in a sequencespecific manner. Nucleolin is a phosphoprotein whose synthesis positively correlates with cell division rates. When cells are synchronized to the G0, G1, S, and G2 phases of the cell cycle to detect nucleolin DNA binding to HPV18 enhancer, nucleolin binding is detected predominantly in S phase of cell cycle. The E6 and E7 oncoproteins of HPV18+ exert their carcinogenic potential by inactivation of the tumor suppressors pRB and p53. E6 and E7 oncogene transcription are blocked by nucleolin downregulation. Nucleolin expression is altered in HPV18+ precancerous and cancerous tissue from the cervix uteri. Nucleolin distributes mostly diffuse in the nuclei of normal squamous and glandular epithelial cells, however, it is speckled in the nuclei of HPV18+ squamous cell carcinoma. Rb is a prototypical ▶tumor suppressor, frequently inactivated in certain types of human cancer. There are many molecular mechanisms of Rb-mediated tumor suppression to understand the cancer cell proliferation. Nucleolin is associated with Rb in intact cells in the G1 phase of the cell cycle, and the complex formation is mediated by the growth inhibitory domain of Rb. Interaction with Rb inhibits the DNA binding activity of nucleolin for the HPV18 enhancer, resulting in Rb-mediated repression of HPV18 oncogenes. Rb controls the interaction of nucleolin with the HPV18 enhancer and the nucleolin-dependent activation of transcription of E6 and E7, oncoprotein of HPV18. Nucleolin could be a target molecule for the therapy of HPV18+ carcinomas of the cervix uteri. Nucleolin’s Interaction with Other Factors Nucleolin has been found to interact with several RNA/DNA/protein targets within the nucleus, cytoplasm, and on the cell surface of several cell lines. From these different interactions, a function for nucleolin in different processes was deduced. Nucleolin also binds to Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, responsible for inhibitory effect

Nucleophile

of tumor growth. AS inhibits tumor growth by binding the nucleolin protein on the surface of cancer cells. Nucleolin interacts with transcription factors and regulates negatively and positively. The mammalian ▶Myb family of oncoproteins is composed of three transcription factors, A-Myb, B-Myb, and c-Myb. c-Myb is expressed mostly in hematopoietic cells and regulates their proliferation and differentiation. Nucleolin binds DNA-binding domain of c-Myb and A-Myb and downregulates Myb-mediated transcriptional activity. Interferon regulatory factor-2 (IRF-2) acts as a positive modulator for interferon-stimulated response element (ISRE)-like sequences such as the promoter H4. The nucleolin acts as a positive modulator of IRF-2-dependent transcriptional activation through an association with IRF-2. IRF-2 plays an important role in cell growth regulation through H4 gene activation and has been shown to be a potential oncogene. IRF2 is acetylated by histone acetylase PCAF or p300 and acetylation of IRF-2 regulates cell growth by activation of H4 promoter in NIH3T3 cells. Nucleolin associates with an acetylated IRF-2, but its binding activity with nonacetylated IRF-2 is very low. For H4 gene regulation by IRF-2, nucleolin acts as an oncogenic activator via transcriptional activation, suggesting the cooperation of IRF-2 and nucleolin in cell growth regulation. IRF-2 binds H4 promoter with PCAF and nucleolin in growing NIH3T3 cells; however, in growth-arrested cells, nucleolin binding to H4 promoter is much lower compared to growing cells. Nucleolin binds to acetylated IRF-2 and IRF-2/PCAF/nuclelin complexes in turn stimulate the activation of gene transcription, which drives cell growth. From proteomic analysis, many proteins are identified from the isolated nucleolin-binding complex. Nucleolin binds to the ribonucleoprotein (RNP) complex mainly through the sequence-specific protein–RNA interaction. As nonribosomal protein which binds nucleolin, B23, hnRNP U, DNA helicase (Ku), eukaryotic initiation factor 2(elF-2), and nuclear factors 90 are identified. Nucleolin is also induced by ▶Myc (c-myc protein product), which is a transcription factor of basic helixloop-helix zipper (bHLH-Zip) family. c-myc is an immediate early response gene following mitogen stimulation and regulated by tumor suppressor genes, tyrosine kinase receptors, and growth factors and known to be induced in S phase of cell cycle. Myc activates the nucleolin promoter via E-boxes located in intron I, resulting in increase of nucleolin. Nucleolin is implicated in the maturation of ribosomal RNAs with other c-myc-target factors such as a cofactor of RNA polymerase III BN51, indicating of the association of the ribosome biogenesis with Myc. Nucleolin is a multifunctional protein which located in the nucleolus, nucleus, cytoplasm, and at the cell surface and is abundant in proliferating and cancer cells. Nucleolin is a cell cycle regulated transcriptional activator. There is much information about nucleolin

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that correlates with normal and cancer cells. Further, function of nucleolin should be clarified in future because of its abundance in the nucleolus.

References 1. Ginisty H, Sicard H, Roger B et al. (1999) Structure and functions of nucleolin. J Cell Sci 112:761–772 2. Srivastava M, Pollard HB (1999) Molecular dissection of nucleolin’s role in growth and cell proliferation: new insight. FASEB J 13:1911–1922 3. Takagi M, Absalon MJ, McLure KG et al. (2005) Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell 123:49–63 4. Gristein E, Shan Y, Karawajew L et al. (2006) Cell cyclecontrolled interaction of nucleolin with the retinoblastoma protein and cancerous cell transformation. J Biol Chem 281:22223–22235 5. Masumi A, Fukayawa H, Shimayu T et al. (2006) Nucleolin is involved in Interfron regulatory factor-2dependent transcriptional activation. Oncongene 25:5113– 5124

Nucleolus Definition Plural nucleoli; Is a sub-organelle of the cell nucleus. A main function of the nucleolus is the production and assembly of ribosome components (RNA, proteins). Within the nucleus are one or more nucleoli. The nucleolus is roughly spherical, and appears as a mass of densely stained granules and fibers under an electron microscope. It consists of nucleolar organizers. They are specialized regions of some chromosomes with multiple copies of genes for ribosome synthesis, along with a considerable amount of RNA and proteins representing ribosomes in various stages of production. An average, healthy cell can produce up to 10,000 ribosomes per minute. ▶G2/M Transition

Nucleophile Definition Is a reagent that forms a chemical bond to its reaction partner (the electrophile). ▶Xenobiotics

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Nucleoporin

Nucleoporin S AI -J UAN C HEN 1 , G UANG -B IAO Z HOU 2 , X UE -TAO B AI 1 , Z HU C HEN 1 1

State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui-jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China 2 Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

Definition Nucleoporins are the main components of the nuclear pore complex in eukaryotic cells, and mediate bidirectional nucleocytoplasmic transport, especially of mRNA and proteins. Defects in nucleoporins, e.g., overproduced or involved in ▶chromosomal translocation, may affect nucleocytoplasmic transport and cell function, leading to cell transformation and cancer.

Characteristics The Nucleocytoplasmic Transport The nucleus is the defining feature of the eukaryotic cell. Unlike their prokaryotic counterparts, eukaryotic

cells separate the nuclear synthesis of DNA and RNA from cytoplasmic protein synthesis with a barrier termed the nuclear envelope (NE). The NE is perforated by large proteinaceous assemblies, called nuclear pore complexes (NPCs), which are multiprotein channels that span the double lipid bilayer of the NE and act as the sole gatekeepers controlling the exchange of material between the two locales (Fig. 1a). In yeast, half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. NPCs are freely permeable to small molecules (such as water and ions), but they restrict the movement of larger molecules (such as proteins and RNAs) across the NE. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. To overcome this barrier, macromolecules carry specific signals that allow them to access the nucleocytoplasmic transport machinery of the cell. In this way the cell ensures that only selected macromolecules can travel between the nucleus and cytoplasm. Factors that are important for nuclear transport can be divided into four categories: the proteins of the NPC (nucleoporins), the Ran▶GTPase, transport receptors called karyopherins (or importins/ exportins) that recognize cargoes for transport, and specialized factors that promote transport of some

Nucleoporin. Figure 1 The NPC and nucleoporins in the NPC. (a) Three-dimensional reconstruction of NPCs (Akey, Radermacher (1993) J Cell Biol 122:1). CF, cytoplasmic filaments; CP, cytoplasmic particles; CR, cytoplasmic ring; CYT, cytoplasm; NR, nucleoplasmic ring; NUCL, nucleus; LR, lumenal ring; LS, lumenal spoke; RA, radial arms; ISR, inner spoke ring; S, spokes; T, transporter; NC, nuclear cage (basket); DR, distal ring. (b) The position of the Nups relative to the NPC cylindrical axis (R) and mirror plane of pseudosymmetry (Z). Each circle represents a Nup position with green for symmetrical FG Nups, blue for strictly nuclear FG Nups, red for strictly cytoplasmic Nups, gray for non-FG Nups, and purple and purple stripes for different integral membrane proteins associated with the NPC (Rout MP et al. (2000) J Cell Biol 148:635).

Nucleoporin

protein/RNA complexes. Macromolecular transport in or out of the nucleus generally begins with recognition of the transported cargo by a receptor, followed by docking and movement through the NPC. Karyopherins can generally be thought of as superhelices with an inherent flexibility. The RanGTPase and its regulators control the rate at which cargo is moved in and out of the nuclear pore, as well as the loading and unloading of cargoes. Nucleoporins Nucleoporins (Nups) are the individual protein components of the NPC (Fig. 1b). Proteomic analyses of the mammalian NPC have estimated 30–50 Nups, many of which are functionally conserved from yeast to mammals. Nups can be divided into two families: pore membrane proteins (Poms) and FG nucleoporins. Poms are membrane-spanning integral proteins specific to the pore membrane. All of the FG nucleoporins share domains consisting of repeated phenylalanine/glycine (FG), which are separated by spacers that are either highly charged or rich in polar residues. The repetitive motifs are probably distantly related to one another and are predicted to assume a β-sheet conformation. Two classes of FG repeats, GLFG and FXFG subtypes, have been identified. A number of nucleoporins contain one or the other or a combination of these repeats. In addition

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to the shared repeats, each of the FG nucleoporins has distinct domains that appear to mediate localization and function, such as the coiled-coil domains found in Nup49, Nup84, the leucine zipper found in Nup214, the zinc finger domain found in Nup153, the RNA binding domain included in Nup98 or Nup145. The Nup98 and Nup96 proteins are encoded by a single gene. Much evidence showed that nucleoporins play important roles in docking, translocation and releasing of the cargo. Except for roles in transport, several studies in yeast have demonstrated that nucleoporins are essential for mediating ▶epigenetic control of transcription. For example, Nup60 and Nup145 are found to be required for full repression of HMR locus in yeast. Nucleoporins and Cancer To date, Nups are reported to be involved in several types of cancers through different mechanisms. Firstly, Nups are shown to be overexpressed in cancer cells. For example, Nup88 was shown to be upregulated in a broad spectrum of tumors. Nup88 was similarly enhanced in severe dysplasias and in situ carcinomas of organs such as colon, stomach, breast, and prostate. Conversely, Nup88 was either sporadic or not detectable in most benign tumors and hyperplasias. In normal adult tissues, Nup88 was occasionally noted in sites such as colonic crypts, bronchial mucosa, and fallopian tubes. However,

N Nucleoporin. Table 1

Chromosomal translocations and nucleoporins fusion genes associated with cancers

Chromosomal translocations t(7;11)(p15;p15.5) t(11;17)(p15;p15) t(11;17)(p15;p15) t(11;12)(p15;q13) t(11;12)(p15;q13) t(2;11)(q31;p15) t(2;11)(q31;p15.5) t(1;11)(q23;p15.5) t(3;11)(q29q13;p15) inv11(p15.5;q22) t(11;20)(p15.5;q11) t(9;11)(p22;p15.5) t(4;11)(q21;p15.5) t(5;11)(q35;p15.5) t(8;11)(p11.2;p15) t(10;11)(q25;p15) t(6;9)(p23;q34) t(9;9)(q32;q34) Amplified episomes

Fusion genes

Associated cancers

Nup98–HoxA9 Nup98–HoxA13 Nup98–HoxA11 Nup98–HoxC11 Nup98–HoxC13 Nup98–HoxD11 Nup98–HoxD13 Nup98–PMX1 NUP98–IQCG Nup98–DDX10 Nup98–TOP1 Nup98–LEDGF Nup98–RAP1GDS1 Nup98–NSD1 Nup98–NSD3 Nup98–ADD3 DEK–Nup214/CAN SET–Nup214/CAN Nup214–ABL1

AML, MDS, t-MDS/AML, CML AML, MDS CML AML AML AML AML, t-MDS/AML t-MDS/AML T-ALL/AML or mixed lineage leukemia AML, t-MDS/AML AML, t-MDS/AML AML T-ALL AML AML T-ALL AML, MDS AML T-ALL

AML, acute myeloid leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; T-ALL, T-cell acute lymphoblastic leukemia; t-MDS/AML, therapy-related acute myeloid leukemia.

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Nucleoside Analogs

whether Nup88 overexpression was simply the result of increased nucleocytoplasmic transport required to meet the increased demand of proteins by transformed cells, or Nup88 might play a role in ▶carcinogenesis, remained obscure. Secondary, Nups are involved in ▶chromosomal translocations which are predominantly found in ▶leukemia (Table 1). The t(7;11)(p15;p15.5) was the first rearrangement discovered in patients with ▶acute myeloid leukemia (AML), while the resultant Nup98– HoxA9 was the first fusion protein that was found to involve a nuclear-pore protein. So far the Nup98 has been reported to fuse with at least 20 partner genes in leukemia having 11p15 translocation. An interesting finding is that, about 50% of Nup98 fusion partners are ▶homeobox domain (HD)-containing transcription factors. In addition, the non-homeobox fusion partners do not have DNA binding domain, but all bear a putative protein-protein interaction motif, a feature also shared by HD and suggestive of a possible common mechanism. Nup214 has also been reported to be fused to several partners, e.g., DEK, SET in AML and ABL1 in acute lymphoid leukemia (ALL). Interestingly, both Nup98 and Nup214 fusion proteins retain the FXFG repeat region that is characteristic of several nucleoporins. Normally, the FXFG repeats help to regulate the nucleocytoplasmic transport of proteins and RNAs as well as function as docking sites for the karyopherins. One model for the mechanism by which Nup98 and Nup214 fusion proteins promote leukemogenesis is that the FXFG repeats act as protein–protein interaction domains that allow the fusion protein to interact with other transcription factors and act as transcriptional activators. Changes in nuclear transport machinery can markedly alter cellular function and potentially promote tumorigenesis. For example, nuclear mislocalization of ▶nuclear factor κB as a result of IκB degradation and improper acetylation by ▶p300, mislocalization by activated AKT, and nuclear localization and activation of the transcriptional activator ▶β-catenin, can facilitate tumorigenesis.

References 1 Rout MP, Aitchison JD (2001) The nuclear pore complex as a transport machine. J Biol Chem 276:16593–16596 2 Kau TR, Way JC, Silver PA (2004) Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer 4:106–117 3 Conti E, Muller CW, Stewart M (2006) Karyopherin flexibility in nucleocytoplasmic transport. Curr Opin Struct Biol 16:237–244 4 Gould VE, Martinez N, Orucevic A et al. (2000) A novel, nuclear pore-associated, widely distributed molecule overexpressed in oncogenesis and development. Am J Pathol 157:1605–1613 5 Alber F, Dokudovskaya S, Veenhoff LM et al. (2007) The molecular architecture of the nuclear pore complex. Nature 45:695–701

Nucleoside Analogs Definition Molecules that are similar to the building blocks of DNA and RNA. ▶Epigenetic Therapy

Nucleoside Diphosphate Kinases ▶NM23 Metastasis Suppressor Gene

Nucleoside Transporters Definition Proteins that traverse the plasma membrane and permit the passage of nucleosides and structurally-related drugs into and out of the cell cytoplasm. ▶Adenosine and Tumor Microenvironment

Nucleosomal DNA Fragments ▶Nucleosomes

Nucleosome Remodeling ▶Necleos omes ▶Chromatin Remodeling

Nucleosomes

Nucleosomes S TEFAN H OLDENRIEDER Institute of Clinical Chemistry, University Hospital of Munich, Ludwig-Maximilians-University, Munich, Germany

Definition Are, complexes formed by a core particle of eight histone proteins which are surrounded by 147 bp of double-stranded DNA, are the basic elements of the chromatin in eukaryotic cells.

Characteristics Structure and Function of Nucleosomes About 99% of human DNA is localized in the nucleus of the cells where it is organized in a multistep manner. In its secondary structure, chromatin is arranged as a chain of nucleosomes. These consist of a central protein component formed by an octamer of the double-represented histone aggregates H2A-H2B and H3-H4. Hundred and forty seven base pairs of doublestranded DNA are twisted around this complex with 14 defined fixing sites and build the disk-like nucleosomes with a molecular weight of ~206 kDa. Thereof nearly half is part of the histones and half of the DNA component. Neighboring nucleosomes are connected by so-called linker DNA, which varies between 10 and 100 bp. A further histone H1 is located at these linking sites outside of the nucleosomes and stabilizes the chromatin chain in its tertiary structure as chromatin fiber. Beyond the organization and stabilization of the DNA, the arrangement in multinucleosomal order plays an essential role for the regulation of the transcription of genetic information, for the replication and for repair processes. The access of transcription factors to relevant DNA sequences is mainly coordinated by nucleosomal histones which can be modified at their tails by acetyl-, methyl-, phosphor-, ubiquitin-, and ADP-ribose groups. Histone acetylation leads to decondensation of the chromatin, unfixing of intra- and internucleosomal connections, and promotes the transcription process, whereas deacetylation (▶Histone deacetylases) and condensation suppress it. Further, genetic and ▶epigenetic modifications of the DNA may contribute to the regulation of the interaction between transcription factors and specific promoter regions, too. The active involvement of DNA in transcription processes requires a flexible and dynamic structure of the nucleosomal organization which is enabled by ATP-dependent chromatin-remodeling factors. These promote the liberation of the DNA from its close connection to the histones by disrupting the contacts,

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transferring the histone octamer to another DNA molecule, or sliding the core particle along the DNA. Origin of Circulating Nucleosomes As small amounts of nucleosomes are also found in blood of healthy adults, ▶Apoptosis during physiological cell regeneration is thought to be a major source of nucleosome release. Although most of liberated nucleosomes are engulfed and digested by ▶macrophages and neighboring cells, a part of them reaches the blood circulation (▶Circulating nucleic acid). In situations of enhanced cell death such as during degenerative, autoimmune, inflammatory, ischemic, traumatic and toxin-mediated diseases, or in malignant tumors, this elimination system seems to be overloaded or impaired resulting in higher levels of circulating nucleosomes in blood. Depending on the type and intensity of the cell damaging stimulus as well as on the type and energy level of the affected cells, various cell death modes can lead to the cellular demise such as apoptosis, oncosis, or mixed forms between these extreme forms. Because most of the DNA is present in blood as small monoand oligonucleosomal fragments of 200, 400, 600, and 800 bp, apoptosis, which is associated with internucleosomal cleavage of the chromatin by activated endonucleases, is assumed as a major mechanism of nucleosome liberation. In contrary, oncosis produces high molecular weight DNA fragments released into blood after acute damaging events. As a further mechanism, active secretion of nucleosomes by lymphocytes is in discussion. Metabolism of Circulating Nucleosomes Generally, cell-free DNA (▶Circulating nucleic acids) can circulate in blood as naked DNA, associated with histones in nucleosomes, bound to other plasma proteins, or packed in apoptotic bodies. Although the exact contribution of each form may vary inter- and intraindividually, the main part of the circulating DNA was shown to be organized in multimeric complexes as mono- and oligonucleosomes. Hemodialysis experiments which produce a considerable release of nucleosomes from lymphocytes have revealed that nucleosomes are removed in vivo from circulation in a biphasic, saturable, and concentrationdependent manner with an initial half-life of 4 min. The degradation and elimination of nucleosomes is thought to be exerted by various systems: . Intraplasmatic degradation by circulating endonucleases . Intraplasmatic immunological complexing by antinucleosome-antibodies . Phagocytosis and lysosomal degradation by cells of the reticulo-endothelial system . Metabolization of nucleosomes in the liver . Direct renal elimination in form of liposomes

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Nucleosomes

During acute ▶inflammation, nucleosomes may bind to acute phase proteins which delay the elimination process. Relevance of Circulating Nucleosomes The role played by nucleosomes in the pathogenesis of diseases is only partially known. In systemic lupus erythematodes, the high antigenic potential of circulating nucleosomes in blood or on the surface of antigen-presenting cells leads to the early production of antinucleosome-antibodies. Complexes of nucleosomes and these antibodies were found to aggregate at the glomerular basal membrane in the kidneys and to promote the disease ▶progression. In tumors, circulating nucleosomes are suspected to carry metastatic information as the transfection of DNA to mice resulted in the generation of new tumors. Further reports suggest an important role of nucleosomes for the evasion of tumor cells from immunosurveillance by inhibition of ▶natural killer cell-mediated tumor cell lysis. Finally, nucleosomes liberated from tumor cells are reported to stimulate the expression of interleukin-8 in endothelial cells which promotes the local ▶angiogenesis in the tumor tissue and supports tumor progression. Clinical Aspects of Circulating Nucleosomes in Blood In recent years, several enzyme linked immunosorbent assays (▶ELISAs) were developed to quantify circulating nucleosomes in serum, plasma, and other body fluids using mainly monoclonal mouse antibodies directed specifically against the DNA and histone component, respectively. It is noteworthy to point out, that a good correlation between a nucleosome assay and the current “golden standard” for DNA quantification by real time ▶PCR was found, suggesting that clinical results obtained by nucleosome or DNA measurements are quite comparable. Although most clinical studies focus on the quantification of DNA amount in plasma and serum, several studies have analyzed the relevance of circulating nucleosomes for diagnostic, staging, and prognostic purposes in cancer diseases as well as in noncancer diseases. As nucleosomes are nonspecific cell death products, they are supposed to appear in circulation in various acute and chronic diseases. Circulating Nucleosomes in Noncancerous Diseases Levels of nucleosomes in plasma and serum were found to be elevated in patients with acute infections as compared with healthy individuals and correlated with ▶C-reactive protein values. Another study reported elevated nucleosomal levels in sera of septic patients and a strong correlation to the severeness of the disease. Further, nucleosome levels were elevated in sera of patients after cerebral stroke, particularly in patients with extended volumes of stroke lesions. During the first week after stroke, the levels increased faster

and stronger in patients with severe functional deficits than in those with only slight deficits. In addition, nucleosomes showed independent prognostic value for the 1-year recovery after acute stroke. High nucleosome levels were observed in patients with various autoimmune diseases, too. Because antinucleosome-antibodies are found only in patients with systemic lupus erythematodes, it was assumed that nucleosomes may undergo specific qualitative processing, e.g., in antigen-presenting cells to achieve its high antigenicity in this disease. Similarly, elevated concentrations cell-free DNA were reported in sera of patients with trauma, stroke, burns, graft versus host reaction (graft-versus-host disease) after transplantation, and exhaustive exercise. Circulating Nucleosomes in Diagnosis and Staging of Cancer Disease Several studies found higher nucleosome levels in individuals with cancer diseases, particularly in those with advanced stages. A comprehensive study analyzed sera levels of 590 patients with cancer disease including colorectal (▶Colon cancer) and various other gastrointestinal cancers (▶Gastrointestinal tumors), ▶lung cancer, ▶breast cancer, ▶ovarian cancer and other gynecologic cancers, renal and prostate cancer (▶Prostate cancer, clinical oncology), and lymphoma, as well as of the relevant organ-specific benign diseases and of healthy individuals. Nucleosome concentrations in sera of healthy donors were generally fairly low. In contrast, pretherapeutic serum levels in various malignancies were significantly higher. However, various benign diseases, particularly infectious diseases, tended to have elevated serum levels of nucleosomes, too, limiting the diagnostic capacity for cancer disease. While nucleosome levels significantly distinguished between healthy donors and malignant diseases, the difference between benign and malignant diseases did not reach the level of significance in general, but only in subgroup of lung cancer. Among the various cancer types, medians, percentiles, and ranges of nucleosome values were comparable, but lung cancer was associated with significantly higher levels and prostate cancer with lower ones. Regarding tumor ▶staging, nucleosome values were found to correlate with tumor stage and the presence of distant metastases only in patients with ▶gastrointestinal cancers, in other subtypes there was only a tendency or no correlation at all. In the contrary, in patients with lung cancer and breast cancer, high nucleosome values often were observed already in early stages. No association was found between nucleosome levels and lymph node involvement, cell differentiation (Tumor ▶grading), age or gender. Similar results concerning the diagnostic capacity of nucleosomes or cell-free DNA were obtained

Nucleotide Base

by other studies which, however, mainly analyzed only the difference between cancer patients and healthy donors. Heterogeneous results were obtained concerning the correlation of nucleosomes or cell-free DNA with tumor stage. Circulating Nucleosomes in Prognosis of Cancer Disease The prognostic relevance of circulating nucleosomes or cell-free DNA is still in discussion. While some studies reported a prognostic value of nucleosomes or DNA in patients with lung cancer and breast cancer, others could not confirm these findings in the same tumor types. Reasons for the discrepant results may be found in the low number and the heterogeneity of the patients investigated as well as in differences concerning the statistical analysis and the consideration of other relevant prognostic factors. In a large study on 300 patients with advanced ▶nonsmall cell lung cancer, pretherapeutic serum levels of nucleosomes had prognostic impact in the univariate analysis, while it did not show independent prognostic information when other clinical and biochemical markers were included in multivariate analysis. Circulating Nucleosomes in Therapy Monitoring of Cancer Disease During systemic cytotoxic therapies such as chemotherapy (▶Chemotherapy of cancer, progress and perspectives) and radiotherapy (▶Ionizing radiation therapy), the changes in the courses of circulating nucleosomes indicate the response to the therapy. Several studies have shown in various cancer types that strongly decreasing levels were mainly found in patients with remission of tumor disease whereas constantly high or even increasing values were associated with progressive disease. Independent from these general observations, nucleosome levels showed an immediate increase during the first days after start of therapy reaching a maximum after 2–5 days followed by a rapid decrease. These kinetics were observed in various cancers during chemotherapy, radiotherapy and ▶immunotherapy. The courses were influenced by the spontaneous, and the therapy-induced release of nucleosomes, and the elimination capacity from circulation of the individuals. Although nucleosomes are not specifically liberated only by tumor cells but may also be the result of the cell death of other cells with high proliferation rates, lung tumor cells have shown to be more susceptible to systemic therapy in vitro than physiological bronchoepithelial cells. The early nucleosomal course showed significant differences between patients with the favorable and nonfavorable outcome after therapy. A comprehensive study on more than 300 patients with advanced nonsmall cell lung cancer patients revealed that patients with clinical remission during chemotherapy initially started from lower nucleosome values, had lower

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maximum values and a more complete elimination of nucleosomes from circulation at the end of the first week of therapy than patients with progressive disease. When nucleosomes were combined with the more specific lung cancer biomarker (▶Serum biomarkers) CYFRA 21-1, the nonresponse could be anticipated in about 30% of progressive patients with a specificity of 100% already after the first course of therapy. This information would help to change the therapy earlier than by currently available imaging techniques in 30% of nonresponding patients and may contribute to increase the therapeutic efficacy. Similar results were obtained in smaller studies on colorectal and pancreatic cancer (▶Pancreas cancer, clinical oncology) during chemotherapy and radiotherapy. Altogether, levels of nucleosomes are elevated in sera of cancer patients when compared to healthy controls. However, due to elevations of nucleosome levels in patients with benign diseases relevant for differential diagnosis, they seem not to be suitable for cancer diagnosis. Although nucleosomes showed prognostic relevance in some univariate analyses, their independent value in multivariate models will have to be elucidated further. Most informative are circulating nucleosomes for the monitoring of anticancer therapy, particularly for the early estimation of therapy efficacy.

References 1. Kornberg RD, Lorch Y (1999) Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98:285–294 2. Luger K (2003) Structure and dynamic behavior of nucleosomes. Curr Opin Genet Dev 13:127–135 3. Ziegler A, Zangemeister-Wittke U, Stahel RA (2002) Circulating DNA: a new diagnostic gold mine? Cancer Treat Rev 28:255–271 4. Holdenrieder S, Stieber P, Bodenmueller H et al. (2001) Circulating nucleosomes in serum. Ann N Y Acad Sci 945:93–102 5. Holdenrieder S, Stieber P (2004) Apoptotic markers in cancer. Clin Biochem 37:605–617

Nucleotide Base Definition One of four compounds residing between the two strands of DNA which makes up the genetic code by sequence arrangement. ▶DNA Damage

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Nucleotide Excision Repair

Nucleotide Excision Repair W. G LENN M C G RE GOR University of Louisville, Louisville, KY, USA

Definition NER; Is a complex biochemical mechanism that recognizes alterations in the chemical structure of DNA due to base modification by physical agents (most notably ▶ultraviolet radiation) or endogenous or exogenous chemicals. After recognition of the alteration in the structure of DNA, the damage is removed by excision of the oligonucleotide that contains the damage. The resulting gap is filled in by DNA polymerase using the complementary (undamaged) strand as template and finally ligated. The process is essentially error-free. There are at least 27 polypeptides required to complete the recognition, excision and gapfilling phases.

Characteristics NER was initially characterized in E. coli in the 1960s and was identified by the ability of wild type bacteria to remove ultraviolet light (UV)-induced photoproducts from large molecular weight DNA. The kinetics of this process correlated well with the resumption of DNA replication and led to the idea that removal of such damage was directly related to the recovery of DNA synthesis and improved survival. Since that time, NER processes in mammalian cells have been documented by a great number of cellular and molecular biology studies. The dissection of the pathway in eukaryotic cells has been greatly facilitated by the availability of mutant cell lines, many of which are derived from individuals who are defective in some aspect of NER and are consequently cancer-prone. During the 1990s, all of the central factors involved in NER were cloned, and the basic “cut and refill” reactions have been reconstituted in vitro from purified components. Despite these advances, however, many important aspects of the regulation of NER and its integration with other basic cell biology processes remain to be elucidated. For review, see [1,2]. Cellular Regulation NER is a versatile and sophisticated pathway for the removal of DNA damage induced by a variety of environmental and endogenous factors. One of the most relevant, and best studied, DNA damaging agents is UV light, which induces dimerization of adjacent pyrimidine bases. The major products of this photochemical reaction (hence the term “photoproducts”) are cyclobutane

pyrimidine dimers (CPD) and 6–4 pyrimidine-pyrimidones (6–4s). Both lesions induce structural distortions in DNA, and if left unrepaired, can cause errors in replication that can lead to mutations. In addition to photoproducts, a wide variety of bulky chemical adducts are removed by NER, and the common denominator amongst these diverse lesions is distortion of the DNA helix, which in turn interferes with basic nuclear transactions such as transcription and DNA replication. These lesions present such a potential threat to the integrity of the cell that the basic mechanism for removing them has diverged surprisingly little throughout evolution. In all eukaryotic cells that have been studied, two modes of NER have been identified. These are global genomic repair (GGR) and transcription-coupled repair (TCR). These mechanisms are distinguished primarily by the way in which damage is detected. After the damage is detected, the pathway for excision, removal and gap filling is common to both GGR and TCR. Many of the proteins were identified by cloning the genes responsible for the NER-defective syndrome ▶xeroderma pigmentosum (XP). There are seven distinct groups of XP termed XP-A through XP-G, and the general molecular defect associated with each group is outlined in the Table 1. Global Genome Repair The global genome is defined as that part of the genome that is not transcribed, which is estimated to be 95% of the human genome. The XPC protein is specifically required for repair of lesions in the global genome, and cells with nonfunctional XPC are completely deficient in such repair. XP-C cells, however, are capable of removing lesions from the transcribed strand of active genes. The exact role of XPC in GGR has been obscure, but recent advances have elucidated its function more fully. XPC is a 125 kDa protein that acts as a heterodimer with one of two homologs of the yeast protein Rad23 (these homologs are termed hHR23A and hHR23B). It is now thought that the XPC-hHR23B (or A) complex acts at the very earliest stages of NER in the global genome in a damage recognition fashion. In ways that are not yet understood, this complex senses and binds to the damaged DNA. In this process, it locally distorts the double helix and then recruits the core repair apparatus. The rate of GGR is strongly dependent on the type of lesion, which presumably reflects the degree of helical distortion and the affinity of the XPC heterodimer. The XPC complex has been shown to have high affinity for DNA that contains a 6–4 photoproduct, and these lesions are repaired very rapidly. Recognition of CPD in the global genome probably requires the XPE protein; the XPE-CPD complex is then recognized by the XPC complex.

Nucleotide Excision Repair Nucleotide Excision Repair. Table 1

Molecular defects in XP complementation groups

Proteina XPAb XPBc XPC XPDc XPE XPF/ERCC1 XPGd

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Defect No lesion recognition Reduced helicase activity that unwinds DNA 3′–5′ of the lesion Reduced recognition of lesions in global genome, normal transcription-coupled repair Reduced helicase activity that unwinds DNA 5′–3′ of the lesion Reduced recognition of cyclobutane pyrmidine dimers in the global genome Mutant endonuclease that cuts 5′ to the lesion Mutant endonuclease that cuts 3′ to the lesion

a

Cells from complementation groups signified with a hyphen (XP-A); the corresponding proteins are not hyphenated (XPA). Patients are very severely affected and may have developmental defects. c Proteins also found in transcription factor TFIIH. Patients are likely to have developmental abnormalities. d Defective transcription-coupled repair of oxidative damage suggests a second, non-endonucleolytic function for this protein. These patients may also have developmental defects, possibly due to reduced repair of oxidative damage. b

Due to this two-stage mechanism, repair of CPD in the global genome is much slower than that of 6–4s. Transcription-Coupled Repair Elongating RNA polymerase II stalls at a wide variety of lesions and must act as a damage sensor, although the mechanism by which it does so remains obscure. In addition to the stalled polymerase, at least two other proteins are required for TCR. These are termed CSA and CSB, and are thought to act in a structural rather than in a catalytic fashion. Depending on the type of lesion, damage in the transcribed strand of an active gene can be repaired either by TCR or GGR. For instance, UV-induced 6–4s in active genes is primarily repaired by GGR because of the very high affinity of the XPC complex for these lesions. UV-induced CPD on the other hand, is repaired by TCR. TCR is highly conserved in both prokaryotes and eukaryotes, presumably because blocked RNA polymerases seriously interfere with cellular metabolism. TCR has also been shown to specifically reduce the frequency of mutations in active genes; mutations in such genes would also be expected to have a deleterious effect on cellular biochemistry and cell-cycle control. Excision and Gap Filling After the damage is initially sensed, the XPA protein plays a critical early role in NER. XPA is a DNA binding protein with high affinity for damaged DNA, and nonfunctional XPA leads to a complete loss of NER capacity. The protein was long thought to be the initial sensor of DNA damage, but more recent data implicates the XPC complex in this process. Current thinking is that XPA verifies that the DNA is damaged and acts to position the repair machinery correctly around the lesion. The human single strand binding protein, RPA, acts in concert with XPA and probably helps to

maintain the open conformation. Through its extensive network of protein binding sites, XPA recruits the basal transcription factor TFIIH to the lesion. TFIIH is composed of five polypeptides, including the XPB and XPD helicases, which unwind the DNA on both sides of the lesion. Two structure-specific endonucleases, XPF-ERCC1 and XPG cut the DNA on either side of the lesion to release an oligonucleotide 24–32 bases in length that contains the damage. The 3′ terminus acts as a primer, and the gap is filled in by either DNA polymerase d or e, together with cofactors; the relative contribution of each polymerase remains to be determined. The final step in the process is ligation of the 5′ terminus, which is probably carried out by DNA ligase I. Since the gap to be filled is short, and the fidelity of the polymerase complex is very high, NER is considered to be an error-free process. Clinical Relevance The central role played by DNA repair in the prevention of cancer was first appreciated in 1968 when Cleaver recognized that patients with the skin cancer-prone disorder xeroderma pigmentosum (XP) had a defect in the repair of UV-induced damage (3). XP patients have a greater than 1,000-fold increase in the incidence of sunlight-associated skin cancer, and if they are exposed to any sunlight they usually succumb to metastatic skin cancer. The cells of XP patients are also defective in the repair of bulky chemical adducts, such as those induced by cigarette smoke and endogenous metabolic processes. Evidence is emerging that XP patients who live longer, because they have compulsively avoided the sun, have an increased risk of internal cancers, particularly if they smoke. Many XP patients also undergo progressive neurological deterioration, and this is thought to be due to the accumulation of damage induced by reactive oxygen species. Repair of such

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damage is thought to require some of the NER proteins acting in pathways that are independent of NER. In addition, patients in XP groups that have a molecular defect that affects transcription in addition to NER (groups B and D), are likely to have developmental abnormalities that are presumably related to subtle defects in transcription. There are two additional syndromes that manifest defects in excision repair but are curiously not cancerprone. These are ▶Cockayne syndrome (CS) and ▶trichothiodystrophy (TTD). CS patients exhibit severe mental retardation and varying degrees of photosensitivity. Cells from these patients are defective in TCR because they lack a protein that couples the blocked RNA polymerase to the repair machinery. It has been postulated that the reason these patients do not develop skin cancer is the increased level of apoptosis induced in these cells following UV. The exaggerated apoptotic response may be due to the persistently blocked RNA polymerase II molecules, which cause accumulation of p53. TTD patients exhibit many of the same symptoms and signs of CS patients, but the molecular defect affects the same protein responsible for the XPD phenotype, namely the XPD helicase in TFIIH. The reason that one syndrome is cancer-prone while the other is not is puzzling, but recent studies have shown that mutations in the XPD gene occur in different regions in TTD and XP-D cells. It has been postulated that the mutations in TTD patients differentially affect transcription to a greater degree, and that subtle defects in transcription prevent cellular transformation. It has also been shown that TTD cells are quite proficient in repairing 6–4s while XP-D cells are not. The development of cancer in the latter cells may be related to the greater mutagenic potential of 6–4 photoproducts.

References 1. Freidberd et al. (eds) (2006) DNA repair and mutagenesis, 2nd edn. ASP Press, pp 267– 371 2. Watson NB et al. (2006) Translesion DNA replication proteins as molecular targets for cancer prevention. Cancer Lett 241:13–22 3. Cleaver JE (1968) Defective repair replication of DNA in xeroderma pigmentosum. Nature 220:652–656

Nude Mice Definition Lack a thymus, they are highly immunocompromised and tolerant for engrafted cells or tissues of other species.

NUP98-HOXA9 Fusion M ALCOLM A. S. M OORE Department of Cell Biology, Memorial-SloanKettering Cancer Center, New York, NY, USA

Definition The t(7;11)(p15;p15) chromosome translocation is found in human leukemic bone marrow cells in a subpopulation (1–2%) of patients with acute myeloblastic leukemia (AML), ▶myelodysplastic syndrome (MDS) and blastic crisis of ▶chronic myeloid leukemia (CML-BC). The translocation involves the association of the C-terminal GLEBS and FG (phenylalanineleucine) regions of the ▶nucleoporin protein NUP98 with the N-terminal region of the ▶HOXA9 protein containing the ▶PBX heterodimerization domain and the DNA interacting homeodomain (Fig. 1). The clinical course of this subset of leukemias is aggressive, and prognosis is poor.

Characteristics

NUP98 is a component of the ▶nuclear pore complex (NPC), an evolutionarily conserved structure made up of multiple copies of 30 different NPC proteins (nucleoproteins) embedded in the nuclear envelope. The NPC mediates transport of proteins (>40 kDa) and RNA species between the cytoplasm and nucleus and helps organize nuclear architecture. The 98 kDa NUP98 protein is generated by an unusual biogenesis pathway that involves synthesis and autocleavage of precursor proteins yielding NUP98 and NUP96, another stable component of the NPC. The mRNA export factors ▶TAP and ▶Rae-1 associate in a mutually exclusive manner with different binding sites on NUP98 to form stable ternary compounds. The Rae1–Nup98 complex functions in both the export of mRNA and import (recycling) of mRNA export factors into the nucleus indicating that this complex is an active player in the gene expression pathway. The human NUP98 gene, located on chromosome 11p15.5, has been found fused to 21 different genes as a consequence of chromosomal translocations in acute leukemia. The resulting chimeric transcripts encode fusion proteins that juxtapose a common N-terminal region of NUP98 to the C-terminus of the partner gene. The most frequent partner genes are members of the ▶Homeobox (HOX) and HOX-related family, particularly HOXA9. HOXA9 plays an important role in normal hematopoiesis, being expressed in early ▶hematopoietic stem cells (HSC) together with Meis1, downregulating with differentiation. Meis1 is a HOX cofactor that alters HOX-DNA binding specificity and affinity, and increases

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NUP98-HOXA9 Fusion. Figure 1 Comparison of the structure of HOXA9 and the NUP98-HOXA9 fusion protein. The transcriptional regulatory region of HOXA9 is replaced by the C-terminal FG region of NUP98. Note the retention of the N-terminal DNA binding homeodomain and the PBX-interaction domains of HOXA9 in the chimeric protein

HOX-transcriptional activity. ▶Thrombopoietin (Tpo), a key regulator of HSC proliferation, enhanced HOXA9 nuclear import and interaction with Meis1 in HSC in a MAPKinase-dependent fashion. Targeted disruption of HOXA9 in mice leads to reduced numbers of progenitor cells, and a profound defect in HSC. Conversely, enforced expression of HOXA9 promotes enhanced proliferation of HSC with impaired differentiation. These data highlight the importance of precise control of HOXA9 protein levels during hematopoiesis. The ubiquitin ligase ▶CUL4A (▶Cullin4A) regulates HOXA9 protein levels by ▶ubiquitination and subsequent proteosome degradation of the protein. Knockdown of CUL4A by siRNA in myeloid cell lines extended the HOXA9 protein half-life 6–10-fold and enhanced proliferation and blocked differentiation in response to ▶granulocyte colony stimulating factor (G-CSF). HOXA9 is one of the most upregulated genes in AML, particularly in the ▶CD34+, CD38-leukemic stem cell fraction. HOXA9 behaves as an oncogene in leukemia following mutations that induce its persistent expression or that convert it to a persistent transcriptional activator.

4–5 months. NUP98-HOXA9 has been detected in blastic crisis of ▶BCR-ABL positive ▶CML indicating that cooperation between NUP98-HOXA9 and the BCR-ABL signaling pathway via receptor tyrosine kinase may be important in progression from chronic disease to ▶blastic crisis. Oncogenic interaction is seen between NUP98-HOXA9 and BCR-ABL chimeras in retroviral mediated gene transfer experiments in mice leading to rapid development of AML.

Mouse Models of Leukemogenesis Expression of NUP98-HOXA9 in murine bone marrow by retroviral gene transfer results in enhanced proliferation in vitro resulting in development of an oligo- or poly-clonal ▶myeloproliferative disease (MPD) upon transplantation into mice, with neutrophil leukocytosis and extramedullary hematopoiesis, progressing to a mono or bi-clonal AML by 7–8 months. In a transgenic NUP98-HOXA9 model created with a myeloid restricted cathepsin G promoter, ~20% of mice developed MPD and eventually AML by 2 years. Retroviral insertional mutagenesis identified a number of co-factors that interacted with NUP98-HOXA9 in leukemia progression, the most frequent being Meis1. Collaboration of Meis1 with NUP98-HOXA9 reduced the median latency of AML development to

Proposed Mechanism of Leukemic Transformation by the NUP98-NUP98 Fusion Aberrant Transcriptional Activity Unlike HOXA9, NUP98-HOXA9 has a strong but aberrant transcriptional activating potential mediated through NUP98FG binding of the transcriptional coactivators ▶CBP/p300. Transduction of NUP98-HOXA9 into a human myeloid leukemic cells line significantly altered expression of over a hundred cytoplasmic mRNAs, and in studies with human CD34+ stem and progenitor cells 50–60 genes were upregulated and 4–15 downregulated. The transcriptosome of NUP98HOXA9 showed little overlap with that of HOXA9. The most striking feature was upregulation of homeobox genes (HOXA5, HOXA6, HOXA7, endogenous HOXA9 HOXB2, HOXC6, HOXB5,

Human Models of NUP98-HOXA9 Leukemogenesis Retroviral transduction of NUP98-HOXA9 into human HSC and progenitor cells from umbilical cord blood or G-CSF mobilized adult peripheral blood confers a proliferative advantage in vitro in long term cytokine stimulated or stromal co-culture assays, and in vivo following transplantation in irradiated immunodeficient NOD/SCID mice. The mechanism involves acquisition of serial myeloid progenitor colony recloning capacity and enhanced HSC self-renewal as confirmed by quantitative in vitro limiting dilution assay. Granulocytic and erythroid differentiation is also impaired.

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NUP98-HOXA9 Fusion

and TALE homeodomain genes Meis1 and PBX3). The “HOX code”, minimally defined by expression of the HOXA5-A9 cluster, is reported to be central to leukemogenesis associated with ▶mixed lineage leukemia protein (MLL) fusion genes and is probably a significant mechanism in NUP98-HOXA9 transformation. Overexpression of these HOX genes favors self-renewal and blocks differentiation. Downstream pathways involving the HOXA9 component of the fusion protein involve binding to the promoter region of the threonine kinase ▶Pim-1 promoter. Pim-1 is a protooncogene overexpressed in the transcriptosome of HOXA9 and NUP98-HOXA9 transduced human hematopoietic cells. Pim-1 increases phosphorylation and inactivation of pro-apoptotic ▶BAD protein and since unphosphorylated BAD normally binds to and inactivates anti-apoptotic proteins such as ▶BclXL and ▶Bcl-2, this would be an anti-apoptotic event. NUP98-HOXA9 upregulation of ▶hepatic leukemia factor (HLF) is consistent with upregulation of HLF reported in normal CD34+ and CD133+ and leukemic HSC. HLF is a basic leucine zipper protein defined by a PAR domain that plays a critical role in hematopoietic specific expression of the LMO2 gene. A role for HLF in

HSC self-renewal is supported by studies showing that ectopic expression of HLF enhanced HSC engraftment and inhibited apotosis. The transcription factor CCAAT enhancer binding protein-α (▶C/EBPα), a tumor suppressor gene and a crucial regulator of granulopoiesis through inhibition of c-JUN, is down modulated by NUP98-HOXA9. Disruption of C/EBPα, including dominant negative mutations of CEBPα, are found in AML and conditional C/EBPα knockout in mice blocked the differentiation of the common myeloid progenitor with myeloblast accumulation in marrow, absence of neutrophils and enhancement of HSC competitive repopulating capacity and self-renewal. Co-expression of an estrogen-inducible C/EBPα-ER protein together with NUP98-HOXA9 in CD34+ cells showed that re-expression of C/EBPα counteracted the stem cell proliferation and cell expansion associated with C/EBPα downregulation. Stabilization of HOXA9 Protein Expression Fusion to NUP98 to HOXA9 results in persistent expression of the homeobox protein due to its reduced sensitivity to CUL4A mediated ubiqitination.

NUP98-HOXA9 Fusion. Figure 2 NUP98 protein localization in normal and NUP98-HOXA9 leukemic cells. (a) NUP98 immunostaining of normal CD34+ cells. Note localization of the protein (purple) in the nuclear membrane and absence of nuclear aggregates. (b) NUP98 immunostaining of a leukemic cell from the bone marrow of a patient with the NUP98-HOXA9 translocation. Note the presence of numerous NUP98+ nuclear aggregates (purple) and impairment in NUP98 association with the NPC. (c) LOH analysis performed by PCR for NUP98 allelic markers on CD34- non-leukemic and CD34+ leukemic cells from the AML patient shown in 2B. Note NUP98 LOH is restricted to the CD34+ leukemic population.

Nutraceuticals

This may be achieved by steric hindrance, dimerization or by the NUP98 FG domains binding CBP/ p300 transcriptional co-activators leading to acetylation of the HOX lysine residues required for ubiquitination. The prolongation of the HOXA9 protein half-life in turn leads to persistence of aberrant transcriptional activation. Depletion of NUP98 from the NPC in Cells Expressing NUP98-HOXA9 NUP98 protein normally moves between the NPC and the nuclear interior where it associates with novel 0.2 μm diameter nuclear bodies termed ▶GLFG bodies because the GLFG domain of NUP98 is required for targeting of these structures. Immunostaining for Nup98 and HOXA in normal CD34+ cells transduced with NUP98-HOXA9 also reveals localization of staining in nuclear aggregates with loss of NUP98 from the NPC. In patients with t(7;11)(p15;p15) AML large NUP98+ nuclear aggregates are observed with absence of NUP98 associated with the nuclear membrane (Fig. 2). The chimeric NUP98-HOXA9 may be irreversibly associating with GLFG bodies and may have a dominant negative effect by sequestrating the remaining wild type NUP98 in heterodymeric colocalization bodies. The loss or depletion of NUP98 likely mediates a leukemogenic action by disrupting nuclear export of mRNAs that are critical to a normal balance of HSC proliferation and differentiation. While disruption of NUP98 by NUP98 translocations is a relatively rare event in normal human leukemogenesis there is growing evidence for a wider role for NUP98 disruption in oncogenesis, supported by the observation that there is ▶loss of heterozygosity (LOH) at the p11.5 NUP98 locus in nearly a third of human AML (Fig. 2) and this is associated with adverse prognosis.

References 1. Romana SP, Radford-Weiss I, Ben Abdelali R et al. (2006) NUP98 rearrangements in hematopoietic malignancies: a study of the Groupe Francophone de Cytogenetique Hematologique. Leukemia 20:696–706 2. Moore MAS (2005) Converging pathways in leukemogenesis and stem cell self-renewal. Exp Hematol 33:719–737 3. Nakamura T (2005) NUP98 fusion in human leukemia: dysregulation of the nuclear pore and homeodomain proteins. Int J Hematol 82:21–7 4. Chung KY, Morrone G, Schuringa JJ et al. (2006) Enforced expression of NUP98-HOXA9 in human CD34+ cells provides a proliferative advantage and enhances stem cell self-renewal. Cancer Res 66:11781– 11791 5. Moore MA, Chung KY, Plasilova M et al. (2007) NUP98 dysregulation in myeloid leukemogenesis. Annals New York Acad Sci 1106:114–142

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NUPR1 ▶p8 Protein

Nur77 Definition

An orphan member of the ▶nuclear receptor superfamily that is expressed in various types of cells and mediates diverse biological processes. It regulates gene transcription by binding to the Nur77-binding response element (NBRE) as a monomer. It is rapidly induced by various stimuli, including growth factors and ▶phorbol ester- and ▶cAMP synthesis-dependent pathways. ▶Retinoid Receptor Cross-talk

Nutraceuticals B LANCA H ERNANDEZ -L EDESMA , B EN O. DE LUMEN Dept of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA

Synonyms Functional foods; Designer foods; Medical foods; Pharmafoods; Food for specified health use

Definition Nutraceutical is a food (or part of food) that provides medical and health benefits, including the prevention and/or treatment of a disease beyond providing nutrition. It has also been defined as a product produced from foods but sold in powders, pills, and other medicinal forms not generally associated with food and demonstrated to have physiological benefits or to provide protection against chronic disease.

Characteristics The term Nutraceuticals was created by Stephen DeFelice in 1989 by combining the words “nutrition” and “pharmaceuticals.” The name nutrition defines the important difference between nutraceuticals and pharmaceuticals. While the pharmaceuticals do not come

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Nutraceuticals

from nature and they are chemically created, nutraceuticals come from food naturally. Food provides calories needed to perform daily functions and maintain normal metabolic processes. Moreover, food contains nutrients and other components that are essential to maintain health and reduce the risk of disease. Some examples of relationships between nutraceuticals and health benefits include the importance of calcium in preventing osteoporosis, folate in the prevention of neural tube defects in infants, and vitamin C in the prevention of the disease scurvy. Mounting evidence from epidemiological studies, animal research, clinical trials, and research in nutritional biochemistry suggests that many nutraceuticals may be beneficial in diabetes, osteoporosis, and other chronic and degenerative diseases such as Parkinson and Alzheimer diseases, coronary heart disease, and cancer. Nutraceuticals and Cancer It has been estimated that 30–40% of all tumors can be prevented with a correct lifestyle and diet. It has been demonstrated that ▶carcinogenesis is inhibited by factors such as ▶retinoids, vitamins E, D, C, polyphenols, fibers, calcium, selenium, and polyunsaturated fatty acids such as ▶Omega-3 fatty acids. Other factors, such as lipids, sodium chloride, nitrite, and nitrate tend to favor it. Numerous epidemiological studies have shown a strong correlation between frequent consumption of fresh fruits and vegetables and a decreased cancer risk. These foods are low in fat, rich in vitamins and minerals, and often contain significant amounts of fiber. The benefits of these are well known, but fruits and vegetables contain a variety of naturally occurring compounds, called phytochemicals that have biological activity in humans. It is the combination of nutrients and phytochemicals working together that benefits health, and helps in reducing the risk of cancer, as well as other important disease including heart disease. Scientists have identified thousands of phytochemicals, although only a small fraction has been studied closely. It has been demonstrated that they may exert physiological effects by different mechanisms of action. They may incite the immune system, contribute to reduce the toxicity of adverse chemical products, influence hormone levels, and control cell growth. They may also involve the activation of antioxidant defenses, signal transduction pathways, cell survival associated gene expression, cell proliferation and differentiation, and preservation of mitochondrial integrity. Furthermore, many of these substances exert antiinflammatory actions through inhibition of oxidative stress-induced transcription factors, cytotoxic ▶cytokines, and ▶cyclooxygenase-2. Four main types of phytochemicals are found in fruits and vegetables, terpenoids, phenolics (including

polyphenolics), nitrogen-containing alkaloids, and sulfur compounds. The terpenoids include ▶carotenoids, many of which are precursors to retinol (▶vitamin A). Carotenoids are organic pigments naturally occurring in plants and some other photosynthetic organisms like algae, some types of fungus, and some bacteria. Carotenoids are important factors in human health. Protective effects of carotenoids against serious disorders such as cancer, heart disease, and degenerative eye disease have been recognized, and have stimulated intensive research into the role of carotenoids as antioxidants and as regulators of the immune response system. Of the phenolic compounds, the ▶flavonoids are the most numerous. They are found in a wide variety of fruits and vegetables, tea, and coffee. In addition to their antiallergic, antiinflammatory, and antimicrobial properties, flavonoids have an important role in the prevention of cardiovascular disease and cancer. They are potent antioxidants and also can influence the expression of ▶phase I enzymes and ▶phase II enzymes. These enzymes metabolize potentially carcinogenic substances to make them more water soluble and hence readily excreted. This ▶detoxification process has a potential protective effect against cancer. ▶Isoflavones are a class of organic compounds and biochemicals related to the flavonoids. They can be found in many foods but the best known source is the soybean. Due to their similar structure to the hormone ▶estrogen, isoflavones act as ▶phytoestrogens in mammals with potent hormonal activity. Other beneficial effects on human health such as antioxidant, antiatherogenic, hypolipidemic, antiosteoporotic, and anticarcinogenic effects have been well demonstrated. Several mechanisms have been proposed for the anticarcinogenic activity of isoflavones. These include upregulation of ▶apoptosis, inhibition of angiogenesis, inhibition of ▶DNA topoisomerases II, and protein ▶tyrosine kinase. Their weak estrogenic activity may be involved in its putative activity against some kinds of cancer, such as ▶prostate cancer and ▶breast cancer. Isoflavones appear to work in conjunction with the ▶soy proteins to protect against cancer, as well as against cardiovascular disease and osteoporosis. Consumer interest in the relationship between diet and health has increased the demand for information about nutraceuticals and functional foods. Found in a mosaic of products emerging from the food and pharmaceutical industries, and the newly merged herbal and dietary supplement market, nutraceuticals are in high demand among consumers looking for specific health benefits. Credible scientific research now makes it fairly evident that diet can help in reducing the risk of chronic diseases and promotes general good health. ▶Phytochemicals in Cancer Prevention

Nutrition Status

References 1. Divisi D, Di Tommaso S, Salvemini S et al. (2006) Diet and cancer. Acta Biomedica 77:118–123 2. Hardy G, Hardy I, Ball PA (2003) Nutraceuticals – a pharmaceutical viewpoint: part II. Curr opin clin nutr metab care 6:661–671 3. Kalra EK (2003) Nutraceutical – definition and introduction. AAPS Pharm Sci 5:Article 25 4. Mandel S, Packer L, Youdim MBH et al. (2005) Proceedings from the “Third International Conference on mechanism of action of nutraceuticals”. J Nutr Biochem 16:513–520

Nutrition Assessment Definition Thorough evaluation of medical history, dietary history, physical examination, anthropometric measurements, and laboratory data with the intent of developing an individualized nutrition care plan. ▶Nutrition Status

Nutrition Screening Definition Use of objective and subjective data in a easy to use, cost effective, valid, quick tool to identify the presence or risk of malnutrition. ▶Nutrition Status

Nutrition Status M AUREEN B. H UHMANN Department of Primary Care, School of Health Related Professions, University of Medicine and Dentistry of New Jersey, The Cancer Institute of New Jersey, New Brunswick, NJ, USA

Definition The definition of nutrition status varies by discipline. In general it refers to the presence or absence of malnutrition. The term “malnutrition” applies to both underweight and overweight populations. Malnutrition

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is defined as “any disorder of nutrition status including disorders resulting from deficiency of nutrient intake, impaired nutrient metabolism, or overnutrition.” Although no singe index can accurately indicate poor nutrition status, weight and weight history are the parameters most commonly used. This method has limitations. Weight alone does not indicate the nature and extent of tissue loss in patients with cachexia. It also does not indicate specific metabolic or biochemical nutritional issues.

Characteristics Weight loss is common in patients with cancer, with 31–100% of oncology patients experiencing weight loss depending upon tumor site, stage, and treatment. As little as 5% weight loss is associated with increased mortality and poor prognosis. Unfortunately the weight loss is multifactorial and commonly requires multiple interventions by a variety of disciplines. Loss of body weight in patients with solid tumors is attributed to losses of fat, water, and ▶lean body mass (LBM). Patients with lung, gastrointestinal (GI), and head and neck tumors experience weight loss >10%, which manifests as a loss of both muscle and fat. Individuals with GI malignancies seem to experience the largest decreases (>50%) in muscle mass and protein content, as well as 30–40% loss in body fat. Despite this, even in severe wasting patients retain some body fat. Visceral mass is also preserved to an extent and skeletal muscle loss is the primary form of lean body mass loss. ▶Cancer cachexia syndrome (CCS) manifests as the characteristic wasting seen in some cancer patients. Symptoms associated with CCS include weight loss, anorexia (loss of appetite), fatigue, early satiety, and asthenia. CCS is a complex metabolic state which results from a intricate, and yet to be fully elucidated, interaction of pro-inflammatory cytokines such as tumor necrosis factor, interferon-γ, and interleukins 1 and 6 and tumor byproducts such as proteolysis inducing factor, lipid mobilizing factor, and mitochondria uncoupling proteins 1, 2, and 3. CCS results in depletion of energy and muscle stores unresponsive to aggressive nutrition support (i.e. enteral nutrition or parenteral nutrition). Provision of nutrients in excess of estimated needs does not reverse this weight loss. Treatment of the underlying disease is the only current effective therapy for CCS. Mechanisms Nutritional decline is often accepted as part of the cancer course and its treatment. Anti-cancer treatment modality choice (i.e. surgery, ▶chemotherapy, radiation therapy) can dictate this decline in nutrition status. The metabolic stress caused by surgery can lead to a hypercatabolic state. This hypermetabolism contributes to muscle and fat breakdown leading to weight loss

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Nutrition Status

post-operatively. Diet restriction (i.e. liquid diet, nothing per oral) and poor appetite lead to decreased dietary intake further adding to this weight loss. Patients with ▶GI malignancies that have lost LBM experience increasing rates of severe complications associated with surgical intervention. Surgically induced anatomical changes to the ▶GI tract may present mechanical barriers to food ingestion or digestion. Bowel, esophageal, gastric, hepatic, and pancreatic resections can lead to diarrhea, malabsorption, and ultimately dehydration. Side effects related to chemotherapy administration vary greatly. Chemotherapy is highly toxic to rapidly dividing cells such as those that line the GI tract. Chemotherapeutic agents may directly impair food intake via stomatoxic reactions such as mucositis, diarrhea, and ▶emesis, or indirectly via fatigue, pain, food aversions, and taste changes. Nausea and vomiting are frequently reported as the most distressing side effects associated with chemotherapy. Chemotherapy induced symptoms can occur prior to treatment, during treatment or several weeks later and can last from several hours to days. Aggressive ▶supportive care is essential to minimize nutrition related side effects. Radiation to any portion of the GI tract can cause extreme susceptibility to malnutrition. Radiation to the pelvic and abdominal area causes an acute inflammatory response in bowel tissue. This ▶inflammation produces diarrhea, abdominal pain and nausea. Chronic radiation enteritis develops in certain cases necessitating lifelong medication and diet changes. Radiation to the head and neck can also lead to nutrition related side effects, such as mucositis, xerostomia, taste change, and dysphagia. These side effects peak two-thirds of the way through treatment and can become chronic. Radiation therapy in the area of the thyroid can cause permanent thyroid damage leading to changes in metabolism. Gastrointestinal symptoms induced bay any of the above therapies, such as nausea, vomiting, diarrhea, and constipation; and oral symptoms, such as xerostomia and mucositis may lead to weight loss early in the course of cancer. Fatigue and psychological symptoms such as, depression and anxiety, also influence weight loss. Assessment of Nutrition Status ▶Nutrition screening of all oncology patients facilitates the early recognition of malnutrition. Screening is intended to quickly identify individuals at nutritional risk. ▶Nutrition screening tools should be easy to use, cost effective, valid, reliable, and sensitive. Screening tools incorporate objective and subjective data to identify the presence or risk of malnutrition. Objective measures commonly included in nutritional screening tools include height, weight, weight change, primary diagnosis, disease stage and the presence of comorbidities. Individual objective measures, such as a laboratory value or current weight, alone are not

specific enough to indicate nutrition risk so multiple objective measures are combined with subjective measures that may impact nutrition. The American Society for Parenteral and Enteral Nutrition (ASPEN) and the American Dietetic Association (ADA) recommend that all patients receive nutrition screening as a component of their initial evaluation. Many nutrition screening tools have been developed, with several specifically validated in an oncology population. The ▶patient generated subjective global assessment (PG-SGA) and the mini nutritional assessment (MNA) are commonly used in the outpatient oncology setting. Many acute care facilities have designed facility specific screening forms to be completed by the nursing staff upon admission. These acute care forms commonly combine nutrition screening with other disciplines. The PG-SGA is a modification of an earlier screening tool called the Subjective Global Assessment (SGA) developed by Detsky. Faith Ottery, a surgical oncologist, modified the SGA specifically for the oncology population. The PG-SGA is comprised of two sections; a patient completed section and a section completed by the healthcare professional. Patients provide data regarding weight history, symptoms, dietary intake, and activity level. Healthcare professionals evaluate metabolic demand, disease in relation to nutritional requirements, and perform a physical assessment. The nursing staff, a nurse practitioner, a registered dietitian, or a physician can complete this section. A numeric score is calculated by adding the points obtained in sections one and two. The numeric scores can be used as a triage system to initiate nutrition intervention and guide followup. The PG-SGA numeric score, when repeated at subsequent time points, is useful in illustrating small improvements or deteriorations in nutrition status. The Nestle Mini Nutritional Assessment (MNA) is a commonly used nutritional screening tool in the elderly population. The 18-item MNA, developed by Guigoz with Nestle Nutritional Corporation, is comprised of two main components; screening and assessment. The six-item screening takes approximately three minutes to complete and includes questions related to decline in food intake, weight loss, mobility, stress and body mass index. The healthcare practitioner is directed to complete the assessment section of the MNA if the screening provided a score of 11 or less. The assessment component includes specific medical history and eating habits as well as some anthropometric measurements. The Malnutrition Screening Tool (MST), although not commonly used in the United States, is a short nutritional screening tool. This three-item tool utilizes data on weight history and appetite to predict nutrition risk The MST, developed by Maree Ferguson, has been validated in both acute care patients and oncology patients receiving radiation therapy.

NWTSG

Nutrition screening should lead to a more in-depth ▶nutrition assessment. Nutrition assessment differs from screening in that it is a thorough evaluation which assimilates data obtained from the medical history, dietary history, physical examination, anthropometric measurements, and laboratory data. The nutritional assessment integrates the review of anthropometrics with data on disease and clinical status to evaluate impact on metabolism and nutrient need. Appraisal of disease and treatment related symptoms is also necessary to plan nutrition interventions. This process leads to the identification and diagnosis of nutritional issues. The nutritional assessment is usually completed by a registered dietitian (RD) or nutrition professional, however other members of the healthcare team can complete it as well. Nutrition Intervention Nutrition intervention refers to the specific activities required to address and correct the nutrition diagnosis. The nutrition intervention is selected, planned, and implemented with the intent of improving nutrition status. Planning of the nutrition intervention requires the input of all disciplines involved in patient care. Reflection must be taken on the causes of weight loss or weight gain. In taking into consideration these factors the patient should remain at the center of the intervention, with patient preferences of paramount importance. The goals of the intervention must be documented and reevaluated frequently. The intervention must be individualized to the patient and consideration should be taken for patient comfort and wishes. Although variable between and among patients, common nutritional goals include symptom management, weight maintenance, and preservation of functional status. Attaining these goals may require modified diets, the addition of oral nutritional supplements, or the initiation of enteral or parenteral nutrition. Supportive care measures should be employed as needed These modifications to dietary intake can be costly and flexibility is required on the side of the individual designing the intervention with respect to specific formulas and administration. The services of financial planners and social workers are very beneficial in this situation. Benefit of Maintenance of Nutrition Status Health-related ▶quality of life (QOL) refers to the physical, psychological and social domains of health that are influenced by an individual’s experiences, beliefs, expectations and perceptions. Cancer treatments influence the social, psychological and emotional aspects of patients’ lives. The presence of malnutrition can also impact a patient’s QOL. Difficulty with eating

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resulting from the side effects of treatment or disease may cause patients to pass up experiences of social interactions with family and friends. This in turn leads to further depression of appetite. Poorer overall QOL has been observed in patients experiencing symptoms that impact socializing such as odynophagia, weak voice, and unclear speech. A relationship has been established between weight loss and QOL in oncology patients. Changes in body composition affect symptom control and complication rates. Increased incidence of nutritional symptoms is correlated with decreases in quality of life. To date, there is little information relating changes in specific body compartments to QOL. Preliminary research relating to the use of an anabolic steroid indicate that increases in body cell mass are associated with improved QOL and ▶ECOG performance status scores in head and neck and lung cancer patients. However, there has been little quantification of the impact of loss of LBM on QOL.

References 1. American Society for Parenteral and Enteral Nutrition Board of Directors (Jan–Feb 2002) Guidelines for the use of parenteral and enteral nutrition in adult and pediatric patients. JPEN J Parenter Enteral Nutr 26(1 Suppl):1SA138SA 2. Bloch A, Charuhas P (2001) Cancer and cancer therapy. In: Gottschlich M (ed) The science and practice of nutrition support. Kendall Hunt, Dubuque 3. Bozzetti F (2002) Rationale and indications for preoperative feeding of malnourished surgical cancer patients. Nutrition 18:953–959 4. Elliott L, Molseed LL, Davis McCallum P (2006) The Clinical guide to oncology nutrition, 2nd edn. American Dietetic Association, Chicago 5. Ottery F, Bender F, Kasenic S (2002) The design and implementation of a model nutritional oncology clinic. Oncology Issues (Integrating Nutrition into Your Cancer Program) 2–6

NWTSG Definition National Wilms Tumor Study Group (NWTSG) is an organization that has co-ordinated clinical trials for the treatment of Wilms tumor in North America since the early 1970s. They have data on epidemiological, genetic and clinical features together with treatment outcome on over 6,000 children treated for ▶Wilms tumor.

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O/E ▶Early B-cell Factors

O6-Alkylguanine-Alkyltransferase Definition Is a member of the family of alkyltransferase enzymes that catalyzes alkylation of O6 position of the guanine residue in DNA. ▶Alkylating Agents

Oat Cell Carcinoma ▶Extrapulmonary Small Cell Cancer

healthy. It is a major risk factor for type 2 diabetes, cardiovascular disease, and some types of cancers. ▶Obesity and Cancer Risk

Obesity and Cancer Risk S USANNA C. L ARSSON Division of Nutritional Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Definition Obesity is a condition in which excess fat has accumulated in the body. The only widely accepted criteria for obesity are based on body mass index (BMI, also called Quetelet index), which is defined as the weight in kilograms divided by the square of the height in meters. According to the World Health Organization (WHO) classification, ‘normal weight’ is defined as BMI between 18.5 and 25 kg/m2, ‘overweight’ is BMI between 25 and 30 kg/m2, and obesity is defined as a BMI of 30 kg/m2 or greater.

Characteristics

Oat Cell Lung Cancer Definition

▶Small Cell Lung Cancer

Obesity Definition Is a status of excess fat accumulation in the body, to a degree that is much greater than what is considered

Trends in Obesity Over the past few decades, the proportion of people with excess body weight has been increasing in most developed and developing countries along with an adoption of a westernized lifestyle characterized by excessive energy intake and lack of physical activity. There are large between-country and within-country differences in the ▶prevalence of obesity. The prevalence of adult obesity is high in North America (particularly in the United States), the Eastern Mediterranean, Central and South America (particularly in Mexico, Paraguay, Argentina, and Chile), Eastern Europe, and some Western European countries (Finland, Spain, Germany, and the United Kingdom). In the United States, the prevalence of obesity doubled over the past two decades, and currently about one-third of the

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Obesity and Cancer Risk

US adult population is obese and an additional onethird is overweight. Smaller increases in the prevalence of obesity have been observed in many European countries. The MONICA project – a large international study of the monitoring risk factors for cardiovascular disease, led by the WHO – showed a prevalence of obesity above 20% in many parts of Western Europe in the 1990s, and it was has high as 35% in some Eastern European countries. Obesity is still uncommon in China and Japan and most parts of Africa. Obesity and Cancer Risk Being overweight or obese is a well-known risk factor for ▶type 2 diabetes mellitus and cardiovascular disease, but ▶epidemiologic studies are also providing mounting evidence for a link between body weight and cancer risk. In 2002, the International Agency for Research on Cancer (IARC) Working Group on the Evaluation of Cancer Preventive Strategies published an extensive review of the literature on body weight and cancer. The Working Group concluded that, based on data from epidemiologic studies, there is sufficient evidence of a cancer-preventive effect of avoidance of weight gain for cancers of the colon, breast (in postmenopausal women), endometrium, kidney (renal cell), and esophagus (adenocarcinoma). Many studies have been published since the IARC report, and the accumulated evidence indicates that obesity may be a risk factor also for pancreatic, liver, gallbladder, and hematopoietic cancers, and possibly for aggressive prostate cancer. Obesity is not associated with risk of lung cancer, and is probably not a risk factor for ▶bladder cancer or noncardia ▶gastric cancer. Findings for cancer at other sites are sparse or inconsistent. Obesity-Related Cancers There is an extensive body of epidemiologic data showing a relation between obesity and ▶colon cancer risk. In general, obesity has been found to be associated with an approximately 30–90% increased risk of colon cancer in men and with a 10–50% increased risk in women. The association between obesity and risk of rectal cancer is less clear, with a generally weak positive association observed in men but no association in women. The first evidence of an association between excess body weight and increased risk of breast cancer came in 1974 from a Dutch ▶cohort study of postmenopausal women. A large number of subsequent studies have found that obesity increases the risk of breast cancer in postmenopausal women by about 30%. Conversely, there is consistent evidence that obesity is associated with a decreased risk of breast cancer in premenopausal women. This reduction in risk may be related to the increased tendency of young obese women to have

anovulatory menstrual cycles and reduced circulating concentrations of progesterone and ▶estradiol. Several studies have shown a stronger association between BMI and postmenopausal breast cancer risk in women who have never used postmenopausal hormones. This observation is consistent with the hypothesis that the effect of obesity on postmenopausal breast cancer risk may be mediated through increased endogenous estrogen production. Obesity has been associated consistently with an increased risk of ▶endometrial cancer in both pre- and postmenopausal women. The risk increased about linearly with increasing BMI in most studies. In different studies, the risk of endometrial cancer was approximately 2–3 times higher in overweight and obese women than in normal weight women. An association between excess body weight and risk of kidney (mainly renal cell) cancer has been consistently observed in epidemiologic studies. The majority of studies have reported a linear increase in risk with increasing BMI. The increase in risk generally ranges from 1.5- to 3-fold in overweight and obese individuals. The ▶incidence rates of adenocarcinoma of the esophagus and gastric cardia have been rapidly increasing in developed countries over the last three decades. In contrast, the rates for squamous cell carcinoma of the esophagus and noncardia gastric adenocarcinoma have remained stable or decreased slightly during the same period. Compelling evidence indicates that overweight and obesity increase the risk of adenocarcinoma of the esophagus by about 2–3-fold and of gastric cardia by about 1.5–2-fold. However, excess body weight is not associated with an increased risk of esophageal squamous cell carcinoma or noncardia gastric adenocarcinoma. Cancers Likely Related to Obesity Several recent cohort studies have observed a 1.2–2fold increased risk of pancreatic cancer in obese men and women. While some studies found a dose-response relationship with increasing BMI, other studies showed an increase in pancreatic cancer risk only among obese individuals. Seven cohort studies in the United States and Europe have observed a 1.5–3.5-fold increased risk of liver cancer in obese individuals, whereas a ▶case–control study in Canada reported no association. Obesity may contribute to the risk of liver cancer by promoting the development of non-alcoholic fatty liver disease. To date, eight cohort studies and three case–control studies have examined the association between obesity and risk of gallbladder cancer, with most studies showing an increased risk in obese men and women. The increase in risk generally ranges from about 1.5–2-fold in obese individuals. Obesity may increase

Occult Cancer

gallbladder cancer risk indirectly by increasing the risk of gallstones, which, in turn increase the risk of gallbladder cancer. Several studies published during the last 5 years have found that obesity is associated with an increased risk of hematopoetic cancers, including ▶non-Hodgkin lymphoma, ▶leukemia, and ▶multiple myeloma. Overall, obesity has been associated with an approximately 20–40% excess risk of these cancers. Other Cancers Results from a large number of studies of the association between obesity and incidence of overall prostate cancer have been largely null. However, obesity has been related to increased risks of more advanced prostate cancer and prostate cancer mortality, and with higher recurrence rates after radical prostatectomy or radiotherapy treatment. These findings suggest that obesity may influence prostate cancer aggressiveness and progression. Obesity has been found to be inversely related to risk of lung cancer in several studies, but this association appears to be due to ▶confounding by smoking (smoking is the main cause of lung cancer and is inversely associated with body weight). There is no association between obesity and lung cancer in nonsmoking populations. Studies of the relation between obesity and ▶ovarian cancer risk are inconclusive. Although some studies found an increased risk (1.5–2-fold) of ovarian cancer in obese women, several studies observed no association. Results on obesity in relation to cervical cancer risk are limited and inconsistent. Mechanisms Linking Obesity to Cancer Risk Several biological mechanisms have been postulated to explain the relation between obesity and cancer risk, including alterations in the metabolism of insulin, ▶insulin-like growth factor-I (IGF-I), and sex steroids (▶estrogens, androgen, and progesterone). Obesity leads to a state of relative insulin resistance, chronically increased insulin concentrations, and an increase in bioavailable IGF-I. Both insulin and IGF-I act as growth factors that stimulate cell proliferation and inhibit ▶apoptosis. Epidemiologic studies have shown that increased circulating concentrations of insulin, C-peptide (a marker of pancreatic insulin secretion), and IGF-I as well as type 2 diabetes mellitus are associated with an increased risk of several cancer types, especially cancers of the colon, endometrium, and pancreas. Sex steroids are involved in the control of cell differentiation, proliferation, and apoptosis, and may also favor the selective growth of preneoplastic and neoplastic cells. There is considerably evidence that the increase in breast and ▶endometrial cancer risk with increasing body weight in postmenopausal women is

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largely the result of the associated increase in circulating concentrations of estrogens, particularly bioavailable estradiol. The relationship between obesity and endometrial cancer in premenopausal women may in part be related to chronic anovulation and decreased progesterone concentrations in young obese women. Adipocytes (fat cells) secrete a number of fat cell hormones/cytokines or adipokines, such as leptin, ▶adiponectin, resistin, tumor-necrosis factor-α, and ▶interleukin-6. These ▶adipokines have important roles in the regulation of energy balance, lipid metabolism, and insulin resistance as well as in ▶inflammation and immune response. It has been proposed that in addition to growth factors and sex hormones, obesity-related alterations in the concentrations of adipokines may be linked with cancer risk.

References 1. International Agency for Research on Cancer (2002) Weight control and physical activity. IARC handbooks of cancer prevention vol 6. IARC Press, Lyon 2. World Cancer Research Fund and American Institute for cancer Research. Food, nutrition, physical activity, and prevention of cancer: a global perspective. Washington DC: AICR, 2007. 3. Larsson SC, Wolk A (2006) Epidemiology of obesity and diabetes: prevalence and trends. In: Mantzoros CS (ed) Obesity and diabetes. Humana Press, Totowa, NJ 4. Larsson SC, Adami HO, Wolk A (2006) Obesity, diabetes and risk of cancer: a review of epidemiologic studies. In: Mantzoros CS (ed) Obesity and diabetes. Humana Press, Totowa, NJ 5. Larsson SC, Orsini N, Wolk A (2007) Body mass index and pancreatic cancer risk: a meta-analysis of prospective studies. Int J Canc 120:1993–1998

Observational Study Definition A study of changes in distribution of exposures, cancer, and other factors, without the intervention of the investigator. ▶Cancer Epidemiology ▶Epidemiology of Cancer

Occult Cancer ▶Dormancy

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OCP

OCP Definition Oral Contraceptive Pill.

Octreotide

processes reach from the pulp chamber through the dentin tubules to the dentinoenamel junction (primary odontoblasts). Their competence lies in secretion of (physiologic) secondary dentin and reparative dentine due to caries, trauma or tooth preparation. Secondary: Derive from mesenchymal precursor cells when primary odontoblasts become destroyed. Secondary odontoblasts (or “odontoblast-like” cells) form irregular masses of tertiary dentine. This capability to produce such calcifications plays an important role as a barrier mechanism after a carious, traumatic or iatrogenic insult to the tooth. ▶Dental Pulp Neoplasms

Definition Is a chemical compound used in nuclear medicine for localization of gastrinoma. Analog of ▶somatostatin. ▶Gastrinoma

Odynophagia Definition Is pain during swallowing.

Oculodermal Melanocytosis

▶Nasopharyngeal Carcinoma

Definition

Nevus of Ota; ▶Hamartoma of melanocytes giving a characteristics blue-gray appearance to the periocular skin and sclera. In Caucasians and Asians, there is an increased incidence of ▶uveal melanoma as well as ▶glaucoma. In African Americans, there is only an increased risk of glaucoma.

8-OHdG Definition 8-Hydroxydeoxyguanosine. ▶UV Radiation

ODC Definition Ornithine Decarboxylase.

Okadaic Acid Definition

Definition

Is a toxin isolated from the marine sponge Halichondria okadai. Okadaic acid accumulates in bivalves and causes diarrhetic shellfish poisoning. The molecular formula of okadaic acid is C44H68O13. Okadaic acid strongly inhibits protein serine/threonine phosphatase 1, 2A, and 2B. The inhibitory effect of okadaic acid is strongest for 2A, followed by 1, and then 2B.

Primary: Single cell line of highly specialized cells which are located inside the dental pulp, but whose

▶Doublecortin

Odontoblasts

Oligoastrocytomas

OKT3 Definition

A monoclonal antibody that targets mature ▶T cells.

Oleanolic Acid

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Oligoastrocytomas M ARICA E OLI 1 , G AETANO F INOCCHIARO 2 1

Unit of Clinical Neuro-Oncology, Istituto Neurologico Besta, Milano, Italy 2 Structure of Experimental Neuro-Oncology, Istituto Neurologico Besta, Milano, Italy

Synonyms Mixed gliomas

Definition

Definition

A triterpenoid from plant species.

Are tumors located mainly in supratentorial subcortical brain areas showing composite histological features and including cells similar to oligodendrocytes or astrocytes.

▶Betulinic Acid

Characteristics

Olefins Definition Chemicals with a vinyl bond. ▶Carcinogen Metabolism

Olf ▶Early B-cell Factors

Olfactory Neuronal Transcription Factor ▶Early B-cell Factors

Olfactory/Early B-cell Factors ▶Early B-cell Factors

Oligoastrocytomas and pure ▶oligodendrogliomas constitute a subgroup of ▶brain tumors receiving increased attention because of reports of chemosensitivity and of favorable survival rate when compared with ▶astrocytomas. This is also reflected in the increasing rate of diagnosis of tumors with an oligodendroglial component raising from about 5% in the past decade to approximately 15% in present years. The morphological borderlines between astrocytomas, oligoastrocytomas, and oligodendrogliomas are difficult and controversial issues. However the accurate distinction between these tumors has important prognostic and therapeutic implications. Patients with pure oligodendrogliomas are more likely to respond to chemotherapy and have a longer survival than patients with diffuse astrocytomas or oligoastrocytomas of the same grade. Oligoastrocytomas show a conspicuous mixture of two distinct neoplastic cell types, resembling the tumor cells in oligodendrogliomas and diffuse astrocytomas; the two components may be separated into distinct areas or be diffusely admixed. A pathological diagnosis based on the assessment of the individual component is difficult, since in most instances the true extent of such component can be hardly determined because of incomplete tumor sampling. Furthermore, precise diagnostic criteria are still lacking, the proposed minimum astroglial component ranging from 1 to approximately 50%. A subclassification of oligoastrocytomas, based on histological features, into oligodendroglioma-predominant, astrocytoma-predominant, or equivalent oligodendroglial and astroglial component has been proposed. However, prognosis and response to therapy were not found to be significantly different for such subtypes.

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Oligoastrocytomas

Oligoastrocytomas are graded, based on the WHO classification, as either low-grade (Grade II WHO classification) or high-grade tumors (anaplastic, grade III WHO classification). The diagnosis of anaplastic oligoastrocytoma requires the presence of histological features of malignancy such as increased cellularity, nuclear atypia, pleomorphism, and increased mitotic activity. Signs of histological malignancy may be present in the oligodendroglial component, the astroglial component, or in both cell types. The histological grading is both of prognostic and therapeutic use. Glioma genotyping can provide important information complementing the histological analysis. Until now no specific abnormalities associated with oligoastrocytomas have been found. Genetic alterations of these tumors include loss of heterozygosity (LOH) on chromosome 1p and/or 19q, typically found in oligodendrogliomas, but also LOH on chromosome 17p or 10q, frequently found in astrocytomas and associated with the progression to glioblastoma. ▶LOH on 1p and/or 19q are present in approximately 50% of oligoastrocytomas, independently of their histological grade; LOH on 17p is seen in 20% and LOH on 10q in 15% and is significantly associated with anaplasia; the observation of 1p loss with intact 19q (or vice versa) is more frequent in oligoastrocytomas than in oligodendrogliomas. Similar to oligodendrogliomas, LOH on 1p and/or 19q has been associated with chemosensitivity and prolonged survival, while LOH on 10q has an unfavorable impact on prognosis. Therefore, genomic changes may permit the division of tumors into more homogenous groups: oligoastrocytomas with LOH on 1p behave like WHO grade II or III oligodendrogliomas with 1p LOH and they are associated with longer survival; on the other hand, oligoastrocytomas without LOH on 1p behave like grade II or III astrocytomas and have shorter survival. Despite the clinical relevance of 1p and 19 q losses, the genes targeted on these two chromosomes are unknown. Some very recent progress, however, may help pinpointing the genetics underlying these alterations. Combined loss of on 1p and 19q is mediated by the ▶chromosomal traslocation (t(1,19)(q10;p10). The high sequence homology of chromosome 1 and 19 centromeric regions could be the mechanisms favoring translocation, facilitated by tumor-related alterations of normal DNA methylation or histone modification affecting the centromeric region. Chromosome engineering was used to generate mouse models with gain and loss of a region corresponding to human 1p36. By this strategy chromodomain helicase DNA binding domain 5 (Chd5) was identified as a tumor suppressor located on 1p36 that controls proliferation, apoptosis, and senescence via the p19(Arf)/p53 pathway. Future studies will clarify the relevance of Chd5 in the formation of different gliomas, including oligoastrocytomas.

Due to the high variability of morphological criteria used for their classification, oligoastrcytomas incidence rate is highly variable, accounting from 1.8 to 10% of brain tumors. Males are affected slightly more frequently than females. Oligoastrocytomas typically occur in young adults (mean age at diagnosis, 35–45 years) and epilepsy is the most common initial symptom. Less commonly, patients may present with headaches, progressive paresis, cognitive impairment, signs of increased intracranial pressure, or have no symptoms at all. Supratentorial subcortical areas are the most frequent locations of oligoastrocytomas. The frontal lobes are most commonly affected. Lesions are usually single, but multiple lesions have been reported. There may be a correlation between the profile of molecular alterations and tumor location. Also, oligoastrocytomas and oligodendrogliomas with LOH on 1p appear preferentially located in the frontal regions. The issues concerning the diagnosis are similar to those for other low-grade tumors. At neuroimaging oligoastrocytomas do not show features allowing a reliable distinction from oligodendrogliomas. Computed tomography (CT) scans reveal a well-demarcated hypodense subcortical mass. Calcifications may be present, but they are less common than in pure oligodendrogliomas. In about half of low-grade oligoastrocytomas CT scans show contrast enhancement. Magnetic resonance (MR) shows a hypointense mass in T1 weighted images and a hyperintense mass in T2 weighted images. The abnormal T2 signal represents both vasogenic edema and the infiltrating tumor. Enhancement is associated with anaplasia, but especially in older people, the absence of enhancement does not exclude the diagnosis of anaplasia; when present, enhancement is irregular and nodular. In most studies patients with oligoastrocytomas are combined with patients with oligodendrogliomas or astrocytomas. However in cohorts of WHO grade II oligoastrocytomas a median survival time of 6.3 years and survival rate of 70% at 5 years and 49% at 10 years have been reported. In WHO grade III oligoastrocytomas the median survival time of 2.8 years and survival rates of 36% at 5 years and 9% at 10 years have been referred. Clinical parameters associated with prolonged survival are young age (50 Gy) that conventional radiation approaches would lead to too much damage to the surrounding normal cells. Radiation is used in cases of osteosarcoma of the pelvis when surgery has not been able to remove the entire tumor. Recent advances in radiation delivery techniques such as intensity-modulated and proton-based therapy may increase survival in these patients. Radiation is also used in palliation, in cases to relieve pain due to impingement of the tumor against vital sites.

(▶Retinoblastoma, cancer genetics) and those with ▶Li-Fraumeni syndrome. These syndromes occur due to mutations in the tumor suppressor genes Rb (▶Retinoblastoma protein, biological and clinical functions) and p53 (▶p53 Protein, biological and clinical aspects), respectively. Analysis of patient tumor samples reveals that over 50% of samples have mutations in either Rb or p53. Other populations that have increased risk of developing osteosarcoma are geriatric patients with ▶Paget disease and children with ▶RothmundThomson syndrome who have truncating mutations in the RecQL4 gene. When karyotypes of patient samples are prepared, all samples show ▶aneuploidy (more than the normal chromosome number of 46), suggesting that a defect in DNA replication may prove important in the etiology of osteosarcoma.

Metastases Twenty percent of patients have detectable spread of disease at the time of diagnosis. Unfortunately, these patients have only a 25% chance of cure. The management of these patients is similar to those of patients with non-metastatic osteosarcoma, removal of the primary tumor and all sites of tumor spread, in conjunction with chemotherapy. There have been many efforts to increase the intensity of chemotherapy drugs given to these patients in an attempt to increase the survival rate. As expected, the number of side effects increased greatly, but there has not been an increase in survival.

Future Research in osteosarcoma has focused on metastatic disease due to the poor prognosis associated with both metastases and recurrence. The futility of efforts aimed at targeting the tumor through intensification of chemotherapy has led to efforts aimed at other targets. In particular, agents that decrease the blood supply to the tumor (angiogenesis inhibitors) (▶Antiangiogenesis) and agents that target the reactive cells that surround the tumor (stromal inhibitors) are receiving increased focus, both in the laboratory and in clinical trials.

Recurrence Another group of patients who do poorly are those who have recurrence of the tumor following treatment. These patients have only a 20% chance of cure. Over 90% of recurrences are in the lung. Patients who do not achieve a second surgical remission have greatly diminished prognosis. Those who are able to have all of their tumor nodules removed have a 40% chance of cure. Again, attempts to increase survival by giving more chemotherapy or radiation to the lung have not been very successful. If any of the four chemotherapy drugs that have activity against osteosarcoma have not been used in the original treatment, those agents should be given in conjunction with surgery. Unfortunately there is still no effective salvage regimen for these patients. Genetics The cause of osteosarcoma remains unknown with most patients having no defined genetic risk factors. The risk of developing osteosarcoma increases two-fold over the general population for patients who receive radiation or chemotherapy (especially alkylating agents and anthracyclines) for treatment of other cancers. Two groups of patients have a much higher risk of osteosarcoma however, those with hereditary retinoblastoma

References 1. Malawar M, Helman LJ, O’Sullivan B (2005) Sarcomas of bone. In: DeVita VT, Hellman S, Rosenberg SA (eds) Cancer: principles and practice of oncology, 7th edn. Lippincott Williams & Wilkins, Philadelphia, PA, 1638–1686 2. Bielack SS et al. (2002) Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol 20:776–790

Osteosynthesis Definition The surgical fixation of fractured bone fragments, or the prophylactic reinforcement (internal fixation) of bone (to prevent for fracture) by plates, screws or intramedullary nails. ▶Cryosurgery in Bone Tumors

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Ototoxicity

Ototoxicity

Ovarian Cancer

Definition

PAT J. M ORIN 1 , E LLEN S. P IZER 1

Ear poisoning.

Laboratory of Cellular and Molecular Biology, National Institute on Aging, NIH, Baltimore, MD, USA

▶Chemoprotectants

Outer Edge of the Tumor Mass Definition Consists of the path-leading cells that interact with the surrounding tissue and form the invasive front of the tumor. The molecular expression profile of cells at the outer edge of the tumor can differ from that of cells in the interior. Nuclear localization of ▶β-catenin, and upregulation of β1-integrin, the L1 ▶adhesion molecule and ▶podoplanin were specifically reported in cells of the tumor rim. ▶Podoplanin

Ovarian Ablation Definition

▶Ovarian Function Suppression.

Ovarian Adenocarcinomas Definition Are malignant ovarian neoplasms classified according to the histology of the tumor. Surface epithelial-stromal tumors are the most common and prototypic ovarian cancers, and include serous cystadenocarcinoma and mucinous cystadenocarcinoma. ▶Ovarian cancer staging is by the ▶FIGO staging system.

Definition A heterogeneous group of malignant tumors derived from the ovary (there are also a wide assortment of benign tumors derived from the ovary). Approximately 90% of malignant ovarian tumors originate from the ovarian surface celomic epithelium and are therefore designated epithelial ovarian cancer (EOC). Germ cell tumors and stromal tumors account for the remaining cases. EOC can be divided according to histological appearance: . Serous, the most common type (composed of epithelium resembling that of fallopian tubes) . Endometrioid (composed of epithelium resembling that of the endometrium) . Clear cell (composed of clear cell epithelium and resembling gestational endometrium) . Transitional cell tumors (composed of epithelium resembling urothelial cells) . Mucinous (composed of epithelium resembling that of the endocervix) . Squamous cell (squamous epithelial cells) . Mixed epithelial tumor (mixture of two or more of the above subtypes) . Undifferentiated (no recognizable differentiation features) Ovarian cancer is staged according to the following International Federation of Gynecology and Obstetrics (▶FIGO) guidelines. Grading of EOC is based on degree of differentiation, cytologic ▶atypia and mitotic index. . Stage I: restricted to the ovaries . Stage II: involvement of ovaries and pelvic extension . Stage III: involvement of ovaries with peritoneal implants outside the pelvis . Stage IV: involvement of ovaries with distant ▶metastasis, such as liver, lung or brain

Characteristics In westernized countries, ovarian cancer is the sixth most common cancer in women. Over half of the women diagnosed with ovarian cancer are over 65 years of age. The age-adjusted incidence for ovarian cancer is 15 per 100,000 and the total number of cases is expected to increase as the overall population ages. Overall, ovarian cancer prognosis is poor. In spite of the

Ovarian Steroid Hormones

recent introduction of aggressive treatments, five year survival rates for patients with advanced ovarian cancer (stage III and IV) has remained low and ranges between 5 and 30%. However, the outcome is much more favorable for stage I patients where 5-year survival can reach 90–95%. Unfortunately, because of lack of symptoms, only 25% of the women with ovarian cancer are diagnosed with stage I disease. These facts make ovarian cancer a disease for which early detection represents an intervention of choice in reducing morbidity. Several putative ovarian cancer serological markers have been identified, including ▶CA125, ▶TATI, ▶CEA and ▶CA19-9. Of those, CA125 has proven to be the most clinically useful. Unfortunately, studies have exhibited mixed results, with some studies detecting only 23% of stage I ovarian carcinoma with CA125. CA125 may be useful when used in combination with pelvic ultrasound assessment, and CA125 has been used in monitoring recurrence in patients with CA125-positive tumors. Overall, CA125 lacks the specificity and sensitivity required for the screening of the general population. The etiology of ovarian cancer is not well understood but the following risk factors have been identified: . Age: half the cases occur in women of 65 years or older . Menstrual history: ovarian cancer risk increases with increasing numbers of menstrual cycles . Birth control pills: the use of which, for at least five years, lowers the risk of ovarian cancer . Pregnancy and breastfeeding: lowers the risk of ovarian cancer . Family history Typical treatment for ovarian cancer is surgery followed by chemotherapy. The exact details of treatment depend on the tumor type, grade and stage, the age, as well as the general health of the patient. In spite of recent advances in the field of cancer genetics and molecular biology, little is known about the mechanisms of ovarian tumorigenesis. Chromosome abnormalities are frequent in ovarian cancer and allelic losses have been observed in chromosomes 4p, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 11q, 12p, 12q, 13q, 16p, 16q, 17p, 17q, 19p, 19q, 22q and Xq. These observations suggest the presence of several tumor suppressor genes important in ovarian cancer, but very few have been unambiguously identified. The tumor suppressor ▶p53 is inactivated in a large number of ovarian cancers. Although the tumor suppressor genes ▶BRCA1 and ▶BRCA2 have been implicated in familial breast and ovarian cancer syndromes, the vast majority of ovarian cancers are sporadic and may have a different natural history. The K-▶Ras, ▶Her-2/neu and c-▶myc ▶oncogenes have all been implicated in EOC. The frequencies of

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alteration of these different oncogenes varies according to the subtype of ovarian cancer. No clear association has been reliably observed between the activation of these oncogenes and survival. Indeed, stage and grade have been the only factors consistently shown to predict outcome of ovarian cancer.

References 1. NIH consensus development panel on ovarian cancer (1995) NIH consensus conference. Ovarian cancer. Screening, treatment, and follow-up. JAMA 273:491–497 2. Markman M (2000) The genetics, screening and treatment of epithelial ovarian cancer: an update. Cleve Clin J Med 67:294–298 3. Mazurek A, Niklinski J, Laudanski T et al. (1998) Clinical tumour markers in ovarian cancer. Eur J Cancer Prev 7:23–35 4. Pejovic T (1995) Genetic changes in ovarian cancer. Ann Med 27:73–78 5. Scully RE (1999) Histological typing of ovarian tumors, 2nd edn. Springer, Heidelberg

Ovarian Function Suppression Definition Synonym Ovarian ablation; Includes the use of surgery, radiation therapy, or a drug treatment to stop the functioning of the ovaries. ▶Adjuvant Chemoendocrine Therapy

Ovarian Steroid Hormones Definition Refer to the female sex steroid hormones 17β▶estradiol and ▶progesterone, secreted by the ovary and by the placenta during pregnancy. Steroid hormones are lipid molecules derived from a common cholesterol precursor (cholestane, C27). Based on the distance between the site of hormone synthesis and secretion and the target tissue, their mechanism of action can be classified as ▶endocrine (distant

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Ovarian Stroma

target), ▶paracrine (neighboring cells), or ▶autocrine (same cell).

Ovarian Tumors During Childhood and Adolescence

▶Progestin

D OMINIK T. S CHNEIDER 1 , U LRICH G O¨ BEL 2 1

Ovarian Stroma

Clinic of Pediatrics, Klinikum Dortmund, Dortmund, Germany 2 Clinic of Pediatric Oncology, Hematology and Immunology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Synonyms Definition Solid inner part of the ovary consisting of various secretory cells. ▶Granulosa Cell Tumors

Dysgerminomas – Dysgerminoma, Embryonal carcinoma, Yolk sac tumor, Choriocarcinoma, Teratoma, mature/immature (cystic teratoma, syn.: dermoid cyst); Sex cord stromal tumors – Granulosa cell tumor, juvenile type, adult type, Sertoli-Leydig cell tumor (syn. gynandroblastoma), Sclerosing stromal cell tumor, Sex cord stromal tumor with annular tubules, Steroid tumor, Brenner tumor; Ovarian small cell carcinoma, hypercalcemic type; Cystadenoma; Ovarian carcinoma

Definition

Ovarian Surface Epithelium

Primary ovarian neoplasms derived from the germinative, stromal or epithelial component of the ovary during infancy, childhood and adolescence.

Definition The tissue covering the outer surface of the ovary, from which epithelial ovarian tumors arise. ▶Endometriosis

Ovarian Teratoma Definition Synonym dermoid cyst; most frequent ovarian germ cell tumor. Mature ovarian teratomata develop from postmeiotic germ cells and present as cystic tumors limited to the ovary with no metastatic spread. In contrast, immature teratomata may show considerable infiltration into neighboring organs and may be a component of mixed malignant ▶germ cell tumors. ▶Ovarian Tumors During Childhood and Adolescence ▶Teratoma

Characteristics Ovarian tumors of childhood and adolescence include a broad variety of clinically, histologically and biologically different entities. The epidemiological patterns differ significantly from those in adult patients. The classical epithelial ▶ovarian carcinoma of adults constitutes a rarity in childhood and adolescence. In contrast, ▶germ cell tumors (GCT) prevail in young patients and represent the most frequent ovarian tumor during childhood and adolescence. The histologic variability of ovarian tumors is related to the varying histogenesis of the different entities, because ovarian tumors may develop from all different histologic components of the ovary. Among the largest group of ovarian tumors, the GCTs, ovarian cystic ▶teratomas are distinguished from malignant GCTs, which may sometimes include mature or immature teratomatous components. Less frequent histologic entities during childhood and adolescence include ▶sex cord stromal tumors (SCST), ▶granulosa cell tumors, ovarian small cell carcinoma of the hypercalcemic type (OSCC) as well as epithelial tumors such as cystadenomas, borderline tumors and classical ovarian carcinomas (▶Ovarian cancer).


Epidemiology Among ovarian tumors of childhood and adolescence, GCTs constitute the largest group, with a peak incidence during adolescence and young adulthood. Ovarian tumors account for ~1% of all registered childhood cancers. In addition, an unknown number of unregistered ▶ovarian teratomas and rare ovarian tumors must be considered. In contrast to ▶testicular GCTs, which show a bimodal incidence distribution according to age with a first peak during infancy and a second during young adulthood, ovarian GCTs show their peak incidence during adolescence and young adulthood, with no distinct cohort during infancy. ▶OSCSTs represent the second largest groups of ovarian tumors during childhood and adolescence. During infancy, the almost exclusive histology is juvenile granulosa cell tumor. With the onset of puberty, ▶Sertoli-Leydig cell tumors become more prevalent. The other histologic subentities are too rare to allow for the evaluation of epidemiological patterns. Epithelial cancers such as OSCC and borderline lesions and the classical carcinoma are diagnosed almost exclusively during adolescence and are rarely reported to pediatric registries.

Histology and Biology Ovarian tumors may develop from any histologic component of the ovary. The histologic appearance of ▶GCTs is interpreted in the light of the holistic concept first proposed by Teilum. Accordingly, GCTs may show pure germ cell differentiation on one hand (▶dysgerminoma, in analogy to the seminoma and germinoma of the testis and CNS, respectively) or somatic differentiation on the other. Morphologically, dysgerminomas resemble immature germ cells and express immature stem cell markers such as c-kit, human placenta like alkaline phophatase and OCT3/4. They may develop in the presence of gonadoblastomas; thus indicating underlying disorders of gonadal development, e.g. related to Ullrich-Turner syndrome of testicular feminization with streak gonads. Among the non-seminomatous GCTs, immature embryonal structures may be observed in embryonal carcinomas (EC) that morphologically remind of the embryoid bodies of embryonal stem cell cultures. The distinct histologic subentities of nonseminomatous GCTs can be distinguished by clinical, histopathologic and immunohistochemic analysis. Embryonal carcinoma may be CD30 and OCT3/4 positive. In contrast, yolk sac tumors express alpha fetoprotein (AFP) and may stain positive for CD34, while choriocarcinoma is detected by staining for ß-human chorionic gonadotropin (ß-HCG). These two markers may be measured in

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the patient’s serum and may therefore be valuable for diagnosis and follow-up of such secreting GCTs. Teratomas show a higher degree of organoid differentiation with presence of mature or immature structures that may be derived from all three germ layers such as hair, squamous and mucous epithelium teeth, cartilage, bone, glial or thyroid tissue. Characteristically, such mature teratomas contain large tumors cysts and have therefore been named dermoid cyst. These tumors are biologically distinct in that they show an isodisomic chromosome count, related to their development from rather mature germ cells that have already undergone meiotic recombination. Immature teratomas present with varying components of immature tissue that mostly resembles immature neural tissue with neurotubular structures. In immature teratomas, the grade of immaturity can be determined according to the relative amount of immature tissue. In conventional cytogenetic analysis, differences between GCTs in children and in adults can be detected: The ▶isochromosome 12 p (designation: i(12p)), which is the result of a centromeric junction of two short arms of chromosome 12 and loss of the long arms, constitutes the pathognomic cytogenetic abnormality in adult GCTs and can be seen in more than 80% of tumors. The remaining “i(12p)-negative” GCTs show gain of regions from 12p, either as double minutes or homogeneously staining regions. In contrast, the i(12p) can not – or only rarely – be detected in GCTs that arise before the onset of puberty. During childhood, deletions (resulting in ▶LOH) of the short arm of chromosome 1 and the long arm of chromosome 6 can be found frequently. According to the unimodal age distribution of ovarian GCTs, no such childhood genetic pattern can be detected in ovarian GCTs of young girls, but the characteristic pattern of adult GCTs including i(12p) may be detected in girls as young as four years of age. GCTs may arise in the context of constitutional sex chromosomal abnormalities. Ovarian GCTs can be associated with Ullrich-Turner syndrome (45,X0), gonadal dysgenesis (Swyer Syndrome) and/or testicular feminization. Characteristically, these tumors arise in the presence of gonadoblastomas, which has therefore been proposed as the ovarian equivalent of the testicular intratubular neoplasia (TIN, syn. carcinoma in situ). Sex cord stromal tumors (SCSTs) may include granulosa-, Sertoli-, Leydig- and theka-cells as well as their respective immature progenitor cells and fibroblast that are derived from the specialized gonadal stroma Granulosa Cell Tumors. In the developing gonads, granulosa and Sertoli cells show a sex-specific differentiation. As a morphologic correlate of their hormonal activity, a positive immunohistochemical staining constitutes the diagnostic hallmark of SCSTs.

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SCSTs may develop in association with several defined hereditary disorders. In juvenile granulosa cell tumors, there is an association with multiple enchondromatosis, syn. Ollier disease. In addition, there is a pronounced association of ▶Peutz-Jeghers syndrome with sex cord tumors with annular tubules. These tumors usually develop at a younger age than in otherwise healthy patients, and they may develop bilaterally. In contrast, testicular large cell calcifying Sertoli Cell tumors can be found in boys with Peutz-Jeghers syndrome. Genetic analysis of sporadic juvenile ovarian granulosa cell tumors with ▶comparative genomic hybridization has not revealed frequent or characteristic chromosomal imbalances. The majority of tumors show balanced karyotypes, and in approx. 25% of patients chromosomal imbalances such as gain of the whole chromosome 12 can be found. The ovarian small cell carcinoma of the hypercalcemic type (OSCC) represents a rare ovarian neoplasm with enigmatic histogenesis that most commonly develops after the second decade of life. The histologic diagnosis is complicated by the pronounced morphologic similarity between JGCT and OSCC; however, the lack to demonstrate Inhibin by immunohistochemistry strongly argues for the diagnosis of OSCC, if additional characteristic morphologic features are present. The clinical presence of hypercalcaemia in approximately two thirds of patients does not substitute the histologic diagnosis, since a variety of other ovarian neoplasms such as clear cell carcinoma or serous carcinoma may also be associated with hypercalcemia. Clinically, OSCC are characterized by a usually very aggressive

clinical behavior associated with fatal outcome in the vast majority of patients. Adolescent girls may rarely present with large cystic serous tumors, histologically resembling either a simple or papillary cystadenoma. These tumors show a clinically benign behavior after complete resection, but serous adenocarcinoma, which represents the malignant form of papillary serous cystadenoma, must be distinguished histologically. In analogy, mucinous adenocarcinoma constitutes the malignant counterpart of the mostly huge but clinically benign mucinous cystadenomas. The majority of Brenner tumors are benign and occur as small solid and well-circumscribed nodules. Again, a malignant variant (malignant Brenner tumor, pure transitional cell carcinoma) has to be distinguished. Ovarian adenocarcinomas rarely develop during adolescents; however these tumors often take a malignant clinical course. Particularly in young patients and in patients with frank familial history, these tumors are associated with inherited BRCA mutations. CA125 can be used as tumor marker following tumor resection, but the vast majority of patients will require adjuvant chemotherapy. Clinical Diagnosis Ovarian tumors are commonly detected as large tumors which may have led to a visible swelling of the abdomen or fatigue. In some patients, tumor related torsion of the ovary or tumor rupture may result in severe acute abdominal pain and require emergency laparatomy. Yolk sac tumors secrete alpha1-fetoprotein (AFP) and choriocarcinoma human chorionic gonadotropin (HCG), which can be detected in the serum. In

Ovarian Tumors During Childhood and Adolescence. Table 1 correlated with histology Histology Germinoma Embryonal CA Yolk sac tumor Choriocarcinoma Teratoma SCSTs Ovarian small cell carcinoma, hypercalcemic type Cystadenoma a

Clinical behavior Malignant Malignant Malignant Malignant Benign (potentially malignant) Malignant Malignant (presumably fatal) Benign (potentially malignant)

Tumor markers in Serum

Sensitivity to

AFP

HCG

Inhibin

Ca++

Chemo

Irradiation Gy

− − +++ − −/(+)

(+) − − +++ −

− − − − −

− − − − −

+++ +++ +++ +++ >54b

24 45 45 45 >54b

−/(+)a −

− −

(+)b −

− ++

++ ++*

n.d.b n.d.b

−

−

−

−

−

−

Some poorly differentiated Sertoli-Leydig cell tumors may secrete AFP. Under evaluation. Abbreviations: n.d., no data.

b

Clinical and biologic characteristic of ovarian tumors


combination with characteristic radiographic findings such as calcifications in mixed tumors with teratomatous elements, these markers can be indicative of malignant GCTs, after other rare diagnoses that may be associated with elevated tumor markers have been excluded. Pure embryonal carcinomas and teratomas are usually not associated with specific serum tumor markers. In ~20% of dysgerminomas, serum levels of placenta like alkaline phosphatase may be elevated. Syncytiotrophoblastic cells in germinoma may produce as well human chorionic gonadotropin (ß-HCG), which therefore can also be elevated (▶Serum biomarkers). SCSTs often induce clinical symptoms that are related to the production of sex hormones by the tumor. Characteristically, infants and children present with signs of isosexual precocity, including breast enlargement, pubarche and vaginal bleeding. In (post-)pubertal girls, tumors may lead to primary or secondary amenorrhea and unspecific signs of virilization. As other steroid hormone producing cells SCSTs also produce Inhibin. Free Inhibin can be measured in the serum and may serve as a serological tumor marker during follow-up. In rare but well documented cases of poorly differentiated ovarian Sertoli-Leydig cell tumors, ▶AFP production has been reported, which may interfere with clinical diagnosis. Histologically, most of these tumors resemble SLCT with retiform, often hepatoid differentiation and heterologous elements. ▶CA125 constitutes a rather unspecific marker of ovarian tumors that may be elevated in different histologic entities. CA125 may provide useful information regarding the response to treatment and for followup in tumors that are otherwise tumor marker negative. Ovarian small cell carcinoma may present with significant hypercalcemia, which is not related to skeletal metastasis. However, hypercalcemia has also been reported in some patients with ovarian GCTs, SCSTs and cystadenoma or other epithelial tumors. The staging system of the World Health Organization and the International Federation of Gynecologic Oncology (▶FIGO) can be applied. Tumors confined to the ovary (stage I) are distinguished from tumors with locoregional spread in the small pelvis (stage II) and the abdominal cavity (stage III) and distant metastases (stage IV). Microscopic spread may have occurred even in stage I tumors, if malignant ascites is noted or if pre- or intraoperative violation of the tumor pseudocapsule occurred (stage Ic). Tumors with no microscopic spread on complete staging are categorized as stage Ia (unilateral tumor) or stage Ib (bilateral tumors). Peritoneal gliomatosis constitutes a specific phenomenon in teratomas and mixed malignant GCTs that refers to glial nodules in the peritoneal surface. Although gliomatosis imposes like wide-spread metastases, it represents a reactive and non-neoplastic disorder so that upstaging is inadequate.

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Surgical Therapy All ovarian tumors require surgical resection. In most patients, a primary tumor resection constitutes the standard approach and will result in complete tumor resection, because most tumors are encapsulated. In metastatic tumors with peritoneal spread, an up-front chemotherapy followed by delayed tumor resection is recommended as it may significantly facilitate complete tumor resection. Therefore, diagnosis must be made on the basis of imaging and tumor markers, and only in tumor marker negative tumors, an initial biopsy will be required. A median ▶laparatomy is considered the classical standard approach to ovarian tumors in childhood and adolescence, as it will allow for preparation and exstirpation of the tumor in one piece. With the modern advances in laparascopy, more patients will undergo laparoscopic surgery. However, the safety of this approach has not been prospectively evaluated in ovarian tumors of childhood. By no means, tumors should be punctured or resected in separate pieces in order to facilitate removal through the laparoscope, because this would not comply with the oncological criteria of complete resection and may thus be associated with an increased risk of relapse. For the same reasons, organ sparing surgery (e.g. enucleation of a cystic tumor) should be avoided, and might only be reserved to those rare patients with bilateral tumors, which are additionally treated with consolidating chemotherapy. Tumor resection may be restricted to ovarectomy, if surgical staging indicates a stage I tumor. Only in stage II or III tumors, unilateral adnectomy is indicated. In general, hysterectomy is considered unnecessary. Surgical staging limited to resection of suspicious lymph nodes only does not adversely affect outcome in the presence of cisplatin containing combination chemotherapy. Thus, routine sampling of unsuspicious nodes is not recommended. In non-metastatic tumors, omentectomy and appendectomy are not required for oncological reasons, but in specific situations may be performed on the basis of individual judging by the surgeon. Last, resection of glial nodules in gliomatosis peritonei is not generally recommended due to the non-malignant nature of this disorder. However, resection of large nodules may become necessary if they result in local complications such as mechanic ileus. Adjuvant Therapy After resection, a choice regarding the indication for adjuvant therapy has to be made. ▶Adjuvant therapy may be chosen from a broad and risk-stratified panel of chemotherapeutic strategies, which may include two to four cycles of a two- or three-agent chemotherapy

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as well as strategies with locoregional or systemic treatment intensification. The adequate choice of treatment does not only require the individual skill and experience but must always consider a careful clinical and histopathologic assessment, preferably by a central reference pathologist. Therefore, all children and adolescents with ovarian GCTs should be enrolled into cooperative protocols, which will provide the treating physician with the required infrastructure for diagnosis and risk-assessment. While previously, adjuvant chemotherapy has been widely used even in completely resected stage I tumors, recent experience has shown that a careful watch-andwait strategy may be justified in stage I tumors. In analogy to testicular GCTs, it is advisable to administer adjuvant chemotherapy to those patients with histological evidence of vessel-invasion. A watch-and-wait strategy must always include a careful and close followup schedule, which specifically includes the assessment of the paraaortal lymph nodes, the most frequent site of relapse. In addition, the patients should be informed that there is a general risk of relapse under a watch-and-wait strategy that is ~20–30%. However, with delayed chemotherapy, there is an excellent salvage rate, so that the overall survival exceeds 90%. Nevertheless, although ▶chemotherapy can be avoided in two thirds of patients, those who relapse will have to bear a higher therapeutic burden. Adjuvant therapy generally consists of platin-based combination chemotherapy. Previously, irradiation has also been administered in dysgerminomas, however, compared to chemotherapy, it will result in infertility of the contralateral ovary without better cure rates. Therefore, irradiation is reserved to salvage treatment only. Current modern platin-based chemotherapy usually consists of a three-agent combination including ▶cisplatin (100 mg/m2/cycle) and ▶etoposide (300–500 mg/m2/cycle) in combination with ▶bleomycin (15–30 U/m2/cycle) or ifosfamide (7500 mg/m2/cycle) ▶platinum drugs. The comparably effective ▶vinblastin is less commonly used because of vascular complications, in particular in combination with bleomycin. Cisplatin can be substituted by ▶carboplatin (600 mg/ m2/cycle) with similar cure rates. However, carboplatin at lower doses (400 mg/m2/cycle) results in unacceptably high relapse rates. In general, carboplatin is assumed to have a better toxicity profile than cisplatin with lower nephro- and ototoxicity but higher hematotoxicity. Intensification of cisplatin with 200 mg/m2/ cycle) results in significant ototoxicity, however also yields a survival advantage in high risk patients. The other drug consistently administered in GCTs is ▶etoposide. In general, etoposide is associated with an increased risk of secondary malignancies, in particular acute myelogeneous leukemia. However, at the doses usually administered for GCTs and in the absence of

concomitant radiotherapy, the risk is acceptably low and does not exceed 1%. In the different national protocols, different choices of the third drug have been made. In the UK and the USA, platin-compounds and etoposide are combined with bleomycin, thus resembling the traditional ▶BEP chemotherapy for testicular GCTs. In contrast, the national groups in France, Brazil and Germany are administering ifosfamide. The toxicity profiles of these two drugs differ significantly. Bleomycin in combination with cisplatin may result in pneumopathy, which in rare patients may be lethal. In contrast, ifosfamide has a more pronounced hematotoxicity and may result in chronic renal tubulopathy. As for both drugs, the risk is highest in neonates and infants, both have not been administered in this age group. Thus, the two-agent regimen has been extended to a larger group of patients with a moderate risk profile, with promising treatment response. Some tumors may show an only insufficient response to conventional three agent chemotherapy and should therefore be selected for treatment intensification. In tumors with locoregional (i.e. peritoneal) spread, treatment intensification may be achieved by combination of chemotherapy with locoregional hyperthermia ▶Hyperthermia. Metastatic tumors however may be selected for dose intensified chemotherapeutic strategies (▶Platinum-refractory testicular germ cell tumors). In contrast to the more frequent GCTs, the experience regarding the adjuvant chemotherapy in the rare tumor entities such as SCSTs and OSCCs is much more limited and awaits prospective evaluation. The overall prognosis of SCSTs is favorable and cure rates exceed 80%. Patients with stage I tumors are followed expectantly, while patients with peritoneal spread are selected for adjuvant chemotherapy (e.g. cisplatin, etoposide, ifosfamide). Patients with stage Ic tumors and malignant ascites of preoperative tumor rupture should also be selected for chemotherapy, since the outcome of this specific group was comparable to that of stage II and III tumors. Experience regarding the optimal treatment of OSCC is extremely small and mostly derived from retrospective clinico-pathological surveys and single case reports, indicating for an extremely aggressive natural course of this disease with an almost invariable fatal outcome. Regardless of stage, all patients with ▶ovarian small cell carcinoma, hypercalcemic type require multi-agent chemotherapy during first-line treatment. High-dose chemotherapy can be used to consolidate the therapeutic success. The guidelines for the treatment of other epithelial tumors such as cystadenoma and adenocarcinomas follow those for the corresponding tumors in adults. If necessary, a combination of carboplatin and ▶taxol is considered the current standard chemotherapy ▶ovarian cancer.

Oxidative DNA Damage

References 1. Billmire D, Vinocur C, Rescorla F et al. (2004) Outcome and staging evaluation in malignant germ cell tumors of the ovary in children and adolescents: an intergroup study. J Pediatr Surg 39:424–429 2. Distelmaier F, Calaminus G, Harms D et al. (2006) Ovarian small cell carcinoma of the hypercalcemic type in children and adolescents: a prognostically unfavorable but curable disease. Cancer 107:2298–2306 3. Göbel U, Schneider DT, Calaminus G et al. (2000) Germcell tumors in childhood and adolescence. Ann Oncol 11:263–271 4. Schneider DT, Calaminus G, Wessalowski R et al. (2003) Ovarian sex cord-stromal tumors in children and adolescents. J Clin Oncol 21:2357–2363 5. Young RH, Oliva E, Scully RE (1994) Small cell carcinoma of the ovary, hypercalcemic type. A clinicopathological analysis of 150 cases. Am J Surg Pathol 18:1102–1116

Overexpression Definition Gene overexpression refers to the increased expression of a gene. This can occur either endogenously by regulotary pathways, by genomic ▶amplification of a gene or exogenously by experimentally introducing DNA-constructs (plasmids) encoding the respective gene into cells.

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Oxidation Definition The chemical process involving removal of electrons. If dithiols are oxidized with two electrons, they may form disulfides. ▶Thioredoxin System ▶Reactive Oxygen Species

Oxidative DNA Damage A NDREW R. C OLLINS Department of Nutrition, University of Oslo, Oslo, Norway

Definition Represents free radical damage to DNA. Oxidation essentially involves the addition of oxygen or removal of hydrogen atoms from a molecule. Oxidation of DNA may simply result in a small change to one of the bases, or a deoxyribose in the backbone of the molecule may be altered to such an extent that the continuity of the backbone is broken. Single-strand breaks are more common than double-strand breaks.

Characteristics

Ovulation Definition The process by which an ovum is released from within the ovary in a cyclical manner. The fluid-filled follicle containing the ovum distends the surface of the ovary until it breaks down, and the ovum is released. ▶Endometriosis

Oxaliplatin Definition Chemotherapeutic agent; platinum-containing; causes DNA disruption. ▶Peripheral Neuropathy

DNA is thought of as a very stable molecule and yet it readily undergoes damage, by a variety of agents that can be either endogenous or exogenous in origin. Ionizing radiation (e.g. X-rays), ▶ultraviolet radiation and various chemicals, including some present in tobacco smoke (▶Tobacco carcinogenesis), cause the release of free radicals, and if DNA is not protected, oxidative damage can occur. Free radicals (▶Reactive oxygen species) also occur within the cells of the body, arising as a minor product during the cycle of oxidation of carbohydrates in the mitochondria. The hydroxyl radical, *OH, is particularly reactive with DNA. In addition to single- and double-strand breaks, many different oxidation products of the four bases (▶Adducts to DNA) have been identified in DNA treated with radiation or other free radical-generating chemicals. Some of these modified bases are potentially capable of giving rise to a ▶mutation. For instance, ▶8-oxoguanine, if present in the DNA when it is replicating, may lead to the incorporation of adenine rather than cytosine into the newly synthesized complementary strand, thus changing the DNA sequence. Some oxidation is detectable in the DNA of normal human cells. It is usually measured by gas

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chromatography (▶GC-MS) or high performance liquid chromatography (▶HPLC). Normally for GCMS, the DNA is acid-hydrolyzed to bases (guanine etc.), while for HPLC the DNA is enzymically hydrolyzed to nucleosides (deoxyguanosine (dG) etc.). Both of these methods have given relatively high values for the extent of conversion of guanine to 8-oxoguanine, with up to 300 or more 8-oxo-guanines for every 106 unaltered guanines. However, recently it has been recognized that guanine tends to oxidize during preparation of samples for analysis and so the early estimates of 8-oxoguanine are considered to be excessive. Values as low as 3 per 106 have been reported from HPLC analysis of anaerobically prepared samples.

Oxidative DNA Damage. Figure 1 The comet assay. Cells are embedded in agarose on a microscope slide, lysed, and electrophoresed at high pH. This view is of the DNA from one cell, stained with DAPI and visualized by fluorescence microscopy. The percentage of DNA fluorescence in the tail of the “comet” is proportional to the frequency of breaks – such as breaks introduced at 8-oxoguanine sites by the enzyme FPG.

There is another approach to measuring oxidized bases using bacterial repair endonucleases, which recognize the damage and make a corresponding break in the DNA. The enzyme FPG (formamidopyrimidine DNA glycosylase) recognizes 8-oxoguanine. DNA breaks can then be measured in various ways, including the ▶comet assay (Fig. 1). This approach gives values for 8-oxoguanine that are even lower, with around 0.5 per 106 guanines. The extent of background DNA oxidation in normal cells remains an important question. Methodological problems must be solved before a consensus can be reached.

Mechanisms Measurement is made of the steady state level of DNA damage, which is a dynamic equilibrium between input of damage and its repair (Fig. 2). In the case of oxidative damage, input is controlled by ▶antioxidant defenses. The tripeptide glutathione is present at high concentration in the nucleus and “mops up” free radicals before they can cause damage. Superoxide dismutase and catalase are enzymes that convert superoxide and hydrogen peroxide (two reactive forms of oxygen) ultimately to non-harmful products. Other enzymes combine various organic free radicals with glutathione, thus inactivating them. Fruits, vegetables and grains in the diet are a source of ▶antioxidants, including ▶vitamin C, ▶vitamin E, ▶carotenoids and flavonoids. These ▶natural products have the ability to quench or scavenge free radicals; whether they act as antioxidants in vivo depends on whether they are taken up from the gut in sufficient amounts, and has been the subject of recent human intervention trials. In general, it is possible to detect a significant decrease in the steady state level of base oxidation (and/or an increased resistance to in vitro oxidation of DNA) in white blood cells of volunteers taking individual antioxidant

Oxidative DNA Damage. Figure 2 DNA damage: a steady state. There is a constant inflow of damage, caused by free radical attack and attenuated by antioxidants. This is normally balanced by cellular DNA repair processes, which remove the damage and restore the DNA sequence. Little is known about what modulates repair activity.

Oxidative Phosphorylation

supplements or antioxidant-rich foods, ranging from fried onions to kiwifruit. However effective the antioxidant defenses, some DNA oxidation does occur. The turnover of this damage is achieved by ▶repair of DNA. Small base damage, which includes base oxidation, is repaired primarily by base excision repair. Here, the damaged base is removed, followed by the base-less sugar-phosphate residue and perhaps a few neighboring nucleotides. A small repair patch of new nucleotide(s) is inserted and ligated. As well as being present in cellular DNA, 8-oxodG is detectable in urine as free nucleoside. The idea that urinary 8-oxodG represents the accumulated product of DNA repair in all the cells of the body is attractive but flawed; base excision repair releases the base not the nucleoside. Even if this 8-oxodG originates in the oxidation of broken down DNA from dead cells passing through the kidneys, it still reflects ▶oxidative stress, and it has given useful information about oxidative stress related to exercise, smoking and nutrition. Most impressively, consumption of 300 g a day of brussels sprouts led to a decrease of 28% in urinary 8-oxodG concentration. Clinical Aspects It is commonly stated that oxidative DNA damage is a significant cause of cancer, and that fruits and vegetables protect against cancer because the antioxidants they contain decrease the amount of base oxidation in the cellular DNA. However, there is little evidence that this is true. In two large scale human intervention trials, smokers and/or ▶asbestos workers were given β-carotene (a carotenoid antioxidant) daily for several years. The ▶lung cancer incidence was actually higher in these subjects compared with those taking placebo (or other supplement). Other intervention trials have shown no beneficial or harmful effect of supplementation with antioxidant micronutrients in terms of cancer risk – in spite of their ability to decrease oxidative damage. In an experimental animal system, a high level of oxidative DNA damage is not necessarily marked by an elevated cancer risk. In a ▶knockout mouse model, which is defective in the murine equivalent of FPG, there is a slight increase in the steady state level of 8-oxoguanine, but no increase in cancer incidence. It seems that there is a back-up repair pathway that deals (more slowly, but adequately) with oxidative damage. Oxidative stress is a feature of many other diseases, including heart disease, diabetes, cataract, and rheumatoid and arthritic conditions. It may be a cause of the clinical condition, or an effect. A common theory of ageing argues that the accumulation of free radical-induced damage to biomolecules – lipids, proteins and nucleic

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acids – is responsible for the general cellular dysfunction and deterioration of body processes in later life, but the evidence for accumulation of oxidative DNA damage is weak. The importance of fruits and vegetables in a healthy diet is not in doubt. But it is clear that antioxidants are not their only feature, and we should be looking at other effects that phytochemicals might have on metabolism to account for their capacity to prevent disease.

References 1. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–714 2. Collins AR (1999) Oxidative DNA damage, antioxidants, and cancer. BioEssays 21:238–246 3. ESCODD, Gedik CM, Collins A (2005) Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J 19:82–84 4. Collins AR (2005) Antioxidant intervention as a route to cancer prevention. Eur J Cancer 41:1923–1930

Oxidative Metabolism Definition Consists of replacing a carbon hydrogen bond in a drug by a carbon oxygen bond or the breaking of a carbon nitrogen bond (N-dealkylation). ▶ADMET Screen

Oxidative Phosphorylation Definition Biochemical reactions in the mitochondria that generate ATP. Is an oxygen-dependent process by which aerobic cells obtain energy (ATP) in the mitochondria. In this process, electrons from NADH and FADH2 are passed along the electron-transport chain to oxygen. This electron transport generates an electrochemical proton gradient across the inner mitochondrial membrane that is used by ATP synthase to phosphorylate ADP and form ATP. ▶Hydrogen Peroxide

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Oxidative Stress

Oxidative Stress J E´ ROˆ ME A LEXANDRE , C AROLE N ICCO, F REDERIC B ATTEUX

Faculté de Médecine Paris – Descartes, UPRES 18-33, Groupe Hospitalier Cochin – Saint Vincent de Paul, Paris, France

Definition Is caused by an imbalance between the production of reactive oxygen and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. It is defined as a disturbance in the prooxidant–▶antioxidant balance in favor of the former leading to potential damage. Oxidative stress is believed to contribute to the development of several diseases, including cancer.Corresponding author.

Characteristics The complex of oxidative stress involves a number of cell-chemical elements: ▶Free radicals include any species that contain one or more unpaired electrons, generating electrons singly occupying an atomic or molecular orbital. The presence of unpaired electrons confers a considerable degree of reactivity upon a free radical. Dioxygen is the main source of intracellular free radicals. ▶Reactive oxygen species (ROS) is a collective term that includes both oxygen free radicals such as • superoxide anion (O•− 2 ) and hydroxyl radical (OH ) and other reactive molecules as ▶hydrogen peroxide (H2O2). H2O2 is not a free radical but is also reactive and easily converted into radicals. ROS are physiological byproducts of normal aerobic metabolism. 90% of intracellular superoxide anion comes from the mitochondrial electron transport chain. Other significant sources of superoxide anion include ▶NADPH oxidase (NOX), ▶xanthine oxidase, and NADPH-▶cytochrome P450 reductase. NOX are a family of multimeric membrane oxidases tightly regulated by various stimuli including growth factors. Superoxide anion is considered as the “primary” ROS. The chemical reactivity of superoxide anion is weak in aqueous medium. Moreover, its negative electronic charge makes it unable to diffuse through lipid membranes. That’s why superoxide anion by itself has little cellular effects. The conversion of O•− 2 into H2O2 is catalyzed by enzymes of the ▶superoxide dismutase (SOD) family. Three isoforms of SOD have been identified in humans: CuZn-SOD, which is located in the cytosol, Mn-SOD, located in the mitochondria, and extracellular (EC)SOD. H2O2 is able to go trough cellular membranes

and can diffuse relatively far from its production origin. At physiological concentrations, H2O2 reacts mainly with sulfydril radicals, inducing reversible alterations of cysteines. Thus, H2O2 is able to specifically alter activity of proteins containing cysteine residues in their enzymatic sites, especially tyrosine phosphatases. The hydroxyl radicals are formed through the Fenton or Haber-Weiss reaction that converts O•− 2 and H2O2 into OH• in the presence of Fe2+ or Cu2+. OH• is extremely reactive and induces severe cellular oxidative damages as DNA alterations, being thus highly mutagenic. It leads to polyunsaturated fatty acids peroxidation, increasing cellular membrane permeability. OH• is also able to react with proteins, inducing irreversible inactivation. Several antioxidant enzymes can detoxify the whole cascade of ROS and protect normal cells from the potential damages implicated by oxidative stress. SOD catalyses the conversion of O•− 2 into H2O2, which is further detoxified by catalase or by enzymes of the glutathione peroxidase family using reduced glutathione. This complex enzymatic antioxidant system is completed by exogenous compounds such as arginine, vitamins A, C, E, β-carotene, glutathione, polyphenols, and minerals (selenium and zinc). Oxidative Stress is Involved in Oncogenesis There are several evidences that ROS play a key role in the oncogenesis process. In vitro exposure to chronic oxidative stress is associated with oncogenic ▶transformation and tumor growth. Overexpression of NOX1 (the catalytic subunit of NOX) stimulates the generation of O2− and induces the transformation of mouse ▶NIH 3T3 cells. Patients carrying specific polymorphisms on GSH-peroxidase 1 gene associated with decreased antioxidant activity present an increased risk of lung and breast cancers. An excessive production or a defective detoxification of ROS may favor the promotion and the development of cancer during chronic infection or ▶inflammation which is considered to cause one third of world cancers. The tumoral transformation induced by ROS, especially by OH•, is at least in part consecutive to their ▶mutagenic effects. They can act directly on DNA structures leading to single- or double-strand breaks, to point and frame-shift mutations, and to chromosome abnormalities. ROS can also promote proliferation and survival of cancer cells and tumor angiogenesis by means of ▶epigenetic effects. By altering activity of several tyrosine phosphatases, including ▶PTEN and ▶MAPK phosphatases, H2O2 activates mitogenic and survival pathways. This molecule has also been reported to stimulate ▶VEGF production by tumor cells. Increased Oxidative Stress in Cancer Cells Under basal conditions of culture, various cancer cell lines produce more O•− 2 and H2O2 than nontransformed

Oxidative Stress

cells. The presence of oxidative modifications of DNA in primary human cancer cells has also been demonstrated. The oxidative stress observed in cancer cells mostly results from the increase production of superoxide anion. The overexpression of ▶Myc oncogene has been associated with increased production of superoxide by mitochondrial respiratory chain, while ▶RAS oncogene mutations induce NOX activation. A decrease activity of antioxidant enzymes could also contribute to oxidative stress since tumor cells, compared with normal cells, often express lower levels of catalase, glutathione peroxidase, reductase and their respective cofactors. The overproduction of ROS in cancer cells contributes to the genetic instability, leading to nuclear and mitochondrial DNA mutations and more aggressive phenotype. Thus, oxidative stress is part of a positive amplification loop where ROS induce malignant transformation and transformation is itself associated with more ROS production. Therapeutic Perspectives of Oxidative Stress Modulation Differential Effects of Oxidative Stress in Normal and Cancer Cells Growing evidence suggests that ROS can induce a widetype of cellular responses from proliferation to senescence and cell death. Adding increasing amounts of exogenous H2O2 or increasing its intracellular levels by overexpression of SOD leads to a dose-dependent decrease in proliferation and tumor cell death. H2O2 can stimulate proapoptotic signal molecules such as apoptosis signal regulating kinase 1, c-Jun-NH2-kinase, and p38; activation of the p53 protein pathway; startup of the mitochondrial apoptotic cascade. When adding ROS to cells culture media, differential cellular effects have been observed in normal and cancer cells. In normal cells, persistent ROS production leads to activation of the ▶JNK pathway and apoptosis, whereas low concentrations or transient high levels of ROS induce the proliferation of those cells, through the activation of the ERK pathway. In sharp contrast, similar expositions to ROS cause tumor cell growth arrest and apoptotic death because of their high basal level of ROS that is close to the threshold of cytotoxicity. Thus, the fate of tumor cells exposed to oxidative stress is tightly correlated with the duration of ROS stimulation and depends on the basal redox status of the cell. Use of Antioxidant Agents in Cancer Patients Clinical data regarding the effects of antioxidant molecules in cancer patients have been controversial. Because oxidative stress induced DNA damages and is involved in malignant transformation, the hypothesis was made that antioxidant agents could have preventive effect against cancer. Numerous clinical trials were performed using various antioxidants (N-acetylcysteine (NAC), β-carotene, vitamin A, vitamin C, vitamin E, …),

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but failed to show any protective effect. Furthermore, a doubt has been cast on the possibility that some of these molecules, especially NAC might trigger tumor growth. These results should be viewed in light of the biological effects of antioxidants in cancer cells. Indeed, we previously observed that NAC, which is endowed with catalase and glutathione-like activities, decreases intracellular H2O2 concentration and increases the proliferation of tumor cells in vitro and in vivo, probably because tumor cells are submitted to detrimental oxidative stress. Antioxidant molecules probably play a protective role against cancer in healthy individuals by preventing DNA damages linked to the oxidative stress. However, once cancer cells have emerged, a cancer-promoting effect could result from the administration of agents that decrease intracellular H2O2 level. Therefore, antioxidant should be used with caution in cancer patients. Anticancer Agents Increase Oxidative Stress in Cancer Cells The most commonly used anticancer agents, such as ▶cisplatin, ▶adriamycin, ▶fluorouracil, ▶irinotecan, or ▶paclitaxel, are able to induce ROS production in cancer cells. Hydrogen peroxide and superoxide accumulation are observed within few hours of drug exposure and occur before the commitment of the cells into apoptosis. Moreover, antioxidants such as NAC and catalase are able to decrease their cytotoxicity. These results strongly suggest that oxidative stress is involved in the cytotoxicity of most anticancer agents. However, the underlining mechanisms seem to differ from an agent to the other. For example, ▶5-FU and ▶anthracyclins induce ROS production by a p53dependent pathway involving the activation of several mitochondrial oxidoreductases such as proline oxoreductase and ferrodoxine reductase. On the other hand, membrane NOX activation is induced by ▶paclitaxel and ▶arsenic trioxide. Tumor cells have higher levels of ROS than normal cells and could therefore be more sensitive to the additional oxidative stress generated by anticancer agents. This hypothesis offers a new explanation to the observation that anticancer agents are usually more toxic to cancer cells than to normal cells. Finally, cellular antioxidant enzymes may influence the sensitivity of tumor cells to anticancer agents. Thus, high levels of reduced glutathione, the cofactor of glutathione peroxidase, in tumor cells have been associated with a ▶multidrug resistance phenotype. A correlation was found between resistance of breast cancers to docetaxel and the overexpression of several genes controlling the cellular redox environment. Using Oxidative Stress Modulators as Anticancer Agents The fundamental differences between normal and tumor cells in terms of responses to H2O2 overproduction

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provide new possibilities in the treatment of cancer, taking into account the tumor cell susceptibility to ROS-induced apoptosis. Overexpression of SOD in cancer cell lines induces an increase in H2O2 production and reduces tumor growth. However, the SODs are polypeptides of high molecular weight that are not able to cross the cellular membranes and therefore have a limited interest for clinical use. Several nonpeptidyl SOD mimics with lower molecular weight have been developed, such as (Cu(II)(3,5-diisopropylsalicylic acid)2) (CuDIPS), Mn(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP), or mangafodipir. The SOD mimics increase the concentration of H2O2, resulting in the abrogation of tumor cells proliferation in vitro. Similar results have been observed in vivo. Increasing the Therapeutic Index of Anticancer Agents by SOD Mimics Several reports have suggested that compounds that increase intracellular hydrogen peroxide concentration could enhance the activity of anticancer agents. For example, buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, can increase the cytotoxicity of melphalan by inhibiting glutathione peroxidase activity and increasing H2O2 level. Similarly, we previously showed that the antitumor activity of oxaliplatin and paclitaxel is enhanced by SOD mimics. A potential limitation to the clinical development of such compound is that they could also increase the toxicity of anticancer agents on normal cells. As an example, BSO depletes glutathione in both normal and cancer cells, increasing melphalan’s hematologic toxicity and thus abrogating any enhancement of the therapeutic index of this anticancer agent. Mangafodipir has SOD-, catalase- and glutathione reductase-like properties, allowing it to act at multiple steps of the ROS cascade. We showed that mangafodipir protects mice treated with paclitaxel from developing leucopenia and also amplifies the antitumoral effect of paclitaxel on implanted tumor, increasing its therapeutic index. These opposite effects of mangafodipir in normal and cancer cells may be related to the differences in the redox status of these cells, as described above. Clearly, oxidative stress modulators, especially SOD mimics, warrant further development in anticancer treatment.

4. Laurent A, Nicco C, Chereau C et al. (2005) Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res 65:948–956 5. Nicco C, Laurent A, Chereau C et al. (2005) Differential modulation of normal and tumor cell proliferation by reactive oxygen species. Biomed Pharmacother 59:169–174

Oxidized Definition The product of a compound that has gone through a reaction involving an ▶oxidation. ▶Thioredoxin System

Oxidized Phospholipids Definition Are formed by a non-enzymatic (radical attack) breakdown of polyunsaturated fatty acids (arachidonic, linoleic) leading to phospholipids with short (only few carbons) oxidized acyl chains at the sn-2 position of glycerol. ▶Lipid Mediators

Oxidoreductase Definition An enzyme having the capacity to catalyze reactions involving reduction or oxidation, in the case of thioredoxin (Trx) or TrxR usually converting disulfide motifs into dithiol motifs. ▶Thioredoxin System

References 1. Alexandre J, Nicco C, Chereau C et al. (2006) Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir. J Natl Cancer Inst 98:236–244 2. Benhar M, Engelberg D, Levitzki A (2002) ROS, stressactivated kinases and stress signaling in cancer. EMBO Rep 3:420–425 3. Halliwell B, Gutteridge J (1999) Free Radicals in Biology and Medicine. New York: Oxford University Press

8-Oxoguanine Definition Is one of many products of the oxidation of DNA. It differs from guanine by replacement of a hydrogen

Oxygenation of Tumors

atom with a hydroxyl group, 8-oxodeoxyguanosine occurs when combined with deoxyribose. ▶Oxidative DNA Damage

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the microvessels to the cells and the ▶respiration rate (O2 consumption rate) of the parenchymal and stromal cells making up the tissue. O2 supply is mainly influenced by the efficacy of blood flow, diffusional O2 flux and blood hemoglobin (Hb) level.

Characteristics

Oxygen Free Radicals Definition Reactive Oxygen Species (ROS).

Oxygen Partial Pressure Distribution in Tumor ▶Oxygenation of Tumors

Oxygen Tension Distribution in Tumor ▶Oxygenation of Tumors

Oxygenation of Tumors P ETER VAUPEL Institute of Physiology and Pathophysiology, University of Mainz, Mainz, Germany

Synonyms Oxygen partial pressure distribution in tumor; Oxygen tension distribution in tumor; Oxygenation status and tumor oxygen level

Definition Tumor oxygenation, which reflects the distribution of oxygen (O2) partial pressures (pO2 values) or O2 concentrations, results from O2 availability (O2 supply) to the tumor tissue, the ▶diffusional flux of O2 from

Whereas in normal tissues the O2 supply meets the metabolic demands, in many solid tumors the respiration rate may outweigh an insufficient O2 supply and result in the development of tissue areas with very low O2 levels (▶hypoxia, O2 depleted tissue areas) or even areas completely lacking O2 (▶anoxia). Considerable evidence demonstrates that in most human tumors oxygenation is compromised as compared to normal tissues, which are characterized by “normal” O2 partial pressure distributions (▶normoxia). Oxygenation is extremely heterogeneous within an individual tumor (intra-tumor heterogeneity). Furthermore, considerable heterogeneity of oxygen-depleted areas (hypoxic areas) has been shown between tumors of the same clinical size, stage, grade or histological type (inter-tumor heterogeneity). Tumor oxygen supply is not regulated according to the metabolic demand, as is the case in the physiological situation. On average, the median pO2 values in tumors are substantially lower than in normal tissues at the site of tumor growth (Fig. 1). Tumor oxygenation is independent of clinical size, stage, grade, histology and various oncologic parameters or patient demographics. In some cancer entities, the oxygenation status significantly deteriorates in ▶anemia (as a result of a decreased O2 capacity of the blood) and at Hb concentrations above the median Hb level (most probably due to rheological problems within the chaotic tumor microvessels). Metastatic lesions seem to have an oxygenation status comparable to that of the primaries, whereas local recurrences have a poorer oxygenation status than the primary tumors. Pathomechanisms of Tumor Hypoxia Tumor hypoxia results from an inadequate O2 delivery to the respiring neoplastic as well as stromal cells. Limited and even abolished O2 supply is due to severe structural and functional abnormalities of the ▶microcirculation, as well as due to a deterioration of diffusion geometry. In addition, cancer-related and/or therapyinduced anemia and carboxyhemoglobin formation (in heavy smokers) can lead to a reduced O2 content of the blood. As a result, areas with very low (down to zero) O2 partial pressures exist in solid tumors. These very low pO2 microregions are heterogeneously distributed within the tumor mass and may be located next to regions with pO2 values corresponding to the normal tissue from which the tumor has been derived. In

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Oxygenation of Tumors. Figure 1 Oxygenation status of normal tissues (left panels) and solid tumors (right panels). Frequency distributions of measured pO2 values (pO2 histograms) for normal breast tissue and uterine cervix are compared with the respective cancer tissues (clinical stages Ib – IV). Oxygen partial pressures (pO2) in the tissue were measured with a computerized polarographic microsensor technique which enables direct assessment of the pO2 data with an O2 sensitive needle-electrode. Oxygen partial pressures were measured along several electrode tracks in each individual tumor. Pooled data presented here are derived from pre-treatment measurements in conscious patients.

addition, significant temporal variations in the oxygenation status have been observed in tumors. Methods of Measurement Assessment of the tumor oxygenation status in experimental tumors and in the clinical setting has been performed using various techniques. So far, the most direct and often used method for description of oxygenation is the polarographic measurement of O2 partial pressures. With this invasive microtechnique, frequency distributions (▶histograms) of measured intratumor pO2 values can be obtained with a relatively high spatial resolution. Other direct procedures include fiber-optic O2 sensors and electron paramagnetic resonance oximetry. This latter technique is minimally invasive requiring only application of the paramagnetic material. Measurement of intravascular oxyhemoglobin (HbO2) saturation is another method that has the potential to allow

a characterization of the oxygenation status of human tumors. However, it only provides information related to the vascular compartment and thus the situation in the extravascular space can only be inferred (e.g., BOLD-MRI). Tumor oxygenation can be measured in tomographic images in the clinical setting upon inhalation of radiolabeled 15O-gas in ▶positron emission tomography (PET) studies. However, as with magnetic resonance imaging (MRI) procedures, limitations include poor quantification and sparse spatial resolution. The parameter measured with these non-invasive techniques is not directly interpretable as a tumor pO2 value or O2 concentration. Non-invasive methods for detection of tumor hypoxia include the binding and the retaining of radiolabeled ▶bioreductive drugs, such as fluoromisonidazole (labeled with 18F and detected by positron emission


tomography) and iodoazomycin-arabinoside (labeled with 123I and detected with single photon emission computerized tomography, SPECT). Several techniques for assessment of tumor oxygenation require the analysis of tumor biopsy specimens. Using immunohistochemistry with exogenous hypoxia markers (such as misonidazole, pimonidazole, etanidazole or nitroimidazole-theophylline), detailed information concerning hypoxia at the cellular level can be obtained. Disadvantages include the need for injection of a hypoxia marker and possible sampling errors. The role of HIF-1α, GLUT-1 or CA IX as endogenous markers of tumor hypoxia is questionable. Clinical Relevance Tumor hypoxia has been considered to be a therapeutic problem since it renders solid tumors resistant to sparsely ionizing radiation (X- and γ-rays), some forms of chemotherapy (e.g., cyclophosphamide, carboplatin) and photodynamic therapy. Oxygen levels may furthermore influence a series of biological parameters, which in turn may markedly increase the malignant potential of a tumor irrespective of tumor treatment modalities. Hypoxia is a common characteristic of locally advanced solid tumors that has been associated with malignant ▶progression, that is, an increasing probability of recurrence, locoregional spread, and distant ▶metastasis. Emerging evidence indicates that the effect of hypoxia on malignant progression is mediated by a series of hypoxia-induced proteomic (C). Functionally evaluation showed that nonsynonymous polymorphisms (2677G>T, A, or C) at amino acid position 893 (Ala>Ser, Thr, or Pro)

have a great impact on both the activity and the substrate specificity of ABCB1. The polymorphisms of 2677G>T (893 Ala>Ser) is reportedly associated with high risk of lung cancer. ABCC1 (MRP1/GS-X pump) and ABCC2 (MRP2/cMOAT) Human ABCC1 (MRP1) gene is located on chromosome 16p13.1 and spans at least 200 kb consisting of 31 exons. The ABCC1 gene encodes a 1531 amino acid protein which has a molecular weight of 190 kDa in its mature glycosylated form. In the promoter region of the ABCC1 gene, there are a number of putative transcription factor motifs, such as the activator proteins AP1 and AP2 (Sp1), glucocorticoid response element (GRE), and also estrogen response element (ERE) and cAMP response element (CRE). Human ABCC2 (MRP2 or cMOAT) is a 1545 amino acid protein whose exon gene is located in chromosomal region 10q23–24. Although there is limited sequence similarity between ABCC1 and ABCC2 (49%), the primary structure and membrane topology of the two proteins are similar. In addition, the two transporters also have similar substrate characteristics. Both ABCC1 and ABCC2 transport a wide range of organic anions, including glutathione disulfide (GSSG), glutathione-metal complexes, glutathione conjugates, as well as glucuronate and sulfate conjugates. Elevated expression of ABCC1 mRNA and/or protein levels have been observed in many multidrug resistant cancer cells. Transfection of ABCC1 cDNA in cultured cells resulted in enhanced resistance to many cytotoxic agents including doxorubicin, vincristine and VP-16. ATP-dependent transport of these

Pharmacogenomics in Multidrug Resistance

anticancer drugs can be enhanced by the presence of ▶glutathione (GSH) in membrane vesicles prepared from ABCC1-overexpressing cells, suggesting that ABCC1 co-transports anticancer drugs and GSH. In patients, human colorectal cancers frequently overexpress ABCC1 and ▶γ-glutamylcystein synthetase (γ-GCS), a rate-limiting enzyme of GSH biosynthesis, as compared to the surrounding normal tissue. The frequency of ABCC1 expression in carcinoma was higher than that in adenoma (pA) leads to the replacement of the negatively charged glutamic acid residue with a positively charged lysine residue. This polymorphism affects the ATP-binding domain, between the ▶Walker A motif (amino acid residues 83–89) and the signature region (amino acid residues 186–189). The Q141K variant was also detected in all ethnic groups tested: the allele frequency ranged between 0% and 35%, (the Africans in North of Sahara, the Africans sub-Saharan, and African-American subjects with low; the Japanese and Chinese populations with high allele frequencies). The SNP (Q141K) was postulated to cause increased sensitivity of normal cells to anticancer agents that are ABCG2 substrates such as topotecan, diflomotecan, and SN-38. Perspectives Drug transporters as well as drug-metabolism play pivotal roles in determining the pharmacokinetic profiles of drugs and, by extension, their overall pharmacological effects. There are an increasing number of reports addressing genetic polymorphisms of drug transporters. Information is still limited, however, regarding the functional impact of genetic polymorphisms in drug transporter genes. Detailed functional analysis in vitro is critically important to provide clear insight into the biochemical and therapeutic significance of genetic polymorphisms. Functional validation of SNPs and

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Pharmacogenomics in Multidrug Resistance. Figure 2 Nonsynonymous polymorphisms of human ABCG2. Schematic illustration of the structure of ABCG2 protein and the locations of amino acid changes. R482G and R482T are acquired mutations.

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their linkage with clinical data would provide a new approach to individualized pharmacotherapy in the 21st century.

Pharmacokinetics and Pharmacodynamics in Drug Development

References 1. Borst P, Elferink RO (2002) Mammalian ABC transporters in health and disease. Annu Rev Biochem 71:537–592 2. Ishikawa T (2003) Multidrug resistance: Genetics of ABC transporters. In: Cooper D.N. (ed.) Nature encyclopedia of the human genome Nature Publishing Group London pp.154–160 3. Kalow W, Meyer UA, Tyndale RF (eds) (2001) Pharmacogenomics, Marcel Dekker, Inc. New York 4. Sakurai A, Tamura A, Onishi Y et al. (2005) Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications. Expert Opinion on Pharmacotherapy 6:2455–2473

Pharmacogenotype

S IMON PACEY, D EBASHIS S ARKER , PAUL WORKMAN Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Surrey, UK

Definition Pharmacokinetics ▶Pharmacokinetics (PK) is the study of how a drug is absorbed, distributed, metabolized, and excreted over time. Pharmacodynamics ▶Pharmacodynamics (PD) is the study of how a drug affects its target(s) in a dose- and time-dependent fashion.

Characteristics Definition Is the influence of genotype on the therapeutic and toxic responses to a drug. ▶Personalized Cancer Medicine

Pharmacokinetic Profile Definition The study of the fate of a drug in the body as a function of time, in terms of absorption, metabolism, and excretion. ▶Liposomal Chemotherapy

Pharmacokinetics Definition PK; Is the study of how a drug is absorbed, distributed, metabolised, and excreted over time. ▶Pharmacokinetics/Pharmacodynamics

To maximize the chance of successful drug development, a comprehensive knowledge of the compound under investigation is required. Assuming the drug target has been correctly selected, drug efficacy requires delivery into the patient such that adequate drug concentration is achieved within the plasma and tumor (measured by PK studies) to effect target modulation (measured by PD studies) resulting in anticancer effect(s). These principles have been incorporated into a pharmacological audit trail consisting of a series of questions that should be addressed during drug design and development. These can be summarized as: . Is the drug target expressed in the tumor of interest? . Are adequate plasma and tumor drug levels achieved? . Does this level of drug exposure result in target modulation? . Can effects be demonstrated on the biochemical pathway downstream of the target? . Are the desired biological effects achieved, e.g., cell cycle arrest, induction of apoptosis or inhibition of angiogenesis? . What are the therapeutic consequences of drug exposure and target modulation? Related to these questions, it is valuable to determine if biomarkers of sensitivity or resistance can be identified. From these questions it is obvious that PK and PD studies form an important part of the knowledge base required for modern drug development. The advantage

Pharmacokinetics and Pharmacodynamics in Drug Development

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of using PK/PD biomarkers, and of implementing the pharmacological audit trail, is that decisions in drug development can be made on a rational basis, therefore making the process more effective. Pharmacokinetics It is usual to assume that the therapeutic and toxic effects of a drug are related to its concentration at the site of action. Previously it was estimated that around 40% of drugs failed clinical development due to poor PK properties. Inappropriate PK can lead to inadequate or variable drug exposure, and hence to lack of therapeutic activity or undesirable toxicity. This figure has fallen to around one in ten, due mainly to more thorough preclinical PK modeling. Early in drug discovery, physicochemical properties need to be addressed, in order to maximize the chance of the final molecule possessing appropriate drug-like properties. The Lipinski rule of five provides a useful guide. This is based on the fact that most marketed drugs have a molecular weight of less than 500 Da, partition coefficient cLog P < 5 (a measure of lipophilicity), T mutation in VHL gene that reduces but does not abolish HIF-alpha activity. The VHL mutation diminishes HIF-alpha degradation by pathologically upregulating HIF-alpha target genes, including Epo. CP is characterized by congenital erythrocytosis, but has yet to be extensively phenotyped. The CP is a nonbenign hematological disease associated with lower peripheral blood pressures, higher estimated pulmonary artery pressures, varicose veins, vertebral hemangiomas, arterial and venous thrombosis, major bleeding episodes, cerebral vascular events and premature mortality due in part to cardiovascular and thrombotic events. Moreover, erythroid progenitors of CP patients are hypersensitive to Epo by a molecular mechanism totally obscure. This last property is, however, reminiscent of primary hereditary polycythemias, thus rendering, at least in part, uncertain the classification of CP as a primary or a secondary erythrocytosis. Because CP is characterized by a germline mutation in the VHL gene, it has been hypothesized that homozygotes for this mutation might develop certain vascular tumors similar to those associated with the classic VHL syndrome. However, in no case, spinocerebellar hemangioblastomas, renal carcinomas, and pheochromocytomas typical of classical VHL tumor predisposition syndrome have been found, and no increased risk of cancer has been demonstrated. Through experiments conducted on CP patients, it has been demonstrated the potential role of the VHL pathway in cardiopulmonary physiology. In particular, patients with CP have an elevated basal ventilation and pulmonary vascular tone, with extremely high ventilatory, pulmonary vasoconstrictive, and heart rate responses to acute hypoxia. The abnormalities they displayed mimicked those caused by acclimatization to hypoxia at high altitude. It has been estimated that the VHLC598T mutation arose in a single ancestor between 12,000 and 51,000 years ago. It is possible that the wide dissemination from the original founder may be associated with some survival advantages for heterozygotes carrying this mutation. Such an advantage might be related to a subtle improvement of iron metabolism, erythropoiesis, embryonic development, energy metabolism or some other yet unknown effect. An intriguing possibility is raised by the recent demonstration of a protective role for HIF-alpha in protecting against pre-eclampsia, the leading cause of maternal and fetal mortality

worldwide. Another positive role of a mildly augmented hypoxic response is an improvement in the bactericidal action of neutrophils, as recently observed in HIF1alpha knock-in mice. Finally, other VHL mutations have been detected in either homozygotes or compound heterozygotes affected by secondary congenital polycythemias. It is tentative to classify these cases and Chuvash erythrocytosis as VHL-dependent polycythemias. Although several genetic alterations responsible for secondary polycythemias have been identified, more than 50% of these erythrocytoses with normal or increased serum levels of Epo (and other cytokines), do not have a definite molecular basis. These cases include both diseases inherited with a recessive or a dominant fashion. Secondary polycythemias due to acquired conditions include a large array of causes, all resulting in low peripheral pressure of oxygen. Among these, high altitude dwelling, chronic obstructive pulmonary disease, sleep apnea, cyanotic heart disease result in secondary polycythemia as a physiological adaptation. In addition, kidney transplantation has polycythemia as a complication. The cause of this acquired secondary erythocytosis might be correlated to specific pharmacological treatments required during the post-transplantation period.

References 1. Khan J, Wei JS, Ringnér M et al. (2001) Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat Med 7: 673–679 2. Gordeuk VR, Stockton DW, Prchal JT (2005) Congenital polycythemias/erythrocytoses. Haematologica 90:109–116 3. Tefferi A (2006) Classification, Diagnosis and Management of Myeloproliferative Disorders in the JAK2V617F Era. Hematology Am Soc Hematol Educ Program 240–245 4. James C, Ugo V, Le Couedic JP et al. (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434:1144–1148 5. Ang SO, Chen H, Hirota K et al. (2002) Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia. Nat Genet 32:614–621

Polycythemia Vera Definition PV, synonym primary polycythemia, a condition in which there is an overproduction of red blood cells in the body as a result of an abnormality of the bone marrow. ▶Erythropoietin

Polyphenols

Polygenic Definition A mode of inheritance characterized by a sporadic occurrence and variable intensity of manifestation of the phenotype. It is due to the segregation in the same individual of susceptibility/resistance alleles at multiple unlinked genetic loci. ▶Modifier Loci

Polygenic Diseases Definition Are caused by a combination of environmental factors and mutations in multiple genes; each mutation has only a small contribution to the disease susceptibility. These include most of the common chronic diseases like obesity, hypertension, Alzheimer, diabetes, arthritis, and many cancers.

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1%. The major histocompatibility complex is (▶MHC) the most polymorphic gene cluster known in humans. ▶Linkage Disequilibrium ▶Personalized Cancer Medicine ▶Pharmacogenomics ▶MDM Genes

Polymorphonuclear Leukocyte Elastase ▶Neutrophil Elastase

Polyneuropathy Definition Malfunction of multiple peripheral nerves. ▶Peripheral Neuropathy

▶Case–Control Association Study

Polypeptide Polymers

P

Definition Definition Are high molecular weight molecules consisting of a repeating chain of identical base units (called monomers) connected by covalent chemical bonds. Cationic non-biodegradable polymers enhance the internalization of therapeutic molecules by cells. Biodegradable polymers are used to achieve the controlled release of therapeutic molecules.

A chain of amino acids linked by peptide bonds, in the range of 10–100 amino acids; proteins are composed of much longer sequences of amino acids and their functions are determined additionally by the 3-dimensional structure. ▶Gut Peptides ▶Modular Transporters

▶Non-viral Vector for Cancer Therapy

Polyphenols Polymorphism

E LVIRA

DE

M EJIA

Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL, USA

Definition Literally means existing in a variety of different shapes. Genetic polymorphism is variability at a gene locus in which the variants occur at a frequency of greater than

Definition Polyphenols are plant substances possessing more than one aromatic ring bearing one or more hydroxyl

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Polyphenols

groups, including their ▶functional derivatives, and may occur as unconjugated aglycones or as conjugated with sugars, organic acids, amino acids or lipids. Examples of polyphenols are ▶epigallocatechingallate, ▶genistein, ▶resveratrol, quercetin, rutin. Common dietary sources rich in polyphenols include tea, soybean, berries, chocolate, wine, apple and orange juices, black beans, tomato, sweet peppers, broccoli, onion.

Characteristics Polyphenols consist of a family of diverse compounds, which comprises chalcones (butein), dihydrochalcones (tephropurpurin), flavanones (naringenin), flavones (apigenin), dihydroflavonols (engeletin), flavonols (quercetin), flavanols (catechins), ▶isoflavones (genistein), proanthocyanidins (propelargonidins), and anthocyanidins (delphinidin). Their main physiological function is as antioxidants. Therefore, a long-term consumption of a diet rich in plant foods containing polyphenols may offer some protection against chronic diseases, including ▶cancer. Thus fruits, vegetables, tea, red wine and cocoa consumption have been suggested to have the capacity to reduce cancer development. ▶Flavonoids may also exert other effects unrelated to their ▶antioxidant capacity, for example, ▶anti-inflammatory effects and inhibition of tumorigenesis. Cancer has been associated with ▶oxidative stress and mechanisms involving inflammation, aberrant signaling pathways and ▶gap junction intercellular communication. It is possible that the presence in the diet of compounds with the capacity to ▶scavenge free radicals like polyphenols may play a role in oncogenesis. While the free radical scavenging and antioxidant properties of phenolics are well established, emerging literature reports suggest that their chemopreventive effects may also be attributed to their ability to modulate components of cell signaling pathways. Different polyphenols have different degrees of absorption in humans, however, it is believed that isoflavones are the best absorbed even though this parameter is influenced by the matrix of the diet and enhanced by a high fat diet. Studies in humans and animals have indicated that some polyphenols can be absorbed in the small intestine (5–10%), and most of them enter the circulation as methyl, sulfate and glucuronide conjugates; of these, only a very small amount (5–10%) enter the plasma as unchanged plant polyphenols. The 90–95% ingested total polyphenols are fermented in the colon and a variable portion of these (5–50%) is absorbed mainly as conjugates of microbial metabolites. It is clear that the major part of polyphenols consumed never reach the plasma and systemic circulation so the tissues most exposed are those of the oro-gastrointestinal tract. Animal studies have shown that diets rich in polyphenols that reach the colon may protect rodents from carcinogenesis.

Inside the human body, flavonoids themselves are of little or no direct antioxidant value; however, inducing phase II enzymes can help in the elimination of mutagens and ▶carcinogens and may be of value in cancer prevention. Polyphenols could also induce mechanisms that help kill cancer cells and inhibit tumor invasion.

Cellular and Molecular Studies The biological mechanisms related to the chemopreventive activities of polyphenols are believed to occur by the regulation of signaling pathways such as ▶nuclear factorkappa B, ▶activator protein-1 or mitogen-activated ▶protein kinases. By modulating cell signaling pathways, polyphenols activate cell death signals and induce ▶apoptosis in precancerous or malignant cells resulting in the inhibition of cancer development or ▶progression. However, regulation of cell signaling pathways by dietary polyphenols can also lead to cell proliferation/survival or inflammatory responses due to increased expression of several genes. Dietary polyphenols can exert their effects on these pathways separately or sequentially and in addition the occurrence of crosstalk between these pathways can also take place. Polyphenols can also behave as detoxifying enzyme inducers, modulating gene expression including induction of phase II enzymes, such as glutathione S-transferases and quinone reductase, which usually leads to protection of cells/tissues against exogenous and/or endogenous carcinogenic intermediates. Phase II gene inducers also activate ▶MAPK kinases that are involved in the transcription activation of antioxidant response element-mediated reporter gene. Genistein, an isoflavonoid with ▶phytoestrogenic properties, in animal models has shown to have a very complex effect on carcinogen-induced mammary cancer and great care is required in extrapolation of this information to human ▶breast cancer. Some unconjugated isoflavones from fermented soybean and ▶tamoxifen promoted an additive reduction in the number of mammary tumors in rats. Isoflavonoids have biphasic effects on the proliferation of breast cancer in culture; genistein at low concentrations can stimulate the growth of estrogen receptor-positive breast cancer cells but it does not stimulate the growth of ▶estrogen receptornegative breast cancer cells. Phytoestrogen responsive genes characterized from these cells can be used to clarify the role of isoflavones in cancer prevention. Of course many other mechanisms of action have been suggested for isoflavones and in particular for genistein. An important aspect of cancer risk is the involvement of the inflammatory response thus soy isoflavones may have potentially protective benefits at sites of inflammation due to their antioxidant action and could contribute to anticancer ability because ▶reactive oxygen species could initiate signal transduction through the ▶mitogen activated protein kinases.

Polyunsaturated Fatty Acid

Clinical Studies Human studies are still contradictory and not final conclusions can be drawn on the effect of polyphenols and cancer. Flavonoids may be capable of exerting antioxidant effects in humans with the possibility of direct radical scavenging, down regulation of radical production, elimination of radical precursors such as hydrogen peroxide, metal chelation, inhibition of ▶xanthine oxidase and of course elevation of endogenous antioxidants. Dietary polyphenols as health promoting dietary antioxidants have a broader mechanism of action than simple radical scavenging and radical suppression. Studies with pre and postmenopausal women administered beverages and supplements with isoflavones gave no conclusive results on breast cancer risk factors and more studies are needed to clarify the effect of isoflavones and breast cancer in women. In cultures where the intake of soy is high and consequently dietary isoflavones, breast fed infants are exposed to high levels without adverse effects and it has been observed that early exposure may even protect against cancer. Safety and efficacy of isoflavones in humans is a topic that needs further investigation. While experimental models have suggested that flavonoids attenuated cancer risk, epidemiological studies have failed to demonstrate a clear effect for tea, although there is moderate evidence for a slightly positive or no effect of black tea consumption on colorectal cancer. Studies on cancer have been limited by sample sizes and insufficient control of confounder factors. In addition, caution must be exerted when polyphenols bypass the gastrointestinal tract (intravenous injections) or mega doses of these compounds (purified and presented in the form of tablets or capsules) are taken due to possible adverse effects such as nephrotoxicity. The available evidence for tea polyphenols tentatively supports their advancement into phase III clinical intervention trials aimed at the prevention of progression of prostate intraepithelial neoplasia, ▶leukoplakia or premalignant cervical disease. In the case of curcumin and soya isoflavones more studies in premalignacies seem appropriate to optimize the nature and design of suitable phase III trials. There is insufficient evidence from human research as yet to claim benefits of polyphenols in relation to cancer prevention. Epidemiological data that suggest tea consumption contributes to cancer prevention do exist, however, these failed to differentiate between green, black or oolong tea. Studies of colorectal cancer suggested either a slightly positive effect or null effect. In conclusion, further mechanistic insights are needed as well as an accurate knowledge of the concentrations of the chemopreventive agents and their metabolites occurring in humans. Only small amounts

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of flavonoids may be necessary to see medical benefits. In terms of safety, consistent with the expectation that dietary constituents are harmless and well tolerated, unexpected cases of severe toxicity associated with the consumption of polyphenols have been rare. Severe adverse effects have only been reported for a very limited number of cases when consuming daily gram amounts of green tea polyphenols in the form of green tea extracts or when administering high doses of quercetin intravenously in cancer patients.

References 1. Gardner EJ, Ruxton CH, Leeds AR (2007) Black tea – helpful or harmful? A review of the evidence. Eur J Clin Nutr 61:3–18 2. Thomasset SC, Berry DP, Garcea G et al. (2007) Dietary polyphenolic phytochemicals – promising cancer chemopreventive agents in humans? A review of their clinical properties. Int J Cancer 120(3):451–458 3. Fresco P, Borges F, Diniz C et al. (2006) New insights on the anticancer properties of dietary polyphenols. Med Res Rev 26:747–766 4. World Cancer Research Fund American Institute for Cancer Research Food, Nutrition, Physical Activity, and the Prevention of Cancer: A Global Perspective. Washington D.C. AICR, 2007

Polyubiquitination Definition Is a process in which a chain of at least four ubiquitin peptides are attached to a lysine residue on a protein substrate, resulting in the degradation of the protein via proteasome. ▶Ubiquitiuation

Polyunsaturated Fatty Acid Definition PUFA; An unsaturated fatty acid whose carbon chain contains more than one double bond; found chiefly in fish, corn, soybean oil and safflower oil. ▶Melatonin ▶Lipid Peroxidation ▶Fatty Acid Transport

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Population-based Cancer Research

Population-based Cancer Research ▶Cancer Epidemiology

Positional Cloning Definition Cytogenetic and molecular approach to identifying a disease gene based on an identified abnormality in sequence or structure in a specific region of the genome.

Population Candidate Gene Association Study Positive Nodes ▶Case Control Association Study

Definition

▶Lymph Node Metastasis.

Porfimer Sodium Definition

Positive Predictive Value

▶Photofrin

Definition The positive predictive value of a test indicates the proportion of target condition patients that test positive.

Porphyrin

▶Molecular Pathology

Definition Porphyrins (uroporphyrin I and III, coproporphyrkn I and III, protoporphyrin IX, and heme) are colored compounds within the heme biosynthetic pathway and mainly consist of four pyrrole rings with conjugated double bonds. Derivatives of porphyrins are used as photosensitizers in photodynamic therapy and ▶fluorescence diagnostics. ▶Photodynamic Therapy ▶Diagnosis

Positron Emission Tomography A NDREAS K. B UCK , M ARKUS S CHWAIGER Department of Nuclear Medicine, Technical University of Munich, Munich, Germany

Synonyms Hybrid positron emission tomography/computed tomography; Integrated positron emission tomography/computed tomography; Small animal positron emission tomography

Portal Hypertension Definition Definition Portal venous pressure elevation resulting from intrahepatic or extrahepatic venous compression or occlusion. ▶Acites

Positron emission tomography (PET) is a non-invasive nuclear medical imaging modality enabling the visualization and quantification of biological processes. PET provides integral information regarding metabolic activity of the primary tumor and potential lymph node or distant organ metastases. PET can be used for cancer detection (▶early detection), ▶tumor staging and

Positron Emission Tomography

restaging, assessment of response to treatment and anticancer drug development.

Characteristics Principle of Positron Emission Tomography (PET) PET allows non-invasive assessment of the threedimensional distribution of a positron labeled compound within the living body. Positrons are anti-particles of electrons and originate from β+ decays of radioactive isotopes such as 11C, 13N, 15O, 18F, 68Ga, 86Y, or 124I. During β+ decay a positron and a neutrino are emitted, both sharing a certain amount of kinetic energy. Once the positron is slowed down, a positronium consisting of a positron and an electron is created. The positronium has a very short half-life of 10–10 s and the masses of the positron and the electron are finally transferred into energy. This annihilation results in two gamma quants with an energy of 511 keV each. Decay events are detected by coincidence registration enabling the measurement of activity distribution in a specific transaxial section of the body. Activity distribution can be calculated from respective projections after correction for scatter, attenuation, dead time and random coincidences. Attenuation correction can be performed using a radioactive transmission source rotating around the patient. Emission and transmission scanning from scull to mid thigh usually takes 30–45 min, whole body-scans 60–90 min. The radiation dose of a standard PET examination is low with approximately 7.4 mSv and similar to spiral CT of the thorax. Radiolabeled Biomarkers for PET Imaging Specifically Addressing Metabolic Pathways or Target Molecules Depending on the clinical situation, various radiolabeled pharmaceuticals can be utilized for tumor imaging (Table 1). The most important biomarker for functional diagnosis of tumors is the glucose analog 2′-[18F]-fluoro-2′-deoxy-D-glucose (FDG, 18FFluorodeoxyglucose) (Fig. 1). Since conventional imaging modalities such as ▶computed tomography (CT), ▶magnetic resonance imaging or ultrasound detect malignant lesions because of characteristic morphological alterations, FDG-PET enables the diagnosis of malignant tumors due to an increased glucose metabolism in malignant cells. After intravenous administration, FDG is predominantly taken up by tumor cells. After enzymatic conversion of FDG to FDG-6-monophosphate by hexokinase, the metabolite can not be further metabolized resulting in an intracellular “trapping” of FDG (▶metabolic trapping). There are many other radiopharmaceuticals capable of assessing distinct pathophysiological processes (Table 1). As an example, radiolabeled nucleoside analogs such as 3′-deoxy-3′-[18F]-fluorothymidine

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(▶FLT, 18F-fluorothymidine) can be used to noninvasively assess the proliferative activity of tumors. With the positron emitter 15O, H215O can be synthetized and used for assessment of tumor blood flow. A variety of radiolabeled amino acids such as [11C]-methionine (▶MET, 11C-methionine), [11C]-leucine (LEU) or [18F]-fluoro-ethyl-tyrosine (▶FET, 18F-fluoroethyltyrosine) can be used to evaluate transport rates of amino acids and/or protein biosynthesis. Imidazole derivatives such as [18F]-misonidazole (FMISO) can be used to delineate hypoxic tissue areas of the tumor which is particularly useful for radiation treatment planning. Synthesis of phospholipids is increased in many neoplasms leading to increased uptake of [18F]-choline and [11C]-choline (▶CHO, 11C-choline). [68Ga]-DOTATOC (▶DOTATOC, [68Ga]DOTATOC) specifically binds to somatostatin receptors and is therefore highly sensitive for detection of neuroendocrine tumors. [18F]-galactoRGD (▶RGD, 18F-Galacto-RGD) has a high affinity to the vitronectin receptor αvβ3 and can be used as potential surrogate marker of neoangiogenesis. These and many other radiopharmaceuticals specifically address metabolic pathways or bind to specific target structures and therefore enable molecular imaging of cancer. Specific radiotracers are especially helpful for evaluation of new drugs and early response assessment in cancer. Clinical Applications of PET and PET/CT The introduction of PET to clinical medicine has influenced the management of patients with cancer. In most industrialized countries, PET is now accepted as a both useful and economic diagnostic tool for characterization of indeterminate lesions, initial staging, restaging and assessment of response to therapy in a variety of cancers. Combination of a PET scanner with spiral computed tomography in a single examination (▶PET/CT, Integrated positron emission tomography/computed tomography) allows integrated functional (PET) and morphologic (CT) imaging. Additionally to the results returned by individual modalities, coregistration of CT allows precise localization of PET lesions. The addition of PET to CT leads to an increase of sensitivity as well as specificity for tumor imaging. Moreover, CT data can be used for attenuation correction which leads to a significant reduction of scanning time making PET/CT more comfortable for the patient. A standard examination including the head, thorax, abdomen and pelvis can be performed within 25–30 min. Since it’s introduction to clinical medicine in 2001, PET/CT represents the fastest growing imaging modality. The Centers of Medicare and Medicaid Services (CMS) approved a variety of clinical indications including staging and restaging of non-small cell ▶lung cancer, esophageal (▶esophageal cancer), colorectal (▶colorectal cancer), breast (▶breast cancer) and head and neck cancers (▶oral squamous cell

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Positron Emission Tomography. Table 1 unknown primary) Radiopharmaceutical (*under investigation) 18F-fluorodeoxyglucose (FDG)

11C-choline (CHO) 18F-fluorethylcholine (FEC) 18F-fluorocholine 11C-acetate

68Ga-DOTATOC 68Ga-DOTATATE 18F-DOPA

18F-fluoroethyl-tyrosine (FET) 11C-methionine (MET) 18F-galacto-RGD (RGD*)

18F-fluorothymidine (FLT*) 11C-thymidine (THY*) 18F-fluoroazamycine arabinoside (FAZA*) 18F-fluoromisonidazole (FMISO*) 11F-fluoro17-β-estradiol (FES*) 18F-fluoride

Native molecule Glucose

Radiopharmaceuticals (tracer) used for PET imaging (CUP, cancer of

Uptake mechanism in cancer

Clinical applications

Glucose transport/phosphorylation by hexokinase

Diagnosis, staging, restaging (e.g., cancer of the lung, breast, colon, rectum, thyroid, lymphoma melanoma, sarcoma); CUP monitoring of response to therapy (e.g., lymphoma, various cancer types including breast, GI tract, lung*) Choline Uptake by active transport and Staging, restaging (prostate cancer, phosphorylation by choline kinase; bladder cancer*); therapy incorporation into phospholipids monitoring (prostate cancer*, (cellular membrane) bladder cancer*) Acetate Lipid synthesis (key enzyme, fatty Staging, restaging, monitoring acid synthase, FASE) response to therapy in a variety of cancers Octreotide (soBinding to somatostatin receptors Diagnosis, staging, restaging of matostatin analog) (predominantly SSTR-2) neuroendocrine tumors, CUP (neuroendocrine) DihydroxyUptake in tumors capable of DOPA Diagnosis, staging, restaging of phenylanaline decarboxylation neuroendocrine and brain tumors, CUP (neuroendocrine) Tyrosine Amino acid transport, protein Diagnosis, staging, restaging of Methionine biosynthesis brain tumors; differentiation of scar/ recurrence, monitoring response in various tumor types Peptide containing Binding to integrin αvβ3 (vitronectin Assessment of tumor angiogenesis the sequence RGD receptor), expressed on activated (e.g., melanoma, sarcoma, head (arginine, glycine, endothelial cells and neck cancer, breast cancer), aspartate) monitoring response to antiangiogenic treatment Thymidine DNA synthesis, tumor cell Assessment of tumor proliferation proliferation (monitoring response to cytotoxic treatment in lymphoma, sarcoma, breast cancer) Hypoxia markers Passive diffusion into hypoxic cells; Assessment of tumor hypoxia (no biologic analog) reactive intermediates are formed (especially for use in tumors of the by intracellular nitroreductase and head and neck, radiation treatment trapped within the cell planning) Estradiol Binding to estrogen receptors Monitoring of response to antihormone treatment in breast cancer Fluoride Bone mineralization Screening for bone metastases at staging or restaging

carcinoma, oral cancer), ▶malignant lymphoma and ▶melanoma. Monitoring response to treatment in breast cancer is also covered. Just recently, the CMS announced to provide widespread coverage of PET when respective examinations are part of prospective clinical trials.

Differentiation of Benign from Malignant Tumors and Detection of the Primary Tumor (Cancer of Unknown Primary) Due to different glucose consumption of benign and malignant lesions, FDG-PET allows assessment of


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Positron Emission Tomography. Figure 1 The glucose analog FDG (2′-[18F]-fluoro-2′-deoxy-D-glucose) is the most widely used radiopharmaceutical for PET imaging. After i.v. injection, FDG is taken up by glucose transporters Glut-1 (cancer tissue, brain) and Glut-4 (skeletal muscle, heart) and is phosphorylated by hexokinase II. FDG-6-P is not a substrate for hexokinase phosphate isomerase which is the next enzyme of the glycolytic pathway. Consecutively, FDG accumulates in the cytoplasm (metabolic trapping). The figure shows the structure of the native glucose molecule (left) and the [18F]-labeled analog (right).

undefined tumors detected by conventional imaging modalities such as CT or MRI. Furthermore, PET sometimes allows detection of malignant lesions even when no or only minimal morphologic alterations are present. Regarding evaluation of indeterminate pulmonary nodules, prospective studies reported sensitivity values for FDG-PET between 89 and 100%, a specificity of 69–100% and an overall accuracy of 89–96%. FDG is not tumor specific leading also to nonspecific tracer accumulation in benign, predominantly inflammatory lesions. However, surgery may be circumvented in patients with increased perioperative risk if the PET scan is negative. Dynamic data acquisition can further enhance the accuracy of PET imaging. In malignant lesions, a continuous increase of glucose uptake has been described, whereas benign lesions showed an increase of FDG-uptake followed by rapid efflux of FDG. Dual time point imaging or delayed PET imaging after 1 and 2 h contributes to better differentiate between benign and malignant tumors. PET can also be used for detection of the malignant primary (▶cancer of unknown primary, CUP). PET is especially useful in detecting primary tumors in the head and neck region. In case of increased cancer biomarkers (▶clinical cancer biomarkers) or paraneoplastic syndromes, PET can aid in localizing the primary tumor manifestation site. Staging of Cancer, Prognostic Potential of PET For optimal treatment of patients with cancer, precise knowledge of the extent of the disease is crucial (tumor staging). If cancer is detected at a stage in which uncontrolled growth of tumor cells takes place but no tumor manifestations are present in distant organs, surgery is usually performed to obtain ultimate cure.

However, if the tumor has already spread to distant organs, cure can usually not be achieved by surgery alone (Fig. 2). In this situation, surgery has to be replaced or supported by systemic chemo- and/or radiotherapy to entirely destroy the primary tumor and metastatic sites or to induce growth arrest in the tumor. In this context, PET has several advantages compared to conventional imaging modalities. Small tumor manifestation sites such as ▶metastases in the bone, liver, lung, adrenal gland or in rare locations such as soft tissues, thyroid or (sub-) cutaneous lesions can be detected. However, micrometastases or single tumor cells can also not be detected with PET. Also, small lung metastases may appear negative at FDG-PET. In principal, staging of all tumors is possible. With the standard radiotracer FDG, PET is highly accurate for staging of non-small cell ▶lung cancer, thyroid cancer, tumors of the head and neck region (▶oral squamous cell carcinoma, ▶oral cancer), ▶colon cancer and ▶esophageal cancer, ▶malignant lymphoma, sarcoma (▶osteosarcoma, ▶Ewing sarcoma, ▶chondrosarcoma), and ▶melanoma. PET has been demonstrated to cause a change in patient management in 15–40% depending on the type of cancer. Some tumors present without increased glucose consumption such as prostate or neuroendocrine cancer. 11C-Choline PET and 11 C-Choline PET/CT have been demonstrated to be highly accurate for staging and especially restaging of prostate cancer. 68Ga-DOTATOC is a new PET-tracer for imaging neuroendocrine tumors. A variety of molecular probes have been evaluated to address biologic targets or metabolic pathways in vivo (Table 1). In the majority of these compounds, clinical utility remains to be determined. The most important prognostic factor (▶Prognosis, prognostic factor) is the tumor stage at initial presentation. However, risk stratification according to the

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Positron Emission Tomography. Figure 2 (a) FDG-PET/CT in beast cancer (maximum intensity projection); malignoma-associated intense FDG-uptake in primary breast cancer, lymph node metastases in the axilla, liver metastases and bone metastases (arrows). (b) Transaxial PET section showing intense FDG-uptake of the primary tumor. (c) Transaxial section of helical CT indicates the anatomic correlate of primary breast cancer. (d) Fused image. (e) Transaxial PET section, high FDG-uptake in a bone metastasis of the vertebral column. (f) Transaxial section of helical CT indicates a sclerotic lesion corresponding to the PET lesion. (g) Fused image. Intense physiologic uptake of FDG in the brain, the heart, and intestines (*).

TNM-system is also subject to error, because patients with limited disease undergoing definite therapy may also develop recurrent disease. Other factors such as tumor aggressiveness or metabolic activity of tumors may aid in individual risk assessment. Several studies have correlated the intensity of FDG-uptake in the primary tumor to progression free and overall survival in various cancers. In lung cancer, intensity of FDG-uptake turned out to be an independent prognostic marker. The prognostic potential of PET has also been described for colorectal cancer, breast cancer and malignant lymphoma. Assessment of Response to Therapy Therapeutic efficiency of chemo- and radiotherapeutic strategies varies significantly between individual patients. Therefore, non-invasive assessment of the performance of a therapeutic protocol in an individual patient is highly desirable. With conventional imaging modalities such as CT or MRI, response (▶radiological response criteria) to therapy can be detected as early as a reduction in tumor size occurs. On the contrary, PET

allows assessment of response to treatment at an earlier time point before tumor shrinking can be detected by conventional imaging. In responding tumors, metabolism of tumor cells is markedly decreased due to the cytotoxic effect of the respective therapeutic regimen. Concomitantly, accumulation of FDG is reduced. This is a sign of an efficient treatment and has a high prognostic value regarding the success of further treatment. In case of a non-responding tumor, the therapeutic regimen can be altered by changing the combination of cytotoxic drugs or the radiation dose. In ▶breast cancer, rapid decline of FDG-uptake already after one cycle of chemotherapy was demonstrated, whereas in non-responding tumors increasing or unchanged FDG-uptake was described. A variety of other neoplasms including ▶malignant lymphoma, ▶gastric cancer and ▶esophageal cancer, head and neck (▶oral squamous cell carcinoma, ▶oral cancer) or non-small cell ▶lung cancer showed rapid reduction of FDG-uptake in responding tumors. Significantly better disease-free and overall survival was described in responders compared to tumors without


significant reduction of tumoral FDG-uptake (Fig. 3). Clinical studies are needed reporting on the clinical benefit of a PET-guided change of patient management. Restaging of Cancer, Detection of Recurrence After definite surgery or chemo/radiotherapy, examinations and imaging at follow-up is important to early detect disease recurrence originating from residual tumor cells. In daily clinical practice, differentiation between scar tissue and vital tumor tissue is a frequent problem. At anatomically based imaging modalities, both are present as indeterminate tissue formation and, frequently, biopsy is needed for further clarification. Differentiation of scar tissue from vital tumor tissue is a prerequisite of PET imaging. While new onset of cancer tissue is associated with increased metabolism causing increased uptake of, e.g. FDG, scar tissue is frequently associated with reduced metabolism compared to surrounding normal tissue. PET is especially useful in the follow-up of tumor entities such as colorectal (▶colorectal cancer) and ▶esophageal cancer, non-small cell ▶lung cancer, ▶breast cancer, tumors of the head and neck (▶oral squamous cell carcinoma, oral cancer), brain tumors (▶malignant brain tumors), ▶melanoma and ▶malignant lymphoma. Restaging with PET is also approved for differentiated thyroid carcinoma with a negative 131I whole-body scan and elevated tumor marker thyroglobulin. Radiation Treatment Planning The use of metabolic information leads to biological target volumes which can have substantial impact on radiation treatment planning (▶radiation oncology) by increasing or reducing the target volume. The additional

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identification of tumor manifestation sites which are not visible at conventional staging causes an enlargement of respective target volume. On the other hand, the radiation field can be reduced when non-malignant lesions such as atelectatic tissue can be reliably characterized as benign (Fig. 4). Consecutively, radiation dose to surrounding normal tissue can be reduced. The use of PET for radiation treatment planning leads to a change of the target volume in up to 60% of patients. This is in part related to pretherapeutic detection of distant metastases, previously unknown metastases in locoregional lymph nodes or characterization of suspicious lesions as benign. However, PET-based radiotherapy planning is not trivial. Especially the delineation of the primary tumor is subject to a relevant interobserver variability. There is a need for standardized evaluation criteria of PET allowing also the quantification of metabolic changes. The recent introduction of PET/CT hybrid scanners has lead to a reduction of errors concerning image coregistration. In several prospective studies it was shown that overall survival of patients receiving PET-guided radiation therapy was significantly longer compared to patients receiving standard treatment. Prospective randomized studies have to be performed demonstrating that the use of PET positively affects patient outcome and overall survival. PET for Anticancer Drug Development PET imaging has unique properties for use in anticancer drug development. Therapeutic efficiency of a novel drug can be evaluated non-invasively by assessment of specific biologic endpoints such as changes in cellular proliferation (e.g., by the use of [18F]FLT), glucose utilization ([18F]FDG), tissue perfusion ([15O]H2O), metabolism of amino acids ([18F]FET, [11C]MET),

Positron Emission Tomography. Figure 3 (a) FDG-PET for response assessment in malignant lymphoma (maximum intensity projection), intense FDG-uptake of multiple lesions of high-grade non-Hodgkin’s B-cell lymphoma (arrows indicate lymphoma manifestations in the left supraclavicular region, in paraaortic, parailiac and inguinal lymph nodes. (b) Corresponding PET image of the same patient 3 weeks after completion of 8 cycles of chemotherapy with R-CHOP. No pathologic FDG-uptake in residual lymph nodes indicates complete remission of the disease and favorable outcome. (c–f) Corresponding transaxial sections of PET and CT.

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Post Surgical Systemic Therapy

Positron Emission Tomography. Figure 4 FDG-PET/CT for radiation treatment planning of nonsmall cell lung cancer. (a) Transaxial section of spiral CT shows a central tumor in the left lung which can not be discriminated from adjacent atelectatic tissue. (b) Corresponding section of FDG-PET indicates high metabolic activity of the malignant primary but anatomic landmarks are missing preventing precise tumor localization. (c) Fused PET/CT image allows exact delineation of the tumor which can be distinguished from adjacent atelectatic tissue.

or inhibition of ▶angiogenesis ([18F]galacto-RGD). (Over-) expression of the therapeutic target such as thymidylate synthase, VEGF receptor, ErbB2 or ▶estrogen receptor status can be quantified with [11C] thymidine, radiolabeled antibodies specifically binding to VEGF or ErbB2, or 18F-fluoro-17-β-estradiol, respectively. Assessing biologic endpoints further provides proof of principle of the proposed mechanism of action. PET can also be utilized for in vivo-evaluation of gene expression, e.g. by the use of the substrate [124I] fluoro-5-iodo-1-β-D-arabinofuranosyluracil (FIAU) for detection of Herpes simplex virus thymidine kinase type 1- or Na[124I] for detection of sodium iodide symporter expression. Generic endpoints can also be studied by PET. Drugs or biochemical probes can be labeled with positron emitters such as small molecules, proteins, or antibodies. Drugs which have been evaluated so far include 18 F-fluorouracil, 18F-tamoxifen or 13N-cisplatin. Pharmacokinetics of a drug can be investigated in tumors and normal tissues, in animal models or as part of clinical phase I (or phase II) studies. In the future, PET will be increasingly used to assess the efficiency of novel anticancer drugs.

References 1. Juweid ME, Cheson BD (2006) Positron-emission tomography and assessment of cancer therapy. N Engl J Med 354:496–507 2. von Schulthess GK, Steinert HC, Hany TF (2006) Integrated PET/CT: current applications and future directions. Radiology 238:405–422 3. Weber WA (2006) Positron emission tomography as an imaging biomarker. J Clin Oncol 24:3282–3292 4. Grosu AL, Molls M, Zimmermann FB et al. (2006) Highprecision radiation therapy with integrated biological imaging and tumor monitoring: evolution of the Munich concept and future research options. Strahlenther Onkol 182:361–368

5. Lardinois D, Weder W, Hany TF et al. (2003) Staging of non-small-cell lung cancer with integrated positronemission tomography and computed tomography. N Engl J Med 348:2500–2507

Post Surgical Systemic Therapy ▶Adjuvant Chemoendocrine Therapy

Postcode Prescribing Definition A consequence of local decision making within the United Kingdom’s National Health Service that results in patients having differential access to treatments according to their place of residence. ▶National Institute for Health and Clinical Excellence

Postirradiation Sarcoma ▶Radiation-Induced Sarcomas After Radiotherapy

pp60c-Src

Postischemic Reperfusion Definition The restoration of blood flow to an organ or tissue. After a heart attack, an immediate goal is to quickly open blocked arteries and reperfuse the heart muscles. ▶Amine Oxidases

Postlabeling

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subjected to reversible or permanent modifications. They are one of the later steps in protein biosyntheses for many proteins. To identify the specific sites of modifications in the analysis of enzymatically digested proteins, tandem mass spectrometry with collisioninduced dissociation of peptides can be used. More than 300 PTMs have been identified, such as phosphorylation, ▶glycosylation, ▶ubiquitination, deamidation, proteolytic processing, fatty acylation, and glycosylphosphatidylinositol lipid anchor attachment. Analysis of a protein for PTMs is very important for understanding issues such as activity, stability, interaction, and turnover. ▶Proteinchip ▶Histone Modification

Definition 32P-postlabeling is a sensitive method for the detection and quantification of adducts to DNA. DNA is extracted in microgram quantities from tissue samples and enzymatically digested to mononucleotides. These are labeled with radioactive phosphorus (32P) and the resulting 32P-labeled adducts are separated from normal nucleotides by two-directional thin layer chromatography. Quantification can proceed by autoradiography, and identification is possible by co-chromatography with authentic samples. ▶Biomarkers

Postnatal Stem Cells

Pou Transcription Factors Definition (Acronym for Pit, Oct and Unc); Pou transcription factors are a family of proteins with a conserved region of 150 amino acids that comprise a DNA binding/ regulatory region. This DNA binding domain recognizes the octamer ATGCAAAT in specific target genes for the purpose of regulating transcription during development. ▶Stem Cell Markers

▶Adult Stem Cells

Pox Viruses Postreplication Repair ▶DNA-Damage Tolerance

Definition Enveloped DNA viruses that can cause pox diseases in vertebrates. ▶Semaphorin

Posttranslational Modification Definition PTM is the chemical modifications of proteins following their translations. After syntheses, the proteins are

pp60c-Src ▶Src

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pp60v-Src

pp60v-Src ▶Src

ppm Definition Parts per million, ratio to determine the molecular presence of a particular substance per million parts in relation to others.

PPARg Definition Peroxisome proliferator-activated receptor-γ that is associated with cancer expression. ▶Conjugated Linolenic Acids ▶Fucoxanthin

PPARs Definition Peroxisome proliferator-activated receptors; A group of nuclear receptor isoforms that exist across biology. Originally identified in Xenopus frogs as receptors that induce the proliferation of peroxisomes in cells, they are intimately connected to cellular metabolism (carbohydrate, lipid, and protein) and cell differentiation. They function as transcription factors. PPAR-γ forms heterodimers with retinoid X receptors and is believed to be involved in adipocyte differentiation. ▶Arachidonic Acid Pathway ▶Fatty Acid Synthase ▶Peroxisome Proliferator-Activated Receptor

▶Benzene and Leukemia

PPNAD Definition Primary pigmented nodular adrenocortical disease.

pPNET Definition Peripheral primitive neuroectodermal tumor as opposed to the unrelated central ▶primitive neuroectodermal tumor (PNET) mainly comprised by ▶brain tumors; synonym for ▶Ewing sarcoma with at least two neural markers expressed on the cell surface or with presence of ▶Homer-Wright rossettes in the tumor.

PR Definition

▶Progestin receptor. ▶Adjuvant Chemoendocrine Therapy

ppb Definition Parts per billion, ratio to determine the molecular presence of a particular substance per billion parts in relation to others. ▶Benzene and Leukemia

pRb Definition

▶Retinoblastoma Protein; Is the product of a tumor suppressor gene, which is inactivated in ▶retinoblastoma

Pre-mRNA Splicing

and various other tumor types. pRb inhibits G1/S cell cycle progression by interacting with transcription factors, such as ▶E2F, to block transcription of growth regulating genes; ▶retinoblastoma protein, biological and clinical functions, retinoblastoma protein, cellular biochemistry.

Pre-invasive Breast Cancer ▶Ductal Carcinoma In Situ

Pre-mRNA Definition Precursor mRNA, an RNA that has not been processed and contains all of its introns and exons. ▶pre-mRNA Splicing

Pre-mRNA Splicing DAWN S. C HANDLER Department of Pediatrics, Columbus Children’s Research Institute, Center for Childhood Cancer, The Ohio State University School of Medicine, Columbus, OH, USA

Definition Splicing is a tightly regulated mechanism for the control of gene expression that involves the precise removal of ▶introns from the precursor mRNA molecule and the subsequent ligation of the remaining ▶exons.

Characteristics Premature RNAs that are transcribed within the cell nucleus contain both coding sequences (contained within functional units called exons) and non-coding sequences that are eventually excised. Splicing is the mechanism by which the non-coding portions of the RNA (also known as introns) are removed from

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▶pre-mRNAs, via two cleavage-ligation reactions, each involving transesterification at a splice site phosphate (see Fig. 1). Ultimately, the two exons are ligated to generate the spliced mRNA and the excised intron is released in a lariat configuration that is eventually degraded. The splicing reaction has been well-defined and is mediated by the dynamic ordered assembly of numerous ▶spliceosome components directly on the pre-mRNA. To avoid the production of nonfunctional proteins, it is essential that splicing occurs precisely and consistently as introns that are removed incorrectly or not at all can cause prematurely truncated proteins resulting from in frame stop-codons located in the intron or in proteins translated out of frame. Splicing is therefore, by necessity, an extremely accurate and specific reaction. The way in which regulation of splicing is achieved is a complex intersection of distinct recognition sequences for binding of the splicing machinery and accessory molecules with the proteins and ▶snRNP complexes that bind to these elements. Since exons are separated by introns and can be literally thousands of base-pairs away, the splicing machinery acts to recognize the splice sites and to remodel the RNA such that the splice sites are juxtaposed. In the first step of splicing the splicing components U2AF and U1snRNP bind to the 3′ and 5′ splice sites respectively. These proteins, along with accessory molecules known as ▶SR proteins that help to stabilize the reaction, form the splicing commitment complex. Differential splicing regulation can be achieved by the relative concentration of these accessory molecules in the nucleus and by the expression of proteins that enhance or inhibit the splicing reaction. Subsequent steps involve the binding of the U2snRNP to the branchpoint and the U4/U5/U6 trisnRNP complex remodeling the RNA and catalyzing the splicing reaction. Several systems have taken advantage of the precise nature of the splicing reaction and have implemented it as a mechanism for regulation of gene expression and function. Alternative RNA splicing is the process by which mRNAs encoding several distinct proteins are produced from one single pre-mRNA sequence by use of differential splice site choices. There is a large degree of diversity in the ways that cells use alternative splicing as a mechanism for gene regulation (see Fig. 2). Exons can encode discrete functional domains and thus exons that are differentially included may dramatically alter the protein function. The inclusion or exclusion of exons may also change the reading frame of the RNA and thus regulate protein function by affecting whether or not an RNA with an intact open reading frame is produced. Because of the versatility of splicing regulation alternative splicing is prevalent in several developmental processes including muscle development, neurogenesis, meiosis, and spermatogenesis.

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Pre-mRNA Splicing

Pre-mRNA Splicing. Figure 1 The catalytic steps of pre-mRNA splicing. The required elements of the pre-mRNA are shown schematically. The exons are depicted as boxes and the intron as a line. The conserved 3′ and 5′ splice site sequences are depicted along with the relevant phosphate groups (p). The polypyrimidine tract (Py)n and the branchpoint (A) are also shown. The dashed arrows signify the hydroxl group attack of the splice site phosphate. The first transesterification reaction produces two splicing intermediates, the free 5′ exon and the intron/3′ exon in a lariat configuration. The second transesterification reaction results in the ligation of the two exons and the release of the intron lariat that is degraded. The resultant ligated exons make up the mRNA that eventually gets transcribed into protein. Figure adapted from Ref [1].

However, disruption of the developmental regulation or alteration in appropriate RNA splicing can lead to incorrect expression of alternatively or aberrantly spliced isoforms and can lead to disease. Spliced Forms in Cancer For each of the alternative splicing patterns there exists multiple examples that exhibit cancer specific expression. An example of each is discussed below. Mutually exclusive splicing of exons (Fig. 2a) occurs such that only one of a group of adjacent exons is included in an RNA at any one time. This type of splicing regulation is common for control of RNAs that encode proteins with defined tissue-specific functions. In the case of the c-Jun amino-terminal kinase 2 (▶JNK2, ▶JNK subfamily and ▶cancer) that is known to phosphorylate the c-Jun protoconcgene, there is a well-defined tissue specific splicing pattern. In neuronal cells, exon 6A is

included and 6B excluded, while in non-neuronal cells, 6B is included in the absence of exon 6A. There are several examples of alternate splice site usage (Fig. 2b) for genes that are associated with cancer. The ▶KLF6 tumor suppressor gene produces oncogenic variants due to alternative 5′ splice site selection. These splice variants are over-expressed in prostate cancer and seems to negatively affect the tumor suppressor activity of its normally spliced counterpart. In another example, the telomerase reverse transcriptase, TERT, utilizes alternative splicing of a 3′ splice site to generate TERT alpha. TERT is activated in most human cancers and is overexpressed in high grade astrocytic tumors. The alpha TERT isoform, that results from the use of a downstream 3′ splice site, lacks twelve amino acids and is deficient for telomerase activity. TERT alpha is furthermore suspected to cause apoptosis and thus provide cancer protection. In these

Pre-mRNA Splicing

Pre-mRNA Splicing. Figure 2 Examples of possible alternative splicing patterns. Splicing choices are shown schematically with exons depicted as boxes and introns depicted as lines. A single pre-mRNA may employ multiple of these alternative choices in a combinatorial manner and thus greatly increase the diversity of proteins encoded. Figure adapted from Refs. [2] and [3].

two cases of splicing regulation in cancer, the proteins that result from alternative splicing appear to have antagonistic functions to their full-length counterparts, one form promotes tumors and the other protects against cancer. Intron retention (Fig. 2c) is yet another mechanism of regulating the splicing and thus function of genes that are involved in cancer. Intron retention occurs when the 5′ and/or 3′ splice sites are not recognized by the splicing machinery and the intron is therefore not removed from the transcript. A gene that is thought to regulate invasion and metastasis, CCK2, is altered in various cancers by retention of an intron that encodes a large intracellular loop as part of its transmembrane domain. The splice variant promotes growth via interaction with the ▶Src tyrosine kinase. The misregulation of splicing can occur via two general mechanisms. The first way is via point mutations that affect the cis-regulatory elements important for splicing control. Point mutations that create or destroy splicing signals within the RNA itself

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can affect splicing and are a prevalent way to destroy RNA splicing in a number of different cancers. In one example, the breast cancer susceptibility gene, ▶BRCA1 (▶BRCA1/BRCA2 germline mutations and ▶breast cancer risk), is spliced incorrectly and the resultant truncated BRCA1 protein leads to breast cancer. In this case, a single nucleotide mutation was identified in exon 18 of BRCA1. This point mutation disrupts an ▶exonic splicing enhancer (ESE) that is known to bind to the SR protein SF2/ASF. This disruption leads to the inability of exon 18 to be recognized by the splicing machinery and the exon is skipped as in Fig. 2d. The resultant mRNA is in a different reading frame than the normally spliced RNA and a truncated protein is produced. This example underscores the importance of regulatory element sequence fidelity for efficient splicing and is an important example of a cancer-causing point mutations that leads to a splicing deficiency. The second way to express incorrect splice forms is through the altered expression or function of splicing trans-regulators. Changes in expression levels, phophorylation state and sub-cellular localization of splicing accessory molecules are all known to be ways of regulating specific splice site choices. In the case of the ▶Mdm2 (or Hdm2 in humans, ▶MDM genes), the modulator of tumor suppressor p53 activity, splicing is regulated in normal cells as a response to stress. The resultant splice form is also an example of exon inclusion (normally) versus exon skipping (in stressed cells). As shown in Fig. 3, the internal eight exons are skipped (exons 4–11) making this an extreme example of exon skipping. Although the mechanism of MDM2 splicing regulation is still unknown, it is thought to be a result of transfactor control since this represents a normal cellular process that is reversible once the stress is removed. The MDM2 protein derived from the alternatively spliced form has been shown to bind to the full-length MDM2 and interfere with its ability to bind to and regulate p53 (see Fig. 3). In this way, expression of MDM2alt1 facilitates upregulation of p53 activity and promotes the damage response playing an important role in tumor suppression. However, this alternatively spliced form of MDM2 is contradictorily found to be overexpressed in a number of human cancers including gliomas, rhabdomyosarcomas, breast and ovarian cancers. Although the expression of the MDM2 alternatively spliced form is predicted to suppress tumors by activating the trp53 tumor suppressor pathway, its prevalent expression in multiple tumor types has led to much speculation about a possible role of MDM2 splicing in tumor formation. One prevailing hypothesis for the role or MDM2alt in tumorigenesis is a cancer progression model in which the expression of the alternative form of MDM2

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Pre-mRNA Splicing

Pre-mRNA Splicing. Figure 3 Alternative splicing of MDM2. The MDM2 pre-mRNA is shown schematically; exons are depicted as boxes and introns as lines. Under normal conditions the MDM2 is spliced to include all exons and encodes the full-length protein as depicted in the left part of the figure. Under stress conditions and in certain cancers, MDM2 alternative splicing causes skipping of exons four through eleven and results in the expression of a novel MDM2 protein that lacks the p53 binding domain, the nuclear localization and export signals (NLS and NES), and the ARF binding domain (as shown in the right part of the figure). The short MDM2 protein negatively regulates its respective full-length counterpart and ultimately activates the p53 pathway. This example of regulated splicing uncovers a novel mechanism by which cellular injury can control distribution and activity of p53 within the cell and possibly lead to cancer.

initially protects the cells by activating trp53, but sustained expression of the MDM2 form and upregulated trp53 puts a selective pressure on the cells that results ultimately in mutations in the p53 gene itself or other genes in the pathway. It is these secondary mutation(s), then, that induce the cancer phenotype. This predicted model mirrors a similar situation in which perpetual ▶myc (Myc oncogene) expression likewise induces trp53 providing cancer protection initially, but secondary mutations in genes of the trp53 pathway lead to cancer, in the end. Although experimental proof for the exact role the MDM2 alternatively spliced form plays in the initiation of cancer is lacking, the expression of alternatively spliced forms of MDM2 is clearly a marker for many different tumor types. In summary, the expression of many alternatively spliced isoforms is associated with cancer. In many cases, the new proteins formed are ant-apoptotic or

growth-promoting lending clear clues to their roles in tumor formation. In other cases, the role of the alternative forms in tumorigenesis is more elusive. Therapeutic Intervention Since a number of tumor specific isoforms have been identified that block apoptosis and/or promote cell growth or invasion, modification of the splicing profile in tumors is a possible therapeutic intervention point. There have been several approaches that have been successfully utilized to change splice site choices in a variety of human diseases. Low molecular weight drugs such as neomycin, aclarubicin, and sodium butyrate have been successfully utilized to promote exon inclusion. Likewise, heterologous expression of tranacting splicing factors can alter splicing patterns of their target genes. These types of therapies must be carefully tested for non-specific effects as they have the potential

Preclinical Studies

to affect the splicing of many genes in addition to the targeted disease-causing gene. This danger of nonspecificity may be avoided by designing selective agents that only recognize the RNA in question. Antisense oligonucleotides and RNAi technology have been shown to be effective in recognizing specific sequences in the target RNA and successfully modulating splicing. As more becomes known about the splicing of genes and the roles of this process cancer, there is great promise for modulating gene expression by directing splice choices that encode tumor-suppressor proteins and squelch the tumor-promoting alternatively spliced isoforms.

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considered to be a benign growth or a precancerous lesion. Adenomas in colon cancer are representative of this state. ▶Colorectal Premalignant Lesions ▶Preneoplastic Lesions

Preclinical Drug Safety Evaluation ▶Preclinical Testing

References 1. Kramer A (1995) The biochemistry of pre-mRNA splicing. In: Lamond A Pre-mRNA splicing. R.G. Landes Austin, TX, pp 35–64 2. Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336 3. Cartegni L, Chew SL, Krainer AR (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 34:285–298 4. Venables JP (2004) Aberrant and alternative splicing in cancer. Cancer Res 64(21): 7647–7654 5. Chandler D (2007) Splicing of the p53 pathway. In: Venables J (ed) Alternative Splicing in Cancer. Transworld Research Network, Newcastle, UK

Pre-replicative Complex

Preclinical Imaging Definition Imaging of live small-animal disease models (most commonly mice or rats) for biomedical research. The term usually refers to an instrument analogous to a clinical diagnostic imaging technology such as x-ray computed tomography, magnetic resonance imaging, radionuclide imaging, or ultrasound that has been designed to resolve structures smaller than 200 μm. That resolution permits the anatomy of a mouse to be visualized at a level of detail equivalent to a clinical image of a human patient. ▶Ultrasound Micro-Imaging

Definition Is a complex of proteins, originally identified by in vivo footprinting, present at replication origins only during late mitosis and G1.

Preclinical Safety Testing

▶Replication Licensing System ▶Preclinical Testing

Precancerous Lesions

Preclinical Studies

Definition During the process of transformation, cells will accumulate mutations in genes in a step wise fashion. At some point, the cell may proliferate resulting in growth, but does not yet have the capacity to invade surrounding tissues. At this point the cell(s) are

Definition Describe the phase of drug discovery before the first clinical studies start in man. ▶ADMET Screen

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Preclinical Testing

Preclinical Testing P ETER G REAVES Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, UK

Synonyms Toxicity testing; Preclinical safety testing; Preclinical drug safety evaluation

Definitions The process of testing potential new therapies in animal and cell-based test systems prior to their study in patients in order to assure their potential safety and efficacy.

Characteristics It was recognized many years ago that potential medicines were often “poisons” so that some form of screening in animal models was required before new drugs could be tried in patients. For all types of medicines it has been generally agreed that these tests should take the form of both pharmacological and toxicological studies in animals to avoid patients being exposed to drugs that are excessively toxic or without evidence of potential efficacy. Added impetus has come from the Nuremberg Code that was formulated after the trials of the Nazi doctors convicted for the conduct of horrific medical experiments on prisoners. Whilst this code deals primarily with the consent of volunteers and patients, one of its articles places emphasis on the justification for any experiments in humans being based on prior information derived from animal experiments: “The experiment should be so designed and based on the results of animal experimentation and a knowledge of the natural history of the disease or other problem under study so that the anticipated result will justify the performance of the experiment.” Hence, the main concept underpinning the preclinical safety testing is the protection of volunteers and patients in the testing of new drugs. The basic paradigm for testing is similar for all drug types and falls into three main phases. Firstly, toxicity testing is often used in the discovery phase to select the least toxic candidate drug from a series of chemicals. Secondly and most importantly it is used to provide the basic safety data to permit first dosing of a novel agent to volunteers. Finally, further detailed testing using repeated dosing schedules over longer periods and specialist protocols is conducted in parallel with ongoing clinical trials

to complete the preclinical safety data. This permits extended dosing of patients and supports eventual marketing of the drug (Fig.1). Although cytotoxic anticancer drugs represent a special case often needing only modest preclinical testing programmes, it needs to be kept in mind that not all drugs used in the therapy of cancer are conventional cytotoxic drugs. Some of these such as those used to manage pain, nausea or vomiting are developed in a manner similar to non anticancer drugs and consequently often have preclinical testing programme analogous to novel drugs intended for longterm use. The use of preclinical safety testing is primarily screening for serious drug toxicity to permit their safe testing in humans. They are no substitute for careful study of new drugs in patients to test their ability to treat disease and for monitoring of adverse effects. Adverse effects occurring in one out of 10,000 or 100,000 patients can be devastating but would never be detected in the small number of healthy animals used in safety testing. Discovery Phase Basic toxicity testing has an important place in the selection of a potential new anticancer therapy, despite application of a vast array of novel approaches using molecular biology, combinatorial chemistry and bioinformatics. The activity of anticancer drugs is typically assessed using a battery of ▶mouse models for different cancer types. At this stage, informal studies, so called ▶screening toxicity using small numbers of rodents, often mice, might be conducted. This is often performed when series of potential drugs cause toxicity in particular organs such as the liver and kidney to enable selection of the least toxic for progression to the next phase of formal testing prior to administration to humans. Depending on the nature of the toxicity, these studies usually employ simple endpoints – clinical observation, examination of blood values and microscopic examination of important organs. Phase I Clinical Study Following identification of a potentially active new drug, the next major step is testing for effects in humans. However, the transition from the laboratory bench to the bedside represents a large step for which preclinical studies are crucial. At this stage, a small number of carefully planned and conducted animal toxicity and safety pharmacology experiments are performed. Typically, two laboratory animal species are used for each new drug. One of these is a rodent usually rat and the other a non rodent usually dog. These allow the precise characterization of the effects activity of the

Preclinical Testing

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Preclinical Testing. Figure 1 This diagram shows the place of preclinical safety studies within the context of drug discovery and development. So called screening toxicology might be conducted in rodents prior to entry into formal “single dose” and “repeat dose” toxicology studies immediately before testing in humans (red line). In parallel with the conduct of clinical phases I, II and III further testing including “chronic” (6 months or more duration) toxicity and reproductive studies are carried out prior to marketing (green line). Italics show drug kinetics and metabolism studies – performed in both humans and animals and enables effects in animals and humans to be correlated. Cancer drugs usually do not require the testing marked in grey print, although these are needed for drugs of other types intended for long-term use.

drug on body functions and any cellular toxicity. They provide the basis for the design, conduct and safety monitoring of clinical phase I studies of the new drug in volunteers. Whilst healthy volunteers may be studied for cancer drugs that are not cytotoxic, cytotoxic agents are usually studied in volunteer cancer patients. The paradigm for a toxicity experiment is similar for most types of studies. Animals are dosed drug by the similar route to that intended for use in patients. This is usually done at a clinically relevant dose and usually two higher doses on a body weight or surface area basis. The minimum number of animals of each sex in each dose group is mandated in most government guidelines, notably those of the European Union, United States and Japan. These studies are conducted to an agreed international laboratory standard known as ▶Good Laboratory Practice (GLP). This requires regular auditing of studies as well as inspection of laboratories by government agencies. In all these toxicology studies the animals are monitored clinically with particular attention being paid to changes in animal behavior that could signify adverse effects on critical functions such as the nervous system. Blood pressure, heart rate and electrocardiograms are usually monitored in the dog as they are more

difficult to perform in rodents that have very fast heart rates. Eyes are also carefully examined using sophisticated optical equipment similar to that employed by an ophthalmologist or optician. Blood is sampled and tested for any biochemical or hematological alterations in a similar manner to human patients, often using identical laboratory analytical apparatus. Levels of drug circulating in the body are also usually monitored in these experiments, often referred to as ▶toxicokinetics. At the end of the dosing period, or if any ill health intervenes, the animals are humanely killed using an anesthetic or barbiturate and subject to a full autopsy examination. Over 30 tissues from all the organs are taken for microscopic examination by an experienced ▶toxicological pathologist. This is to check for any organ damage that could reflect danger to patients if they were to be treated with the new drug. Organs such as the liver, kidneys, ovaries and testes are particularly important, as are the lymph nodes, thymus and spleen for any deleterious effect on the immune system. Additional special studies are performed if there are findings of concern that need elucidation. As many drugs, particularly cancer drugs are locally irritant to tissues, the gastrointestinal tract is examined closely if

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Preclinical Testing

the oral route is employed. Likewise, injection sites are also studied microscopically if a parenteral route is used. Severe local irritancy might preclude dosing to humans or would dictate particular caution in use. These studies are designed in a particular way for cytotoxic cancer drugs because these are liable to damage rapidly dividing cells such as those in the bone marrow and gastrointestinal tract. For these drugs it has been shown that the ▶maximum tolerated dose (MTD) is often similar in mouse, rat, dog, monkey and man when compared on the basis of mg/m2 of body surface area. Experience has shown that a safe starting dose in humans is one tenth of the MTD based on ▶single dose toxicity studies. This means that a key study for cytotoxic drugs is a single dose toxicity study that has sufficient number of animals per dose to establish an MTD. These single dose studies usually have a follow up period of clinical examination of the animals for at least 14 days after dosing to exclude delayed toxic effects. Usually two animal species, one rodent and one non rodent are used to confirm the MTD. This study is required by ▶government drug regulatory authorities prior to testing cytotoxic cancer drugs in cancer patient volunteers. ▶Repeat dose toxicity studies are also performed with cytotoxic drugs prior to phase I clinical studies. These are designed to mirror the proposed clinical schedule with particular attention being paid to organ toxicity and reversibility of toxic effects. These are of limited duration usually 2–4 weeks or one or two cycles of treatment in two species. However, increasingly many new anticancer drugs are not cytotoxic because they act through other mechanisms such as modulation of hormones or growth factors. These are tested in a more conventional manner similar to most other drugs intended for long-term use. Here, toxicity experiments comprise single dose studies in rodents and repeated dosing over a period of up to 1 month in two animal species. One species is a rodent, usually laboratory rat, and the other a non-rodent species, usually the beagle dog. Experiments in monkeys are avoided where possible unless the compound being studied has particular pharmacodynamic or metabolic characteristics that demand it. Yet another variation on the basic preclinical testing scheme is used for biological products. Again the aim is to mirror the clinical treatment schedule. However, as some biological agents do not possess activity in nonprimate species, monkey studies are sometimes needed to assure safety in humans. Immunogenic potential is also thoroughly investigated in specialized animal models. At this stage determining the safety profile of a new drug is not limited to animal toxicity experiments. There are also in vitro ▶genotoxicity studies, such as the Ames test which uses bacteria and other experiments using cells to study whether the drug can damage DNA. A

standard battery of both in vitro and in vivo genotoxicity experiments is conducted prior to phase II clinical studies for conventional drugs (▶Micronucleus assay). A particularly important component of preclinical study is the investigation of absorption, distribution, metabolism and excretion of drug and their metabolites in the blood and tissues of animal species chosen for toxicity testing. This data helps interpretation of the relevance of adverse findings in animals for humans because it forms a point of comparison of drug handling between animals and humans. It also helps to validate the relevance of the data obtained in animals (▶Pharmacokinetics/pharmacodynamics). At the end of this phase of work scientists and physicians review the assembled data from all these experiments along with information from studies of drug metabolism prior to design and conduct of first studies in humans. In all cases a clear rational based on a trade-off between any potential risks with likely longterm benefit would be generated prior to the conduct of any human experiment. Moreover, there is a mandatory ethical review of the study protocol by a panel not directly involved in the human studies. The review panel would be provided with a summary of the results of the animal safety information. Government agencies in the United States and in the European Union have approval processes for all such human studies. Phase II and III Clinical Studies In clinical studies in patients, dosing periods are usually lengthened and more subjects are involved. These are accompanied by animal toxicology studies of longer duration and when appropriate, other specialist studies. The organization of these toxicity studies is similar to the earlier studies but the period of dosing is extended from a period of up to 1 month to period of dosing of 6 or 9 months or 1 year for most drugs. For anticancer drugs studies are usually conducted with continuous or intermittent dosing for a period equal to the duration of clinical trials although not longer than 6 months. Further drug kinetics and metabolism studies are conducted at this stage as data from humans is available and enables comparison between species. This also serves to validate the preclinical data. Metabolites may be isolated and their toxicity studied if there is particular concern about the adverse effects of metabolites. Enzyme induction potential may also be tested in animals or in vitro hepatocyte test systems (Pharmacokinetics/pharmacodynamics). Although for most drugs special experiments to examine the effects of a new drug on reproductive function and the developing foetus (▶reproductive toxicity studies) are required in to be conducted in two sensitive animal species, conventionally rat and rabbit. However for most cytotoxic anticancer drugs these experiments are not usually performed.

Preneoplastic Lesions

Although in vitro ▶genotoxicity tests are not required for anticancer drugs prior to phase I and phase II clinical trials, tests such as the Ames test which uses bacteria and other experiments using cells to study whether the drug can damage DNA are usually expected prior to clinical phase III trials and marketing applications. A full battery of in vitro and in vivo genotoxicity tests is required before phase II for more conventional drugs (Micronucleus assay). The last preclinical experiments that are usually conducted for most drugs are the so called ▶carcinogenicity studies. Although these are a requirement for most drugs that are going to be used for extended periods in patients, they are not performed for many anticancer drugs. Exceptions exist for drugs such as the selective estrogen modulating drugs such as tamoxifen that are used for extended periods and for cancer prophylaxis The studies have typically been mandated in two rodent species, mouse and rat. Again the organization of the studies is similar to those conducted previously except that the dosing period is for 2 years – most of the lifetime of a rodent (▶Carcinogenesis or ▶toxicological carcinogenesis). More recently, shorter studies using genetically modified, cancer-prone mice have been accepted by ▶government drug regulatory agencies in place of one of these two large studies (Mouse models). At the end of this process, the preclinical information is summarized along with information from clinical trials and manufacturing data and assembled for submission to government drug regulatory authorities to obtain marketing authorization.

References 1. Greaves P, Williams A, Eve M (1997) First dose of potential medicines to humans: how animals help. Nature Reviews Drug Discovery, 3, 226–236 2. Schuster E (1997) Fifty years later: the significance of the Nuremberg Code. New England Journal of Medicine, 337, 1436–1440

Predictive Biomarkers

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Prednisone Definition Is a synthetic steroid with potent antiinflammatory and immunosuppressive activity used in treating acute graft rejection, autoimmune disease, and lymphoid tumors.

Preleukemia ▶Myelodysplastic Syndromes

Premature Menopause Definition Menopause before the age of 40 years. ▶Menopausal Symptoms After Breast Cancer Therapy

Preneoplastic Changes Definition Are phenotypical changes in cells that follow a malignization pathway but are not yet irreversibly malignized.

Preneoplastic Lesions F RANCESCO F EO

Definition Biomarkers to predict whether the drug and other therapies will be effective, or to monitor the effectiveness of treatment; ▶early detection. ▶Oncopeptidomics

Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, Sassari, Italy

Definition The development of primary tumors is often preceded, both in humans and experimental animals (mainly

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rodents), by the appearance of lesions referred to as preneoplastic. These consist of genetically and phenotypically altered cells exhibiting a higher risk of malignant evolution than normal cells. These lesions generally lack one of the principal characteristics of neoplastic lesions: the capacity to grow autonomously after cessation of the stimuli that induced the lesion. Nonetheless, the distinction between preneoplastic lesions and benign neoplasias is sometimes difficult, and the terms “preneoplastic” and “premalignant” are often considered synonyms. However, benign tumors, constituted by autonomously growing cells, cannot be strictly classified as preneoplastic but only as premalignant lesions, whereas, premalignant lesions can include both preneoplastic lesions and benign tumors.

Characteristics Tumorigenesis is considered a multistep process characterized, both in humans and rodents, by the progressive development of preneoplastic, premalignant, and malignant lesions. During this process, genomic instability occurs (▶Microsatellite instability), followed by alterations in oncogenes, oncosuppressor genes, and DNA repair genes, with consequent changes in the ▶signal transduction network (Fig. 1). The progressive accumulation of genetic changes generates autonomously growing premalignant and malignant lesions. Some Experimental Models of Preneoplastic Lesions Preneoplastic lesions have been induced in different organs, including liver, pancreas, lung, colon, skin,

thyroid, mammary gland, gall bladder, prostate, in rodents treated with carcinogens and in transgenic mice. Although a close correspondence of experimental and human preneoplastic lesions does not always occur, the study of experimental lesions allowed discovery of pathogenetic mechanisms and diagnostic and prognostic markers of different tumor types. Only lesions preceding tumor development in some organs (liver, colon, lung) will be given as examples. In hepatocarcinogenesis rodent models, irreversibly initiated cells by a carcinogenic stimulus can undergo clonal expansion by overresponse to the administration of promoting agents, such as phenobarbital, 2,3,7,8tetrachlorodibenzo-p-dioxin, chlorinated hydrocarbons, peroxisome proliferators, for several weeks. Only few initiated cells undergo clonal expansion under promoting stimuli, probably because of irreversible change and apoptosis, giving rise to a heterogeneous population of foci of altered hepatocytes (FAH) expressing specific patterns of marker genes, such as Glutathione Stransferase (GST) 7-7, γ-Glutamyl transpeptidase, and alterations of carbohydrate metabolism defining different lineages of preneoplastic cells. When the treatment with promoters is suspended before the appearance of neoplastic lesions, early preneoplastic liver lesions partially disappear by a process called “▶remodeling.” GST 7-7 positive cells that acquire autonomous growth persist and further evolve to premalignant neoplastic (persistent, atypical, dysplastic) nodules and then to well-differentiated ▶hepatocellular carcinomas (HCCs; ▶Liver Cancer, Molecular Biology), which progress to moderately and poorly differentiated carcinomas. Other

Preneoplastic Lesions. Figure 1 Schematic representation of multistage carcinogenesis. Carcinogenesis initiation is associated with the appearance of genomic instability. Clonal expansion of initiated cells leads to the development of preneoplastic lesions carrying various genomic alterations. The accumulation of these alterations is associated with the acquisition of the capacity of autonomous growth and evolution to neoplastic lesion, which progress to moderately differentiated and poorly differentiated, invasive, carcinomas.


hepatocarcinogenesis models are based on the stable transfection of one or two cancer related genes (i.e., c-myc, c-myc plus Tgf-α genes) in mouse and, less frequently, rat genome, or in inactivating a gene of intact mice (knockout mice). In these models, generally the liver is dysplastic and progressively becomes adenomatous before the development of HCCs. Alterations of several signal transduction pathways occur in rodent HCC and, at a lower extent, in preneoplastic liver lesions. They include upregulation of EGF receptor family, resulting in induction of the Ras-dependent activation [▶Ras] of Mitogen activated protein kinases (MAPK, ▶Map Kinase) and JAKStat signaling, both transducing extracellular signals resulting in phosphorylation of transcription factors targeting various genes (Fig. 2). c-Raf/MAP kinase

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kinase-extracellular signal-related kinases (Mek-Erk) pathway is primarily responsible for responding to cellular proliferation signals, whereas the Mitogen activated protein kinase kinase (Mekk) and c-Jun Nterminal kinases (JNK; ▶JNK Subfamily and Cancer) respond to cellular stress signals and treatment with carcinogens. This leads, trough upregulation of the cfos and c-Jun genes, to the activation of AP-1 and its targets (i.e., Cyclin D1 gene; ▶Cyclin D). As a consequence Odc, a c-Myc (▶Myc Oncogene) target, is overexpressed and its gene product, Ornithine decarboxylase, activates polyamine synthesis required for nucleotide biosynthesis. Upregulation of c-myc, cyclin D1, and other Cyclins and Cyclin-dependent kinases, activates cell cycle and cell proliferation. This is favored by downregulation of cell cycle inhibitors,

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Preneoplastic Lesions. Figure 2 Schematic representation of signal transduction pathways involving Jak/Stat, Ras/Mapk, Wnt/β-Catenin, and Tgf-β1 signaling pathways. Binding to ligands or autologous upregulation of Epidermal growth factor (EGF) family receptors causes phosphorylation of Jak1 and 2 and activation of Stat3 and formation of active GTP–Ras complex, which activates the Raf/Mek/Erk and Mekk/Jnkk/Jnk pathways. This leads to the phosphorylation of nuclear factors that, migrating into nucleus, activate numerous target genes. The inactivation of components of the complex Apc/Axin/Gsk3β/β-catenin complex, or the inhibition of Gsk3β by Dishwelled (Dsh) protein by Wnt1/Freezzled activation, suppresses β-Catenin phosphorylation and ubiquitination followed by disruption by proteasome. β-Catenin accumulation into the cytoplasm causes its nuclear translocation and activation of various target genes. Activation of Tgf-β receptor (TBR) leads to the formation of Smad2/3/Smad4 heterodimers leading to cell death by apoptosis. Overexpression of Smad7 and/or downregulation of Smad2/3 and Smad4, inhibit the TBR signaling pathway. Enhancing and inhibitory effects are indicated by pointed arrows and blunt arrows, respectively.

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such as p16 INK4a and p53, and Tgf-β/Smad signaling. Profound alteration of the Wnt/β-Catenin pathway (▶Wnt Signaling), leading to inactivation of the complex Apc/Axin/Gsk3β/β-Catenin, decreases β-Catenin disruption via proteasome. Consequent nuclear translocation of β-Catenin activates various targets, including c-myc and cyclins. Finally, overexpression of the inducible nitric oxide synthase (iNos) contributes to activation of the MAPK cascade and the hypoxia inducible factor-1, leading to an increase in vascular endothelial growth factor (Vegf ) expression and angiogenesis. The presence of these alterations in preneoplastic lesions indicates their role in early stages of hepatocarcinogenesis. Another largely investigated model of multistep tumorigenesis is represented by chemically induced colorectal cancer (CRC; ▶Colon Cancer) of rodents. Aberrant crypt foci (ACF; Colorectal Pre-malignant Lesions) identified in colonic mucosa of rodents treated with chemical carcinogens are considered preneoplastic lesions. These lesions exhibit increased expression of c-fos, decreased c-myc expression and hexosaminidase activity, and loss of transforming growth factor-α (Tgf-α), whereas mutations of Adenomatous polyposis coli (Apc; ▶APC & ▶β-catenin pathway) gene are absent. K-ras mutations occur in azoxymethaneinduced ACF in rats. However, various observations strongly suggest that ACF are not preneoplastic, but only hyperplastic lesions. A subset of ACF, called “dysplastic ACF,” consisting of fast-growing crypts with altered β-Catenin expression associated, in some cases, with β-Catenin mutation, are considered true CRC precursors. In colonic mucosa of rats, treated with azoxymethane, mucin depleted foci (MDF) have been described. MDF are more dysplastic and more likely to express β-Catenin than common ACF. β-Catenin accumulating crypts (BCAC) with higher cell proliferative activity than ACF significantly increase with time during colorectal carcinogenesis in rodents. The study of the molecular events in early colorectal preneoplastic lesions indicates that various mutations occur and are selected during colorectal carcinogenesis, the main selective factor being represented by those leading to a β-Catenin downregulating function. ▶Lung cancer develops in a gradual and stepwise fashion. Numerous studies have addressed lung tumorigenesis in tobacco smokers. Preneoplastic lesions include focal epithelial cell ▶hyperplasia, squamous metaplasia, and ▶dysplasia. These lesions have been induced in rats by various carcinogens and are followed by the development of adenomas, adenocarcinomas, and squamous cell carcinomas. Exposure of rats to tobacco smoke induces dose-dependent cell proliferation and squamous metaplasia. These effects are

paralleled by activation of MAPK signaling pathways and AP-1 binding to DNA (Fig. 2). This is associated with upregulation of AP-1-dependent cell cycle proteins, such as Cyclin D1 and Proliferating cell nuclear antigen (PCNA). Human Preneoplasia Etiologic factors of HCC include ▶cirrhosis induced by hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic cirrhosis, exposure to Aflatoxin B1, estrogenic steroids, some naturally occurring carcinogens in food, and some rare genetic syndromes (i.e., hemochromatosis, glycogenosis type I, α1 antitrypsin deficiency). About 4% of HBV infections evolve to persistent hepatitis, 30% of which can further evolve to chronic hepatitis and cirrhosis. HBV positive cirrhosis is considered a preneoplastic lesion, although the evolution to HCC has been found in only 10% of cases. Different from HBV infection, HCV-induced hepatitis becomes chronic in the majority of patients. At least 20% of them develop cirrhosis, which in most cases evolves to HCC. In these patients, as well as in individuals with alcoholic cirrhosis, the development of FAH and adenomatous nodules, correspondent to those chemically induced in rats and mice or developing in woodchucks’ viral hepatitis, exhibits several molecular alterations in common with the corresponding lesions of rodent liver. Various preneoplastic lesions have been identified in the gastroenterical tract. About 10% of patients with long-standing gastroesophageal reflux develop the Barrett esophagus, in which the distal squamous mucosal epithelium is replaced by metaplastic columnar epithelium. This lesion is preneoplastic, and the patients with Barrett esophagus develop adenocarcinomas, preceded by dysplastic lesions, with a 30–40 times increased rate over the general population. Dysplastic epithelium exhibits cell cycle deregulation, high proliferative rate, upregulation of TP53, and in more advanced stage, amplification of chromosome 4. Further evolution to carcinoma implicates deregulation of Wnt/β-Catenin pathway and c-ERB-B2 amplification. Preneoplastic conditions of human gastric cancer have not yet been well characterized to date. Ménétrier disease, resulting from profound hyperplasia of mucosal epithelium and glandular atrophy, rarely exhibits epithelial metaplasia, a condition that may favor the development of gastric carcinoma. Interestingly, transgenic mice overexpressing Tgf-α at gastric level, develop a syndrome similar to human Ménétrier disease. A multistep model from chronic active Helicobacter pylori infection through multifocal mucosal atrophy, intestinal metaplasia, dysplasia, and carcinoma has been described. During this process, complex interactions


between several bacterial, host genetic, and environmental factors determine whether H. pylori infected individual develop cancer. H. pylori infection is characterized by upregulation of various inflammationassociated genes, including chemokines, adhesion molecules, surfactant protein D and CD74 in infected stomach. Nitric oxide and reactive oxygen species, which may induce DNA damage (▶DNA Damage Response; ▶Repair of DNA), are overproduced. The role of these and other factors in the evolution of metaplastic and dysplastic lesions to carcinoma is still an object of study. H. pylori infection also predisposes to the lymphomatous transformation of the mucosa associated lymphatic tissue. Most knowledge on multistep carcinogenesis in the gastrointestinal tract derives from colorectal tumorigenesis. Inherited syndromes, occurring in less than 10% of patients, include familial adenomatous polyposis (FAP), hamartomatous polyposis, hereditary nonpolyposis colorectal cancer (▶Lynch syndrome), and common cancer family syndrome, not belonging to aforementioned syndromes, causative germline mutations of high-penetrance genes affect APC gene for familial adenomatous polyposis, LKB1, SMAD4, and BMPR1 genes for hamartomatous polyposis, various mismatch repair genes (e.g., hMSH2, hMLH1, hPMS1, hPMS2, hMSH6) (▶Mismatch Repair in Genome Stability), for Lynch syndrome I, and AXIN2, TGFβR-2, and POLD genes for Lynch-like syndromes. The homologue of human FAP, the Min mouse strain-1, carries the APCMin mutation and develops intestinal neoplasms. The study of these syndromes allowed identifying early human colorectal preneoplastic lesions and the so called adenoma–carcinoma sequence, as well as the molecular alterations underlying the sequence of events leading to CRC. The morphological events include early appearance of preneoplastic ACF, followed by premalignant lesions such as adenomas and adenomatous polyps, and, finally, carcinoma development. The appearance of ACF, however, is preceded by numerous molecular events, such as germline (in the inherited syndromes) or somatic (acquired first “hit” in a “multihit” process, in sporadic cases) mutations of APC or mismatch repair genes. The mucosa harboring these mutations is at risk. Inactivation of normal alleles of tumor suppressor genes (i.e., by promoter methylation of APC, MSH2, β-Catenin) causes hyperproliferation and appearance of early preneoplastic lesions, which following K-RAS mutation evolve to adenomas. The loss of further suppressor genes (i.e., TP53) precedes the appearance of carcinomas whose ▶progression is characterized by additional mutations of various oncogenes, chromosomal aberrations, etc. A four to fivefold increase in the incidence of gastrointestinal tract cancer occurs in patients with

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Crohn disease, in which the development of carcinoma is generally preceded by dysplastic lesions of the ileum and/or colon mucosa. The pathogenesis of neoplastic transformation is unknown. Crohn disease seems to occur in predisposed individuals carrying some susceptibility loci on chromosomes 3, 7, 12 or 16. The ileum localization seems to be linked to mutations of NOD2 and CARD15 genes. However, the relationships between these alterations and cancer development are not known. A 20–30-fold rise in CRC incidence occurs in patients with ulcerative colitis. Cancer development is preceded by premalignant multifocal dysplastic lesions exhibiting DNA instability. Genomic instability has also been documented in the colon mucosa outside of dysplastic lesions and it has been hypothesized that some deficit of DNA repair occurs in these patients. Preneoplastic and premalignant lesions have been described for different other human tumors (Table 1). Epidemiological, clinical, and molecular characteristics of a number of these lesions are often incompletely known, but particular attention must be focused on these parameters because of their importance for prevention and early diagnosis of malignancy. Genetic Predisposition to Neoplasia A body of evidence shows the existence of a genetic predisposition to tumors. Strong predisposition by high penetrance mutations of oncogenes, oncosuppressor genes, and DNA repair genes, occurs for a relatively small number of the so-called hereditary tumors. Genetic predisposition to sporadic tumors depends on the inheritance of several susceptibility or resistance allelic variants, which influence (modify) the behavior of the molecular mechanisms of tumorigenesis and, hence, the phenotypic features of preneoplastic and neoplastic lesions. Several modifier genes have been mapped, but only few genes involved in genetic predisposition have been identified so far. These observations may in some way modify the definition of preneoplastic lesion. Indeed, a cell carrying polymorphic variants of cancer modifier genes responsible for increased susceptibility to malignancy, namely increased cancer risk, could be considered preneoplastic. The identification of modifier genes and the signaling pathways that they influence may lead to the discovery of early diagnostic and prognostic markers as well as therapeutic targets, which may prevent the evolution of preneoplastic cells to full malignancy. Clinical Relevance One of the major challenges of cancer research has been the discovery of tools for efficient prevention of malignancy. In this context, the identification of

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Preneoplastic Lesions. Table 1

Preneoplastic and premalignant lesion of various human tissues

Tissue

Preneoplastic Morphology

Lung

Liver

Fat or papillary mucinous hyperplasia

Oral epithelium

Leukoplakia

Esophagus

Barrett’s epithelium with metaplasia

Stomach

Ménétrier disease with metaplasia H. Pylori atrophic gastritis with intestinal metaplasia Crohn disease

Dysplastic nodules HER-2, Ki-67, P21WAF1, Atypical CYCLIN D1 upregulation,. hyperplasia SMAD 4 underregulation LOH at 3p and 9p Dysplastic leukoplakia TP53 mutations, CDX Barrett’s mutations dysplasia

a

Molecular markers

Upregulation of MAPK, JAK-STAT ODC, C-JUN, C-FOS, C-MYC, RAS family genes, cell cycle HER-2, Ki-67, P21WAF1, CYCLIN D1 upregulation, SMAD 4 underregulation TP53 overexpression; loss of differentiation-related keratins TP53 mutations, APC LOH, p16INK4a hypermethylation or LOH

TGF-α overexpression Dysplastic lesions

Overexpression of Gastrin, COX-2, SURVIVIN, BCL-2

ACF

Epithelial dysplasia Adenoma

Papillary hyperplasia

LOH at 9q

Papilloma VIN I and II Dysplasia (CIN I, CIN II) Atypical hyperplasia Atypical hyperplasia

TP53 mutation, Nitric oxide overproduction β-Catenin activation, APC and K-RAS mutation, LOH at TP53, SMAD2 and 4 LOH at 9q, TP53 mutations ei5A-1 overexpression P16INK4A overexpression, pRb ubiquitination

Endometrium Ovary

Morphology

a

Overexpression of GASTRIN, COX-2, SURVIVIN, BCL-2 NOD2 and CARD15 mutations TP53 mutations, Nitric oxide overproduction β-catenin activation

Ulcerative colitis

Urothelial Vulva Uterine cervix

Molecular markers

Atypical adenomatous hyperplasia; bronchial dysplasia FAH

Pancreas

Gastrointestinal tract

Premalignant

Breast

Metaplastic surface epithelium and inclusion glands Hyperplastic lesions

Thyroid

C-cell hyperplasia

Skin

Lentiginous melanocytic hyperplasia; Lentiginous junctional nevus

Microsatellite instability Overexpression of BCl-2 and BCL-X

Atypical hyperplasia Follicular adenoma (atypical) Dysplastic nevus

Loss of PTEN expression TP53 mutation. Loss of BRCA1 and 2 Microsatellite instability Mutations of RAS family genes

Overexpression of bFGF, and IL-8

The definition of premalignant lesion is often based on morphologic, clinical, and epidemiologic criteria.

very early biochemical, molecular, and morphologic changes, predisposing normal cells to tumorous transformation, plays a pivotal role. In principle, tumor prevention would imply the protection of humans

against tumor initiation. When this is not possible, an efficacious preventive strategy may attempt to block the evolution of initiated cells to malignancy. Of course, early identification of precancerous lesions is

Primary Biliary Cirrhosis

a prerequisite for efficacious prevention. The recognition of these lesions (i.e., polyps, adenomas, dysplastic nevi, leukoplakias) may allow their surgical or medical treatments. The knowledge of early molecular alterations, in preneoplastic lesions, is also important to adopt chemopreventive strategies aimed at contrasting the growth and progression of early lesions to cancer and/or to block the expression of genes involved in cell transformation.

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residue in characteristic carboxy-terminal motifs, giving rise to farnesylated and geranylgeranylated proteins. Such reactions are essential to the anchorage of small GTPases to cell membranes and to protein– protein interactions. ▶Zoledronic Acid

References 1. Brambilla C, Fievet F Jeanmart M et al. (2003) Early detection of lung cancer: role of biomarkers. Eur Respir J 39:36s–44s 2. de la Chapelle A (2004) Genetic predisposition to colorectal cancer. Nat Rev Cancer 4:769–780 3. Gologan A, Graham DY, Sepulveda AR (2005) Molecular markers in Helicobacter pylori-associated gastric carcinogenesis. Clin Lab Med 25:197–222 4. Kumar V, Abbas AK, Fausto N et al. (2005) Pathologic basis of disease, 7th edn. Elsevier Inc, Philadelphia 5. Feo F, De Miglio MR, Simile MM et al. (2006) Hepatocellular carcinoma as a complex polygenic disease. Interpretive analysis of recent developments on genetic predisposition. BBA Cancer Rev 1765:126–147

Prentice Criteria Definition Named after the criteria formulated by Ross Prentice in an influential 1989 article on validation of ▶surrogate endpoints, were developed to ensure that rejection of the null hypothesis under the surrogate endpoint implies rejection of the null hypothesis under the true endpoint. The main criterion, sometimes called the Prentice criterion, is that the distribution of the true endpoint conditional on the surrogate endpoint does not depend on the intervention. In other words, the Prentice criterion says that, for all treatments under consideration, there is a single pathway from treatment to true endpoint that goes through the surrogate endpoint, so once the surrogate endpoint is known, no other information is needed to determine the distribution of the true endpoint.

Preoperative Chemotherapy ▶Neoadjuvant Therapy ▶Induction Chemotherapy

Prevalence Definition A measure of the proportion of people in a population affected with a particular disease at a given time. ▶Obesity and Cancer Risk

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Prevention ▶Cancer Causes and Control

Primary Biliary Cirrhosis Prenylation Definition Definition

An autoimmune liver disease resulting in intrahepatic bile duct destruction leading to liver ▶cirrhosis.

Is a biochemical reaction resulting in the transfer of a farnesyl or geranylgeranyl lipid group onto a cysteine

▶Hepatic Epithelioid Hemangioendothelioma

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Primary Cancer Prevention

Primary Cancer Prevention

Primary Dissemination

Definition

Definition

Prevention of tumor onset through the elimination of risk factors, e.g. chemical carcinogens. ▶Carcinogenesis.

Dissemination at diagnosis.

▶Immunoprevention of Cancer

Primary Cancer Site

▶Leptomeningeal Dissemination

Primary Hepatic Carcinoma ▶Hepatocellular Carcinoma

Definition The organ in which the initial cancer forms. For example, the primary site for metastatic breast cancer is the breast.

Primary Liver Cancer

▶Metastatic Colonization ▶Hepatocellular Carcinoma – Etiology, Risk Factors and Prevention ▶Hepatocellular Carcinoma

Primary Care Trust Definition PCT; Local commissioning organizations within the United PCT Kingdom’s National Health Service whose Trusts (Primary Care Trusts (PCTs)) charged with purchasing specialist services, including cancer treatments, from hospitals. ▶National Institute for Health and Clinical Excellence

Primary Chemotherapy ▶Neoadjuvant Therapy

Primary Cilium

Primary Lymphedema Definition A congenital deficiency in the lymphatic system in which lymphatic vessels are poorly functional, resulting in blocked drainage of fluid from tissues, skin thickening and adipose tissue accumulation. In some patients, this condition is caused by inherited mutations in the FOXC2 or VEGFR3 gene. ▶Lymphangiogenesis

Primary Myelofibrosis AYALEW T EFFERI Division of Hematology, Mayo Clinic College of Medicine, Rochester, MN, USA

Definition

Synonyms

Non-motile, hair-like projection on most mammalian cells involved in signal transduction and chemical sensation.

Myelofibrosis with myeloid metaplasia; Chronic idiopathic myelofibrosis; agnogenic myeloid metaplasia; Idiopathic myelofibrosis

Primary Myelofibrosis

Definition Primary myelofibrosis (PMF) is a stem cell-derived clonal myeloproliferative disorder (MPD) that is characterized clinically by anemia, marked enlargement of the spleen and liver, and severe constitutional symptoms. Peripheral blood findings include the presence of immature myeloid cells including nucleated red blood cells, immature granulocytes, and tear drop-shaped erythrocytes. The bone marrow histology exhibits reticulin and collagen fibrosis, osteosclerosis, and angiogenesis.

Characteristics Background The blood and bone marrow features associated with PMF are discovered either de novo (i.e. PMF) or in the setting of either polycythemia vera (post-PV MF) or essential thrombocythemia (post-ET MF). PMF is also known by many synonyms. However, the use of the term “PMF” was recently endorsed by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT). Historical Perspective The first description of PMF is credited to Heuck (1879). William Dameshek classified PMF as a MPD, along with chronic myeloid leukemia (CML), ET, and PV. In 1960, the Philadelphia chromosome was described in CML, which was later shown to harbor first t(9;22)(q32;q13) and subsequently the BCR-ABL diseasecausing mutation. Accordingly, modern classification systems list PMF, PV, and ET as BCR-ABL-negative classic MPDs. In 1967, the Polycythemia Vera Study Group (PVSG) provided, for the first time, formal criteria for the diagnosis of PMF. Subsequently, a WHO-sponsored committee on classification of hematological malignancies revised the PVSG diagnostic criteria for PMF and reorganized the overall classification system for myeloid neoplasms. Disease Mechanisms In 1978, G6PD-based clonality studies established PMF as a stem cell-derived clonal myeloproliferation. In 2005, a novel gain-of-function (GOF) mutation involving the JAK2 tyrosine kinase (JAK2V617F) was described in ~50% of PMF patients but also in the majority of those with PV as well as ET. In 2006, another GOF mutation involving MPL (MPLW515L/K) was described in ~5% of patients with PMF. JAK2V617F is an exon 14 JAK2 mutation at nucleotide position 1,849 representing a G to T somatic point mutation. The mutation results in the substitution of valine to phenylalanine at codon 617. MPLW515L mutation represents a G to T transition at nucleotide 1,544 resulting in a tryptophan to leucine substitution at

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codon 515 of the transmembrane region of the MPL receptor. Both of the above mutations induce an MPDphenotype in mice, the former a PV-like disease and the latter a PMF-like disease. In addition to clonal myeloproliferation, PMF is characterized by bone marrow stromal aberration including collagen fibrosis, osteosclerosis, and angiogenesis. In mice, similar histological features have been induced by either systemic over-expression of thrombopoietin (TPOhigh mice) or by megakaryocyte lineage restricted under-expression of the transcription factor GATA-1 (GATA-1low mice).17 In both human PMF and experimental myelofibrosis in mice, the bone marrow stromal changes are believed to be secondary to abnormal release fibrogenic and angiogenic cytokines including transforming growth factor-β1 (TGF-β), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), tissue inhibitors of matrix metalloproteinases, and neutrophil-derived elastase and other proteases. Clinical and Laboratory Characteristics The prevalence of PMF is similar in men and women (M:F = 1.6:1) and overall reported incidence figures range from 0.4 to 1.5/100,000. Median age at diagnosis is estimated between 55 and 60 years. Most, but not all, patients with PMF are symptomatic at diagnosis. The typical presentation includes anemia, marked splenomegaly, and constitutional symptoms including fatigue and night sweats. Hepatosplenomegaly in PMF is secondary to extramedullary hematopoiesis (EMH) that might also involve other organs including lymph nodes, pleura, peritoneum, and the paraspinal and epidural spaces. The peripheral blood smear in PMF often shows leukoerythroblastosis (presence of nucleated red blood cells and immature granulocytes) and tear drop-shaped red blood cells. Other laboratory abnormalities at diagnosis include anemia, leukocytosis or leukopenia, thrombocytosis or thrombocytopenia, and increased serum levels of lactate dehydrogenase (LDH). Bone marrow examination reveals both “cellular phase” and “overtly fibrotic” stages of the disease. In both instances, the most characteristic feature is the presence of dense megakaryocyte clusters with atypical megakaryocyte morphology (cloud-like nuclear morphology) that is accompanied by increased granulocyte proliferation and reduced erythropoiesis. Additional histological features of advanced disease include new bone formation and intra-sinusoidal hematopoiesis. Diagnosis Bone marrow examination is essential in the diagnosis of PMF and should be accompanied by mutation screening for BCR-ABL in order to exclude the diagnostic possibility of CML and JAK2V617F in order

P

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exclude the possibility of bone marrow fibrosis associated with non-malignant condition, lymphoid disorder, or metastatic cancer. It should be noted, however, that JAK2V617F can not distinguish PMF from other myeloid disorders such as MDS, ET, PV, or other MPD. Therefore, accurate diagnosis requires careful morphological evaluation. Cytogenetic abnormalities occur in approximately half of the patients with PMF at diagnosis and include del(20)(q11;q13), del(13) (q12;q22), trisomy 8, trisomy 9, del(12)(p11;p13), monosomy or long arm deletions involving chromosome 7, and partial trisomy 1q). Although none of these abnormalities are specific to PMF, the presence of either del(13)(q12;q22) or der(6)t(1;6)(q21–23; p21–23) is strongly suggestive of the specific diagnosis. Prognosis and Treatment Causes of death in PMF include development of blast phase PMF, which occurs in ~10% of patients during the first decade of their disease, and infections. Survival in PMF is estimated by the use of one of several prognostic scoring systems (PSSs) that rely on the presence or absence of well-established adverse prognostic features. Among the latter, the Mayo Clinic PSS has been reported to be superior, compared to other PSSs, in delineating both good risk and intermediate risk disease categories. The Mayo PSS is based on four adverse prognostic variables: hemoglobin < 10 g/dL, platelet count antagonist Agonist > > antagonist Agonist > > antagonist Inactivation Soluble active TNF-α Shedding Active HB-EGF Active IL-1β FGF2 release TGF-β release VEGF release Angiogenic peptides Angiostatin Soluble ectodomain Active IGF release Active MMP-1 Active MMP-3 Active MMP-7 Active MMP-8 Active MMP-9 Active MMP-13 Inactivation Inactivation Inactivation

Stromelysin-1

The additional adenine in the 6A allele increases the binding of a NF-κB p50/p50 dimer leading to a lower level of stromelysin-1 transcription. This polymorphism has been associated with disease severity in diverse pathologies including ▶cancer. (v) A stromelysin-1 PDGF response element (SPRE) at – 1584/–1571 which binds the stromelysin-1 PDGF response element binding protein (SPBP). SPBP transactivates the stromelysin-1 promoter in response to PDGF through a κ/ι ▶PKC-dependent pathway requiring cooperativity with the AP-1 site. (vi) A nerve growth factor response element (NGFRE) at –241/–229, which binds the interferon response element binding factor-1 (IREBF-1). The transcription of stromelysin-1 was shown to be increased by IREBF-1 in response to the nerve growth factor (NGF). Besides these well defined responsive elements, variation in the levels of stromelysin-1 transcription by other compounds has been reported: (i) ▶oncostatin M, which induces stromelysin-1 in human chondrocytes, (ii) protein synthesis inhibitors such as cycloheximide and anisomycin, which induce an increase in levels of stromelysin-1 messenger RNA in human fibroblasts, (iii) ▶transforming growth factor β (TGF-β), which is able to repress the activation of the stromelysin-1 transcription by EGF, Ras and Src in fibroblasts, and (iv) hormones, which are also inducers or repressors of stromelysin-1 transcription. Glucocorticoids are able to inhibit the increase in stromelysin-1 messenger RNA induced by EGF, PMA and IL-1β in fibroblasts. On the one hand, ▶retinoic acid, like glucocorticoids in fibroblasts, is able to repress the stromelysin-1 expression and on the other hand to increase its induction by NGF in pheochromocytoma cells. Oestradiol and progesterone inhibit stromelysin-1 expression during the menstrual cycle. Androgens have been reported to decrease the expression of stromelysin-1 induced by PMA in prostate carcinoma cells. Unfortunately, the mechanism whereby all these compounds exert their effects remains unclear. A post-transcriptional regulation of stromelysin-1 was also described. Indeed, activation of p38 alpha mitogen-activated protein kinase (▶MAP Kinase) enhances stromelysin-1 expression by messenger RNA stabilization. All the different responsive elements and effectors listed above point out the complexity and the sensitivity of stromelysin-1 regulation. It reflects more particularly the equilibrium found at every control level between positive and negative signals. This is a strategy required for all the MMPs to obtain a very subtle-tuned expression in response to various stimuli in different cellular types and biological events. Stromelysin-1 and Cancer It would be simplistic to consider stromelysin-1 just as a proteolytic enzyme hydrolysing components of

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the ECM. Stromelysin-1 is able to generate and release numerous active and latent molecular signals or regulate pre-existing molecular signals. In this way, stromelysin-1, like other MMPs, can modulate the cellular environment at different levels and is therefore able to influence the behaviour and the future of the cells. It can be easily understood that misregulation of such a gene is associated with severe erosive and invasive pathologies such as tumor growth and invasiveness. Stromelysin-1, like numerous other MMPs, is expressed in different tumors and its presence correlates with their aggressiveness. Its expression has been shown in vivo, notably in carcinomas: mammary carcinoma, colorectal carcinoma, lung carcinoma, prostate carcinoma, pancreatic carcinoma, oesophageal carcinoma or cutaneous carcinoma. It is noteworthy that stromelysin-1 has been often shown to co-express with the oncoprotein Ets-1, which is considered, among all the other Ets proteins, as an independent marker of poor prognosis in various cancer types. During the earliest stages of tumor development, stromelysin-1 is predominantly localized at the level of the fibroblasts of the ▶tumor stroma. Stromelysin-1 induces, by its proteolytic activity towards numerous compounds, the release of growth factors which act directly on tumor cells and other surrounding cells, including fibroblasts, inflammatory cells and endothelial cells. Stromelysin-1 in degrading perlecan releases FGF2 (or basic ▶fibroblast growth factor) which in the setting of cancer is known for its potential to induce ▶angiogenesis and might be involved in desmoplasia in tumors, owing to its role in fibrosis. In degrading decorin, another proteoglycan, stromelysin-1 releases TGF-β, a growth factor implied in cell growth and proliferation. Stromelysin-1 is able to cleave extracellular proteins sequestering growth factors such as ▶insulin-like growth factor binding protein 3 (IGFBP-3) which binds to insulin-like growth factor II (IGF II). Stromelysin-1 can also release active ▶heparin-binding EGF-like growth factor (HB-EGF) from cell surfaces by cleaving it at a site just outside the cell membrane. ▶Chemokines are known targets of stromelysin-1 and other MMPs. For example, stromelysin-1 has been shown to transform, by partial proteolysis, the precursor of IL-1β in an active molecule, influencing tumor-infiltrating inflammatory cells. Stromelysin-1 is also involved in the loss of cellular adherence and the phenotypic modification of epithelial cells, which occur during the early stages of carcinomatoid tumor development. Thus, stromelysin-1 cleaves the extracellular domain of the ▶adherens junction protein ▶E-cadherin. The soluble fragment of E-cadherin which is released prompts cells to disaggregate and promotes tumor-cell ▶motility in a paracrine manner, by interfering with the function of other full length E-cadherin

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molecules. Cleavage of E-cadherin also triggers the ▶epithelial-to-mesenchymal transition (▶EMT), promoting cancer cell invasiveness. Acquisition of an invasive phenotype by the tumor often goes with a gain of MMP expression by the tumor itself. Indeed, stromelysin-1 expression is associated with invasive carcinomas. Some ▶squamous cell carcinoma (SCC) tumor cells can express stromelysin-1, providing further activity for a tumor-driven proteolytic cascade, in which MMP-13 can be activated by stromelysin-1 expressed by tumor cells. In addition, many compounds secreted by tumor-infiltrating inflammatory cells as well as by tumor or stromal cells are capable of modulating MMP expression. Tumor cells can also secrete factors, such as extracellular matrix metalloproteinase inducer (EMMPRIN), which enhances the expression of several MMPs, including stromelysin-1 by fibroblasts. Stromelysin-1 has also a role in tumoral neoangiogenesis. Inhibition of the stromelysin-1 activity decreases neovascularization drastically in a murine model of ▶colon cancer. In addition, the release of proangiogenic factors by stromelysin-1 is also in agreement with its known role in angiogenesis: (i) release of FGF2; (ii) release of vascular endothelial growth factor (VEGF) isoform VEGF165, as a consequence of degradation of its natural inhibitor, the connective tissue growth factor (CTGF); (iii) release of active HB-EGF; (iv) production of an angiogenic polypeptide, owing to the cleavage of a matricellular protein, ▶SPARC (secreted protein acid rich in cysteine). Nevertheless, stromelysin-1 can also generate ▶anti-angiogenic factors. Indeed, stromelysin-1 generates angiostatin by cleaving plasminogen and might be involved in the generation of ▶endostatin, a C-terminal fragment of the basement-membrane collagen type XVIII. It indicates that expression of stromelysin-1 in the tumor periphery might also serve to limit or regulate angiogenesis induced by the tumor. Another role of stromelysin-1 as a negative regulator of tumor expansion might exist. In fact, stromelysin-1 is able to cleave and inactivate ▶CXCL12, a ligand for CXC chemokine receptor 4 (CXCR4) on leukocytes. ▶Breast cancer cells express CXCR4 and it has been shown that inhibition of the binding of CXCL12 to CXCR4 by blocking antibodies reduces ▶metastasis in vivo. Therefore, cleavage of CXCL12 by stromelysin-1 might inhibit metastasis. Stromelysin-1 can act as a natural tumor promoter. It can induce premalignant lesions and favor tumor emergence on its own. These observations come from using transgenic mice overexpressing stromelysin-1 specifically in the mammary glands. Mammary tissue of these transgenic mice presents the characteristics of stroma reaction with collagen accumulation, neovascularization, tenascin-C expression and upregulation of endogeneous stromelysin-1. These changes are

hallmarks of cancer ▶progression and may even predispose towards neoplastic epithelial transformation. Thus, overexpression of stromelysin-1 gives rise to changes that could potentially promote mammary ▶carcinogenesis. This is confirmed in older animals from 6–24 months of age, which develop with an important incidence, spontaneous premalignant lesions and mammary cancers. A recurrent genomic instability has been shown by array comparative genomic hybridization (▶ArrayCGH) in these different premalignant and malignant lesions. In addition, stromelysin-1 plays an active role in EMT. Thus, overexpressing this enzyme in normal mammary epithelial cells leads to an important morphological change with a loss of cell-cell adhesions and vimentin synthesis revealing EMT. This was confirmed in vivo using transgenic mice overexpressing stromelysin-1. A recent study indicates that stromelysin-1-mediated EMT is due to the expression of Rac1b, an isoform of Rac1 GTPase, which causes an increase in cellular ▶reactive oxygen species (▶ROS). This increase in the ROS stimulates the expression of the transcription factor ▶Snail, promotes EMT and causes oxidative damage to DNA and genomic instability. This may represent a key event in the stromelysin-1-induced phenotypic and genotypic malignant transformation in normally functioning cells. These results are supported by studies in human cancers where the 5A allele of stromelysin-1 (see above, regulation of stromelysin-1) corresponding to higher levels of stromelysin-1 transcription may represent an unfavourable prognostic feature in breast cancer patients associated with more invasive disease. The 5A allele might also be associated with the increased susceptibility to non small cell lung cancer among smokers and a risk of development and lymphatic metastasis in oesophageal squamous cell carcinoma. On the contrary, other studies using stromelysin-1 transgenic animals showed a reduction in the number of mice developing mammary tumors following treatment by a chemical carcinogen. An ▶apoptosis induction of mammary epithelial cells in transgenic animals overexpressing stromelysin-1 (possibly by degrading laminin) has been reported. In addition, in stromelysin1-deficient mice, topical applications of carcinogens resulted in skin tumors that grew at a faster initial rate than did carcinogen-elicited tumors in wild type mice. This enhanced tumor development was correlated with a reduction in the tumor inflammatory cell infiltrate. Thus, the presence of stromelysin-1 appears to be a protection by an increased influx of inflammatory cells to the area of carcinogenesis. All these data point out that the proteolytic activity of stromelysin-1 and other MMPs towards an always growing number of matrix and non-matrix substrates renders it difficult to predict the effect of cleavage in a complex biological context. This is all the more

Structural Biology

real in cancer progression which involves a continuous interplay between tumor cells, stroma cells, and inflammatory cells. It becomes clear that the biological consequences of this activity can lead to either stimulation or inhibition of tumoral development. Validation of true substrates in vivo and a better understanding of the biological properties of the cleavage products is required to find an efficient therapeutic use for the inhibitors of stromelysin-1 and other MMPs.

References 1. Chakraborti S, Mandal M, Das S et al. (2003) Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem 253:269–285 2. Fingleton B (2006) Matrix metalloproteinases: roles in cancer and metastasis. Front Biosci 11:479–491 3. McCawley LJ, Crawford HC, King LE et al. (2004) A protective role for metalloproteinase-3 in squamous cell carcinoma. Cancer Res 64:6965–6972 4. Overall CM (2002) Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol 22:51–85 5. Radisky DC, Levy DD, Littlepage LE et al. (2005) Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436:123–127

Structural Biology M ICHAEL H ODSDON 1 , E LIAS LOLIS 2 1

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA 2 Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA

Definition Structural biology involves biophysical methods that can determine the three-dimensional structure of macromolecules. These structures can lead to an understanding of the molecular basis of cancer and identify the atomic details necessary for drug design, optimization, and development.

Characteristics The two methods that can elucidate atomic-level structures of biological macromolecules – e.g., proteins, DNA, RNA, and complexes between/among these molecules – are ▶X-ray crystallography and ▶nuclear magnetic resonance spectroscopy (▶NMR). For X-ray crystallography, crystals are necessary for determining structures.

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In contrast, NMR is used to determine structures of molecules in solution. In crystallography, X-rays are diffracted by the crystal creating the data necessary to determine the structure. NMR experiments are based on the property of nuclear spin, inherent to specific nuclear isotopes such as 1H, 13C and 15N. A majority of NMRderived structural restraints are derived from magnetic couplings between nearby nuclei. Structures of the same macromolecule determined by each method are essentially identical. However, each technique presents its own advantages and disadvantages and, together, the two techniques are highly complementary. In practical terms, NMR spectroscopy is limited in the size of the protein investigated, with structures 5 cm), high tumor grade, advanced (metastatic) disease and (higher) patient age. Treatment with chemotherapy (ifosfamide, either alone or in combination with doxorubicin) has been reported to increase the overall survival rates, also

Synovial Sarcoma. Figure 1 The synovial sarcoma specific t(X;18)(p11;q11) chromosomal translocation. The normal chromosomes X and 18 with the respective breakpoints are shown to the left. The derivative-X chromosome, with the breakpoint-associated genes (SS18, SSX1, SSX2 and SSX4) is shown to the right.

of patients with high grade tumors and metastases. Despite these findings, there is ample room for further improvement and optimization of the (differential) diagnosis and treatment of human synovial sarcomas. To enable this, detailed information about the molecular mechanisms underlying synovial sarcoma development is of imperative importance. The tumorigenic nature of the SS18-SSX fusion protein has been established in vitro and in vivo (▶oncogene). Further, functional, analysis of the SS18 and SSX genes has revealed that they encode nuclear proteins that exhibit opposite transcriptional regulatory activities. The SS18 protein functions as a transcriptional co-activator which interacts directly with the transcription factor AF10, the co-activator CoAA, and several members of the epigenetic chromatin remodeling (BRM and BRG1) (▶chromatin Remodeling in Cancers) and modification machineries (p300 and SIN3A) (▶p300/CBP Co-Activators). In contrast, the SSX proteins function as transcriptional co-repressors which interact with the RAB3IP and SSX2IP proteins and the transcription factor LHX4, and are associated with histones and several Polycomb group repressor proteins. The domains involved in these apparently opposite transcription regulatory activities are retained in the SS18-SSX fusion proteins. Therefore, these may function as “activator-repressors” of transcription, which can bind to target DNA through the AF10 and LHX4 transcription factors (Fig. 2). This notion implies that the SS18 and/or SSX protein functions may be impaired in the SS18-SSX fusion protein. Alternatively, the fusion

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Syntenic

Synovial Sarcoma. Figure 2 Model depicting the synovial sarcoma associated SS18, SSX and SS18-SSX (fusion) proteins and their respective interactions. In normal cells (left) the SSX proteins and their interactors SSX2IP and RAB3IP may associate with the Polycomb repressor complex and histones and in addition, through interaction with the LHX4 protein, bind to cognate DNA sites and affect target gene expression. The SS18 protein can interact with several members of the SWI/SNF chromatin remodeling complex (BRM and BRG1), but also with proteins involved in covalent chromatin modifications (p300 and Sin3A) and the co-activator CoAA. In addition, through interaction with the transcription factor AF10, SS18 may bind to cognate DNA sites and affect target gene expression. In synovial sarcoma cells (right) the SS18-SSX fusion proteins have lost the interaction domains for SSX2IP and RAB3IP, but have retained the interaction/association domains for both the SWI/SNF and the Polycomb complexes. Through these interactions, the SS18-SSX fusion proteins may anomalously affect the regulation of these target genes and/or affect the regulation of other (novel) target genes, either through AF10, LHX4, or both.

protein may have gained novel functions. A recent functional analysis revealed that the SS18-SSX fusion protein influences the process of ▶epithelial to mesenchymal transition (EMT) which is commonly observed in tumor cells. This example of a SS18-SSX gain-of-function may underlie the above-mentioned histologic differences among synovial sarcomas. A recent study has indicated that novel treatment options for synovial sarcoma patients may include so-called “epigenetic” drugs (▶epigenetic Therapy). Synovial sarcoma cell lines were shown to be extremely sensitive to the histone de-acetylase inhibitory drug Romidepsin (also known as FK228 or depsipeptide), both in vitro and in vivo. As yet, the exact mode of action of this broad spectrum “epigenetic” drug on synovial sarcoma growth is unknown. However, it is to be expected that detailed knowledge on the molecular mechanisms underlying synovial sarcoma pathogenesis will be instrumental for obtaining insight into its mode of action and, thus, the development of more targeted therapies.

References 1. de Bruijn DRH, Nap JP, van Geurts Kessel A (2007) The (epi)genetics of human synovial sarcoma. Genes Chromosomes Cancer 46:107–117

2. Haldar M, Hancock JD, Coffin CM et al. (2007) A conditional mouse model of synovial sarcoma: insights into a myogenic origin. Cancer Cell 11:375–388 3. Guillou L, Benhattar J, Bonichon F et al. (2004) Histologic grade, but not SYT-SSX fusion type, is an important prognostic factor in patients with synovial sarcoma: a multicenter, retrospective analysis. J Clin Oncol 22:4040– 4050 4. Ito T, Ouchida M, Morimoto Y et al. (2005) Significant growth suppression of synovial sarcomas by the histone deacetylase inhibitor FK228 in vitro and in vivo. Cancer Lett 224:311–319 5. Raney RB (2005) Synovial sarcoma in young people: background, prognostic factors, and therapeutic questions. J Pediatr Hematol Oncol 27:207–211

Syntenic Definition Refers to genes or genetic loci that lie on the same chromosome, i.e. are genetically linked. ▶Amplification

Synuclein

Synthetic Cannabinoids ▶Cannabinoids

Synthetic Chemoprotectants Definition Chemoprotectants that are artificially and chemically synthesized. ▶Chemoprotectants

Synuclein J ULIA M. G EORGE Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, IL, USA

Definition Synucleins are small cytosolic proteins of uncertain function, normally expressed at high levels in the vertebrate nervous system. Increased expression of synuclein proteins, especially γ-synuclein, is associated with progression of a variety of tumors.

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Synuclein Expression in Cancer The first synuclein family member to be associated with cancer was γ-synuclein, which was originally named ▶breast cancer-specific gene 1 (▶BCSG1), due to its specific expression in infiltrating breast carcinoma as compared to normal breast tissue. The other synuclein proteins are also somewhat associated with certain cancers. For example, 87% of ▶ovarian cancers display increased expression of one or more synuclein family members, while 42% express all three. The γ-synuclein isoform is particularly associated with cancer. Overexpression of γ-synuclein is observed in a large percentage of tumors from varied tissues of origin, but not in adjacent non-neoplastic tissues. Expression of γ-synuclein in cancer increases in a stage-specific manner, with moderate expression in stage I tumors, and very high expression in stages III-IV. Increased expression of γ-synuclein in tumor cells apparently results from deregulation of normal expression, as no γ-synuclein mutations or gene amplifications have been associated with cancer. Tissue specific expression of γ-synuclein is mediated by methylation of ▶CpG islands in exon 1, and ▶hypomethylation at these sites has been observed in γ-synuclein overexpressing tumor cells. The increased expression of γ-synuclein observed in advanced cancer is mostly likely the consequence of a loss of ▶epigenetic gene silencing of γ-synuclein expression during tumor progression, rather than a primary event in tumor initiation. Misfolding of α-synuclein protein is associated with both familial and sporadic ▶Parkinson’s disease (PD), and mutations in α-synuclein that alter its sequence or increase its expression cause early-onset PD. PD patients have an increased risk of both ▶melanoma and breast cancer, as compared to the general population, but a decreased risk for many other cancers. This may reflect the involvement of common genes in PD and cancer.

Characteristics The precise function of the synuclein proteins is not well understood. All family members (α-synuclein, ▶SNCA; β-synuclein, ▶SNCB; and ▶γ-synuclein, ▶SNCG) share a conserved domain related to the lipid-binding domains of the ▶exchangeable apolipoproteins, the major lipid transporters in blood. This conserved domain mediates reversible interactions with lipid membranes. α-Synuclein regulates the uptake and incorporation of fatty acids into phospholipids, and mutations in α-synuclein alter the composition of cellular membranes. Each of the synuclein isoforms possesses a unique tail domain, which may mediate distinct physiological actions. α- and β-synuclein are normally expressed in the ▶central nervous system, while γ-synuclein expression is found throughout the central and ▶peripheral nervous systems, and at lower levels in some nonneural tissues. Synuclein family genes have so far been identified only in vertebrate species.

Role in Tumor Progression γ-Synuclein expression in breast cancer cell lines increases cell proliferation. It interacts with the mitotic spindle checkpoint control protein ▶BubR1, promoting its degradation by the proteasome and overriding mitotic arrest. Loss of spindle checkpoint control can result in ▶aneuploidy, the state of having the wrong number of chromosomes. γ-Synuclein augments proliferative signaling through the ▶estrogen receptor pathway, by acting as a molecular chaperone to increase ▶estradiol binding to the receptor. γ-Synuclein also alters signaling via the ▶MAP kinase pathway, which plays a key role in the regulation of cell proliferation. γ-Synuclein enhances cell motility and invasiveness, and its overexpression in established tumors may drive malignant tumor progression. γ-Synuclein promotes production of ▶matrix metalloproteinases, secreted

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proteases that break down the extracellular matrix and allow tumors to invade surrounding tissues and blood vessels, thereby facilitating ▶metastasis. γ-Synuclein expression also increases resistance to certain chemotherapeutic agents that trigger ▶apoptosis via MAP kinase and/or ▶JNK signaling pathways, e.g. ▶taxol, vinblastine, and ▶paclitaxel. Overall, γ-synuclein expression in a tumor correlates with a poor clinical prognosis. Its expression in primary tumors is associated with the presence of distant metastases. This protein may prove to be a useful ▶biomarker for detecting cancer, ▶staging tumor progression, and evaluating metastatic potential.

References 1. Ahmad M, Attoub S, Singh MN et al. (2007) Gammasynuclein and the progression of cancer. FASEB J 21:3419–3430 2. George JM (2002) The synucleins. Genome Biol 3: Reviews3002 3. Lavedan C (1998) The synuclein family. Genome Res 8:871–880

Systemic Antibody-directed Radionuclide Therapy ▶Radioimmunotherapy

Systemic Chemotherapy Definition

Oral or intravenous administration of ▶chemotherapy

Systemic Clearance Synuclein a Definition

A member of the ▶synuclein protein family. Also known as NACP. Encoded by the gene SNCA.

Definition A measure of the efficiency with which a drug is removed from the body. It is proportional to the dose and inversely proportional to the Area under the Curve (AUC). ▶Lead Optimization

▶Synucleins

Synuclein b

Systemic Inflammatory Response Syndrome

Definition

A member of the ▶synuclein protein family. Also known as PNP14. Encoded by the gene SNCB. ▶Synucleins

Synuclein g

Definition SIRS; A syndrome proposed by American College of Chest Physicians in 1992. It is defined as a clinical response to a nonspecific insult of either infectious or noninfectious origin. SIRS is defined as two or more of the following variables:

A member of the ▶synuclein protein family. Also known as persyn or synoretin. Encoded by the gene SNCG.

1. Fever of more than 38°C or less than 36°C 2. Heart rate of more than 90 beats per minute 3. Respiratory rate of more than 20 breaths per minute or a PaCO2 level of less than 32 mm Hg 4. Abnormal white blood cell count (>12,000/μl or 10% bands)

▶Synucleins

▶Sivelestat

Definition

Systems Biology

Systemic Lupus Erythematosus

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Systemic Treatment or Therapy

Definition

Definition

SLE; Systemic lupus erythematosus is an autoimmune disease in which autoantibodies against DNA, RNA, and proteins associated with nucleic acids from immune complex that damage small blood vessels, especially of the kidney.

Therapy theoretically delivered to the entire body, and is distinguished from ▶local therapy. ▶Chemotherapy is the most common form of systemic therapy. Its purpose can be either curative or palliative.

Systemic Spread of Cancer ▶Metastatic Colonization

Systemic Therapy ▶Neoadjuvant Therapy

▶Induction Chemotherapy

Systems Biology Definition A scientific discipline that seeks to understand the dynamic nature of a life by quantitatively integrating the network of interactive sub-processes (metabolic pathways, gene expression, cell-cell interactions, etc.) occurring within an organismal unit in a manner that can accurately predict the net outcome(s) of perturbing or stimulating any of those sub-processes. ▶Drug Design

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T

T Definition

activating macrophages, and are sometimes called inflammatory CD4 T cells. Th1 cells enhance cellmediated immune responses and are essential for controlling intracellular pathogens such as viruses and bacteria.

Testosterone. ▶Cyclin G-Associated Kinase

T Cell Definition

▶Thymus-dependent lymphocyte; A leukocyte, synonym as T lymphocyte, which is part of the adaptive immune system; T cell (CD8+) cytolytic action requires presentation of antigens by antigen-presenting cells (APCs) T cells are capable of forming a memory (CD4+).

▶Chemokine

TH2 Cells Definition Are a subset of CD4 T cells that are characterized by the cytokines they produce. They are mainly involved in stimulating B cells to produce antibody, and are often called helper CD4 T cells. ▶Sjögren Syndrome

▶Natural Killer Cell Activation

T Cell Receptor Complex Definition

TCR complex; Consists of the α and β chain of the T cell receptor heterodimer involved in ▶MHC/peptide recognition and of the CD3 complex that consists of multiple chains involved in intracellular signal generation.

TH3 Cells Definition Are unique cells that produce mainly transforming growth factor-β (▶TGF-β) in response to antigen; they develop predominantly in the mucosal immune response to antigens that are presented orally.

▶Chimeric T Cell Receptors

T Helper TH1 Cells Definition Definition Are a subset of CD4 T cells that are characterized by the cytokines they produce. They are mainly involved in

CD4+ T helper (Th) subsets are characterized by their distinct cytokine production profiles. Th1 cells secrete interleukin-2 (IL-2), IFNγ and TNFβ, which promote cellular immune responses against intracellular pathogens

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and viruses. Th2 cells produce IL-4, IL-5, IL-6 IL-10 and IL-13, which promote humoral immunity by aiding in B cell growth and differentiation. ▶Interleukin-4

T Lymphocyte Definition T cell; type of lymphocyte responsible for cell-mediated immunity; includes cytotoxic cells and helper T-cells. ▶Cancer-Germline (CG) Antigens

T Helper Lymphocytes Definition

Subset of ▶T lymphocytes that express the CD4 surface marker and are capable of enhancing antibody and cellular immune responses. Helper T cells recognize antigen in the form of peptides complex onto class II major histocompatibility complex molecules and produce lymphokines that regulate immune responses.

T-lymphoma Invasion and Metastasis ▶Tiam1

▶Peptide Vaccines for Cancer

T-PLL T-Helper 1 Response

Definition

▶T-Prolymphocytic Leukemia.

Definition Th1 Response; Type of immune response that primarily induces cell-mediated immunity and the activation of cytotoxic effector T cells. Associated with and induced by the release of a characteristic set of cytokines including interleukin-2, interferon-gamma and interleukin-12. It is generally believed that a strong Th1 immune response is required for effective cell-mediated antitumor immunity.

T-Prolymphocytic Leukemia (T-PLL) Definition T-Prolymphocytic leukemia (T-PLL) is a disease that represents 20% of prolymphocytic leukemias, occurs at an advanced age of 70–80 years.

T-Loop

▶Acute Promyelocytic Leukemia

Definition

Of telomeric DNA is formed in each ▶telomere end by invasion of its duplex region by the single-stranded (TTAGGG)-rich 3’-overhang. This structure, which hides the very end of the chromosome, protects chromosome form degradation and loss of vital sequence, block end-end fusion. ▶Stem Cell Telomeres

TAA Definition Tumor-associated Antigen.

Tamoxifen

TACE

TAM

Definition

Definition

Acronym for tumor necrosis factor-α (TNF-α) converting enzyme.

Acronym for Tumor-associated Macrophages.

▶ADAM17 ▶ADAM Molecules

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▶Tumor-Associated Macrophages

Tamoxifen TAK

V. C RAIG J ORDAN Fox Chase Cancer Center, Philadelphia, PA, USA

Definition

Definition

Tat-Associated Kinase, also known as positive-acting transcription elongation factor complex (P-TEFb), is a complex of cellular proteins involved in regulation of gene transcription.

Tamoxifen is a nonsteroidal antiestrogen used for the treatment and prevention of ▶estrogen receptor (ER) positive breast cancer. Tamoxifen is the most studied anticancer agent.

▶TAT Protein of HIV

Characteristics

Takatsuki Disease Definition

▶POEMS Syndrome.

Tall Cell Variant of Papillary Carcinoma Definition Papillary carcinoma of the thyroid characterized by a cell whose height is two- to three times its width and whose cytoplasm is ample and oncocytic. The cell has the characteristic nuclear features of papillary carcinoma. ▶Hurthle Cell Adenoma and Carcinoma

Background Tamoxifen (ICI 46,474) was first described as an effective postcoital contraceptive in rats but the drug induces ovulation in subfertile women. The compound was subsequently reinvented throughout the 1970’s as an agent to be targeted to OER positive breast cancers for use as a long-term adjuvant therapy with potential use for the chemoprevention of breast cancer (▶Estrogenic hormones and cancer, ▶hormones and ▶cancer). Adjuvant Therapy Studies throughout the 1980’s and 1990’s demonstrated that long-term adjuvant tamoxifen therapy (5 years) produced dramatic increases in disease-free survival and overall survival. These data were observed in patients who were classified as Stage I and Stage II breast cancers and the compound is effective in both pre and postmenopausal women. Tamoxifen is cheap and effective with availability in generic form in countries throughout the world. Tamoxifen is credited for increasing survivorship and saving the lives of 500,000 women. The appropriate use of tamoxifen as the gold standard for the endocrine treatment of breast cancer throughout the 1990’s, is credited in contributing to the decrease death rate from breast cancer observed in the United States and other countries around the world. However, concerns about side effects (see below) and the development of drug resistance has led to the development of the aromatase inhibitors as a substitute

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for tamoxifen in the adjuvant treatment of breast cancer in postmenopausal women. Current studies demonstrate that aromatase inhibitors have an improved side effect profile compared to tamoxifen and improved disease-free and overall survival. Chemoprevention Laboratory studies first showed that tamoxifen could prevent the development of carcinogen-induced rat mammary tumors. Randomized clinical trials around the world have demonstrated that tamoxifen can reduce the incidence of breast cancer by 50% in high risk pre and postmenopausal women. High risk refers to the mathematical model (Gail Model) that is available on the internet through the National Cancer Institute in the United States that will determine the 5 year and lifetime risk of a woman developing breast cancer. Some of the risk factors used in the Gail Model are age, start of menses, termination of menses, age when the woman had children, if the woman had children, breast biopsies, ductal hyperplasia, and first degree relatives developing breast cancer. Concerns about the side effects of tamoxifen (see below) have focused the use of tamoxifen in the premenopausal population where the risk/benefit ratio is high. In other words, tamoxifen effectively reduces the incidence of breast cancer but the side effect profile is low. Most importantly, after women take a 5 year course of tamoxifen during their premenopausal years, the beneficial effects of tamoxifen in preventing breast cancer may last for up to another 10 years but the side effects of hot flashes, etc. will disappear. Recent studies of a related compound, Raloxifene that is used for the prevention of osteoporosis, shows that this selective estrogen receptor modulator or (SERM) will prevent breast cancer in women taking raloxifene to prevent osteoporosis. A recent clinical trial in the United States called the Study of Tamoxifen and Raloxifene (STAR) demonstrated in postmenopausal women that tamoxifen and raloxifene were equally effective in preventing an increase in invasive breast cancer but raloxifene had a superior side effect profile. Mechanism of Action Tamoxifen is lipophyllic and well absorbed from the gastrointestinal tract. Patients accumulate tamoxifen over the first 4 weeks of treatment when they reach steady-state. The drug has a long biological half-life so that even if treatment is stopped, the drug can be detected in the blood for up to 6 weeks. Tamoxifen is a prodrug that is metabolically activated by the CYP2D6 enzyme system to the compounds 4-hydroxytamoxifen and endoxifen both of which have a high affinity with the OER. It is important to note that CYP2D6 enzyme system can be blocked by certain selective serotonin reuptake inhibitors (SSRIs) and this may impair the

antitumor actions of tamoxifen. Patients often take SSRIs to reduce the incidence of hot flashes in patients taking tamoxifen. Tamoxifen binds to the ligand binding domain of the OER in the breast tumor and causes a conformational change that is distinct from the natural estradiol OER complex. As a result, tamoxifen is unable to cause gene activation and completely mimic estrogen action. However, tamoxifen is not a complete antiestrogen. The tamoxifen estrogen receptor complex retains estrogen-like actions which can switch on and switch off target sites around a patient’s body. For example, tamoxifen is an antiestrogen in the breast but has estrogen-like properties in bones and in the uterus. Tamoxifen is classified as a selective estrogen receptor modulator. Side Effects Tamoxifen exhibits specific estrogen-like effects and also antiestrogenic effects. Tamoxifen is sufficiently estrogenic to stimulate the uterine endometrium and enhance the growth of OER positive ▶endometrial cancers. Tamoxifen causes a fivefold increase in endometrial cancer compared with women not taking tamoxifen. In other words, if a thousand 60 year postmenopausal women were followed for endometrial cancer, one woman per year would develop endometrial cancer. In contrast, if those same women were taking tamoxifen, five women would develop endometrial cancer (low grade, early stage) per year. Additionally, the estrogen-like effects of tamoxifen are reflected in an increase in thromboembolic disorders in postmenopausal women. It is important to note that endometrial cancer and blood clots are not elevated in premenopausal women. There is also a significant rise in the diagnosis of cataracts and cataract operations. The STAR trial demonstrated that raloxifene does not increase the risk of endometrial cancer. There were fewer hysterectomies, fewer cataracts, fewer cataract operations and a lower overall incidence of blood clots.

References 1. Jordan VC (2003) Tamoxifen: a most unlikely pioneering medicine. Nat Rev Drug Discov 2:205–213 2. Early Breast Cancer Trialists Collaborative Group (2005) Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Lancet 365:1687–1717 3. Jordan VC, Brodie AMH (2007) Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer. Steroids 72:7–25 4. Jordan VC (2007) Chemoprevention of breast cancer with selective oestrogen-receptor modulators. Nat Rev Cancer 7:46–53

Tankyrases

Tamponade Definition The accumulation of a fluid (blood or serous exudate) in thepericardial sac, leading to compression of the chambers of the heart and leading to increasing heart failure. ▶Cardiac Tumors

Tankyrase-2: TNKL, TNKS2, TANK2 ▶Tankyrases

Tankyrases H IROYUKI S EIMIYA Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Koto-ku, Tokyo, Japan

Synonyms TRF1-interacting, ankyrin-related ADP-ribose polymerases; There are two closely related homologues: Tankyrase-1: TNKS, TNKS1, TANK1; Tankyrase-2: TNKL, TNKS2, TANK2

Definition Tankyrases are members of the poly(ADP-ribose) polymerase (▶Parp) family that regulate telomere length in ▶telomerase-positive human cells. There are two related homologues, tankyrase-1 and tankyrase-2. Tankyrase-1 is a protein of 1327 amino acids and 142 kDa. The gene maps to 8p22-p23. Tankyrase-2 is a protein of 1166 amino acids and 127 kDa. The gene maps to 10q23. Tankyrase-1 is relatively abundant in reproductive tissues (i.e. testis and ovary), whereas tankyrase-2 exhibits rather ubiquitous expression.

Characteristics Structure Tankyrase-1 has four characteristic domains: HPS, ANK, SAM, and PARP. The N-terminal HPS domain is a homopolymeric run of histidine, proline, and serine residues, the functional significance of which is unknown. The ANK domain is composed of a long

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stretch of 24 ANK repeats, providing a platform for protein-protein interactions. Distinct from those of ankyrins, tankyrase-1’s ANK domain is further divided into five, well-conserved subdomains, ARC (ANK repeat cluster) I-V. Each ARC works as an independent, ligand-binding site. The SAM (sterile alpha motif) domain is another module for protein-protein interaction and contributes to self-multimerization of the protein. The C-terminal PARP domain catalyzes ▶poly (ADP-ribosyl)ation of acceptor proteins by using NAD as a substrate. This post-translational modification gives drastic negative charges to the acceptor proteins and often disrupts interactions between the acceptor proteins and their target DNA. Tankyrase-2 is a closely related homologue of tankyrase-1. Amino acid identities between ANK, SAM, and PARP domains of tankyrase-1 and tankyrase-2 are 83, 74, and 94%, respectively. The most striking feature of tankyrase-2 is the absence of an N-terminal HPS domain. Tankyrase-1 and tankyrase-2 can associate in intact cells via their SAM domains to form a multimer. Intracellular Distribution Tankyrase-1 is found at various intracellular loci, including telomeres, mitotic ▶centrosomes, Golgi apparatus, and nuclear pore complexes. Telomeric localization of tankyrase-1 is mediated by its interaction with a telomeric repeat-binding factor 1 (TRF1), which directly binds the double-strand telomere DNA, (TTAGGG)n. During mitosis, tankyrase-1 concentrates around the pericentriolar matrices. This accumulation depends on tankyrase-1’s interaction with nuclear/ mitotic apparatus protein (NuMA), which plays an essential role in organizing microtubules at the spindle poles. In the Golgi apparatus, tankyrase-1 is peripherally associated with the Golgi membranes. In adipocytes and myocytes, it is colocalized with GLUT4 (glucose transporter 4) storage vesicles in the juxtanuclear region of the cells, where it specifically binds to insulin-responsive amino peptidase (IRAP). The intracellular localization of tankyrase-2 has been characterized less often than tankyrase-1. So far, it has been reported that tankyrase-2 localizes predominantly to a perinuclear region, similar to the properties of tankyrase-1. Upon subcellular fractionation, both tankyrase-1 and tankyrase-2 are predominantly recovered in the low-density microsomal fraction, which contains vesicular ▶endosomal compartments. Binding Partners To date, various tankyrase-1- and tankyrase-2-binding proteins have been reported with a consensus RXX(P/A) DG motif as a canonical tankyrase-binding site. Such proteins include TRF1, NuMA, IRAP, Grb14 signaling adaptor protein, tankyrase-binding protein of 182 kDa (TAB182), Mcl-1 apoptotic regulator, and Epstein-Barr

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virus nuclear antigen-1 (EBNA-1). TRF1, NuMA, IRAP, TAB182, EBNA-1, and tankyrase-1 and tankyrase-2 themselves have been shown to be poly(ADP-ribosyl) ated by tankyrases. PARP inhibitory compounds, such as 3-aminobenzamide, PJ-34, and 4-amino-1,8-naphthalimide, block this poly(ADP-ribosyl)ation. Functions Depending on the binding partner and subcellular localization, tankyrase-1 is involved in several distinct biological events, including telomere elongation, cell division control, and insulin-stimulated glucose uptake. Also, the coexistence of multiple ARCs and an oligomerforming SAM domain is implicated in the master scaffolding function of tankyrase-1 (and tankyrase-2), which could work as an intracellular “molecular lattice.” Telomere Elongation Classical DNA replication machinery cannot replicate the very ends of linear DNA; ▶end replication problem. Accordingly, native capping structures at the ends of chromosomes, telomeres, gradually erode after each round of the cell cycle in somatic cells. Most immortalized cells, including germ cells and 80–90% of cancer cells, maintain their telomere length by activating telomerase. Telomere elongation by telomerase requires its enzyme activity and accessibility to the substrate, 3′-overhang of telomere DNA (also called as the telomeric G-tail). Telomere access to telomerase is repressed in cis by the telomeric protein TRF1. Thus, TRF1 directly binds an array of double-strand telomere DNA, (TTAGGG)n, and recruits additional telomerebinding proteins, such as TIN2 (TRF1-interacting nuclear factor 2), TPP1 (originally designated PTOP (POT1- and TIN2-organizing protein), PIP1 (POT1interacting protein 1) or TINT1 (TIN2-interacting protein 1)), and POT1 (protection of telomeres 1), to the chromosome ends. The resulting TRF1-TIN2TPP1-POT1 complex diminishes accessibility of telomerase to the telomeres. According to this mechanism, longer telomeres provide more binding sites for TRF1 and therefore become a less reactive substrate for telomerase. Conversely, shorter telomeres provide fewer binding sites for TRF1 and become a more reactive substrate for telomerase. This balance between open and closed states of the telomeres stabilizes the length of telomeric TTAGGG tracts at each chromosome end of telomerase-positive cells. Tankyrase-1 enhances telomere access to telomerase and contributes to telomere elongation: tankyrase-1 binds to the N-terminal acidic domain of TRF1 via its ANK domain. As described above, the ANK subdomain, ARC, plays a role in association with TRF1. While each of five ARCs can independently recognize TRF1, ARC V, the one closest to the C-terminal, is the most important for telomeric function of tankyrase-1. Interaction between

ARC V and TRF1 enables tankyrase-1 to poly(ADPribosyl)ate TRF1. This post-translational modification eliminates telomere binding of TRF1, resulting in dissociation of the TRF1-TIN2-TPP1-POT1 complex from telomeres. Telomere-unbound TRF1 is degraded by ubiquitin-dependent proteolysis; ▶ubiquitination. These alterations induce a telomere open state and facilitate telomere elongation by telomerase. It is notable that tankyrase-1 enhances telomere access of telomerase but does not increase the enzyme activity of telomerase. Indeed, tankyrase-1 overexpression induces telomere elongation in a telomerase-dependent manner. Tankyrase-1 can form a ternary complex with TRF1 and TIN2. In this complex, poly(ADP-ribosyl)ation of TRF1 is prevented by TIN2. So far, however, when and how tankyrase-1 is activated or inactivated is not fully understood. Like tankyrase-1, tankyrase-2 also can recognize and poly(ADP-ribosyl)ate TRF1. However, the extent of functional redundancy and specificity between tankyrase-1 and tankyrase-2 are largely unknown. Cell Division Control Since ▶siRNA-mediated knockdown of tankyrase-1 causes mitotic arrest of multiple cell types, tankyrase-1 is thought to be required for proper cell division. In normal cell division, each pair of sister chromatids is equally divided into two daughter cells. In some tankyrase-1 knockdown cells, however, sister chromatids can separate at the centromeres and arms but not at the telomeres. Accordingly, cell division is interrupted and abnormal chromosome distribution and spindle morphology occur. In other tankyrase-1 knockdown cells, mitotic arrest occurs with intact sister-chromatid cohesion (arrest at the metaphase). Tankyrase-1 is also required for the assembly of bipolar spindles. It recognizes a centrosomal protein NuMA via its ANK domain, and poly(ADP-ribosyl)ates NuMA in intact cells. Consistently, these two proteins are co-localized at the mitotic centrosomes. Meanwhile, tankyrase-1 is phosphorylated during mitosis by glycogen synthase kinase 3 (GSK3). GSK3 inhibitors, such as lithium chloride and indirubin, inhibit this phosphorylation. Currently, the functional significance of NuMA’s poly(ADP-ribosyl) ation and tankyrase-1’s phosphorylation during mitosis remain unknown. Insulin-Stimulated Glucose Uptake The GLUT4 vesicle is an endocytic compartment within adipocytes and myocytes. This storage vesicle contains the glucose transporter protein, GLUT4, and it regulates glucose uptake upon stimulation with insulin; major fractions of the GLUT4 vesicles usually reside in the trans-Golgi reticulum. Insulin stimulation induces exocytic translocation of the vesicles towards the cell surface, where GLUT4 facilitates glucose uptake. In adipocytes, insulin-stimulated translocation of GLUT4

Target Cells

is mediated by IRAP, another GLUT4 vesicle-resident protein. Tankyrase-1 is implicated in regulation of GLUT4 translocation. As mentioned, tankyrase-1 directly binds IRAP via its ANK domain and can poly(ADP-ribosyl) ate IRAP. Tankyrase-1 knockdown by siRNAs attenuates the insulin-stimulated translocation of GLUT4 vesicles and subsequent glucose uptake. This inhibitory effect of siRNA is reproducible with PJ-34, a PARP inhibitor that is effective against tankyrase-1 (and other PARPs). Tankyrase-1 knockdown does not attenuate the upstream insulin signaling, such as phosphorylation of the ▶insulin receptor, IRS-1 (insulin receptor substrate-1), Akt; ▶AKT signal transduction pathway in oncogenesis, GSK3, and p42/p44 ERKs (extracellular signal-regulated kinases; ▶MAP kinase).

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2. Dynek JN, Smith S (2004) Resolution of sister telomere association is required for progression through mitosis. Science 304:97–100 3. Chang P, Coughlin M, Mitchison TJ (2005) Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat Cell Biol 7:1133–1139 4. Seimiya H, Muramatsu Y, Ohishi T et al. (2005) Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell 7:25–37 5. Seimiya H (2006) The telomeric PARP, tankyrases, as targets for cancer therapy. Br J Cancer 94:341–345

TAP Definition

Clinical Aspects Telomere synthesis by telomerase is the Achilles’ heel of unlimited proliferation of most cancer cells. Continuous treatment of cancer cells with telomerase inhibitory drugs (▶Small molecule drugs) shortens telomeres and eventually induces cellular ▶senescence, ▶apoptosis, or both. Thus, telomerase inhibitors have the potential to benefit cancer patients; ▶molecular therapy. One concern is that telomere shortening per se compromises the effect of telomerase inhibitors since shorter telomeres have fewer TRF1 and therefore allow easier access to residual telomerase activity. This phenomenon results from incomplete shutdown of telomerase activity. In addition to enzyme activity, accessibility to telomeres could be a rational target for telomerase inhibition. In fact, tankyrase-1 modulates the impact of telomerase inhibitors on human cancer cells. First, tankyrase-1 overexpression, which removes TRF1 from telomeres, confers resistance to telomerase inhibitors. PARP inhibitors, such as 3-aminobenzamide and PJ-34, reverse this drug resistance. Second, even in cells that do not overexpress exogenous tankyrase-1 (but do express endogenous tankyrase-1) these PARP inhibitors enhance telomere shortening by means of telomerase inhibitors, such as MST-312. Accordingly, the cells undergo earlier crisis. Telomerase inhibitor-resistance caused by telomere shortening per se is also reversed by 3-aminobenzamide. These observations suggest that tankyrase-1 could be a target for cancer therapy; ▶chemotherapy of cancer, progress and perspectives. Pathologically, tankyrase-1 gene expression is elevated in some tumors but not in others.

References 1. Smith S, Giriat I, Schmitt A et al. (1998) Tankyrase, a poly (ADP-ribose) polymerase at human telomeres. Science 282:1484–1487

Is a shuttling mRNA export factor that binds to a GLEBS-like motif on nucleoporin 98 in the nuclear pore complex and functions in both the export of mRNA and import (recycling) of mRNA export factors into the nucleus. ▶NUP98-HOXA9 Fusion ▶Nucleoporin Family

TARC Definition Thymus and activation-regulated chemokine/CCL17 is a Th2 type chemokine expressed by ▶dendritic cells and other cell types that binds to CCR4. ▶Hodgkin Disease, Clinical Oncology

Tarceva ▶Erlotinib

Target Cells Definition The functions of effector T cells are always assayed by the changes that they produce in antigen-bearing target

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cells. These cells can be B cells, which are activated to produce antibody; macrophage, which are activated to kill bacteria or tumor cells; or labeled cells that are killed by cytotoxic T cells.

Target-Based Screen Definition A procedure in which molecules are systematically tested for their ability to activate, perturb, or modify a particular biological molecule. ▶Small Molecule Screens

Targeted Deletion Definition The process used in generation of a mouse embryonic stem cell (ES cell) with a portion of a specific gene deleted on one allele; embryonic stem cells are transfected with a DNA carrying the deleted gene flanked by regions exactly matching the endogenous allele to be targeted, and in some ES cells the transfected DNA recombines with the homologous endogenous allele, replacing the endogenous allele with the deleted allele.

Targeted Drug Delivery D UXIN S UN Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA

Synonyms Drug targeting; Site-specific drug delivery

Definition Targeted drug delivery is to site-specifically deliver or activate the therapeutic compounds in tumor. Thus, the targeted drug delivery is expected to enhance drug efficacy by increasing local active drug concentration in

tumor, and to decrease side effect by minimizing drug exposure in normal tissues.

Characteristics Anticancer drugs possess a greater potential of toxicity and much narrower therapeutic index than any other categories of medication. ▶Chemotherapy is often dose- and toxicity-limited. A delicate dose regimen is usually required to balance drug efficacy, drug toxicity, and drug resistance; a high dose might cause toxicity while a low dose might induce drug resistance. In addition, anticancer drugs are usually designed to act on the fast proliferating ▶cancer cells. However, rapid proliferation is also the feature of some normal cells such as bone marrow, hair follicles, and intestinal epithelium. Although tremendous effort has been explored to improve the protocol of chemotherapy, the success is very limited to enhance drug efficacy and to reduce drug toxicity. Several targeted drug delivery technologies have been studied to improve chemotherapy by enhancing drug efficacy and reducing drug toxicity, which have achieved certain degree of success. Intratumoral Drug Administration The simplest form of targeted drug delivery is local intratumoral drug administration. The therapeutic compounds can be directly injected into tumor tissues to transiently increase local drug concentration with minimal or no exposure to normal tissues. However, this method may not be applicable for many cancers. It is also difficult to maintain effective drug concentration in tumors for a prolonged time, and thus repeated dosing may be required. Liposomal Drug Delivery Chemotherapeutic compounds can be encapsulated in liposome. Liposomal drug formulation achieves passive drug targeting in tumor by enhanced permeability and retention (EPR) effect. Tumor tissues have abnormal vasculature. Since these vasculatures are hyperpermeable with no lymphatic drainage, the liposome will be delivered to the tumor tissues by blood circulation and trapped in the tumors. The drug in the liposome will be gradually released to achieve anticancer efficacy. Therefore, liposomal drug formulation will passively enhance drug accumulation in tumor and decrease exposure to susceptible healthy tissues. The successful liposomal formulation of doxorubicin has been used clinically for cancer treatment. Several generations of liposome formulations have been tested. First generation of liposome passively enhances drug accumulation in tumors and decreases the exposure in normal tissues, but it is rapidly cleared in blood (within minutes). Second generation of liposomes (PEGylated long-circulating

Targeted Drug Delivery

liposomes and ▶immunoliposome) increase half-life in blood circulation and tumor targeting. Tumor-Activated Prodrug Therapy (TAP) The first strategy in TAP therapy is site-specific prodrug activation. Prodrug is an inactive form of a therapeutic compound by chemical modifications. The inactive prodrug can be activated by an overexpressed enzyme in tumor to achieve site-specific activation, while the prodrug remains inactive or less activated in normal tissues to reduce toxicity. The site-specific activation of the prodrug in tumor increases the anticancer efficacy. The clinically used anticancer drug Xeloda (Capecitabine) provides a best example for this strategy. Xeloda is the first oral chemotherapy drug for the treatment of metastatic colorectal cancer. Xeloda is a ▶5-fluorouracil (5-FU) prodrug, which is orally administered. Three enzymes (carboxylesterase, cytidine deaminase, and thymidine phosphorylase) activate Xeloda to produce active 5-FU. Xeloda is hydrolyzed by carboxylesterase into 5′-deoxy-5-flurocytidine (5′-DFCR). Subsequently, 5′-DFCR is converted by deaminase to 5′-deoxy-5flurouridine (5′-DFUR). Finally, 5′-DFUR is converted into 5-FU by thymidine phosphorylase. Due to the high level of thymidine phosphorylase in tumor, more activation of xeloda is observed in tumors than in normal tissues to exhibit better anticancer efficacy than 5-FU. The second strategy of TAP is to directly link the prodrug to a targeting moiety (antibody or other ligands) for targeted drug delivery. The antibodyprodrug conjugate binds to the antigen on the tumor cells. The drug–antibody–antigen complex is rapidly internalized into the cancer cells to achieve high drug concentration in targeted cells. For example, anti-CD33 antibody can be conjugated with calicheamicin to target acute myeloid leukemia cells due to the high level of CD33 on the cell surface. Antibody trastuzumab-taxane conjugate shows 55–200-fold more potent than Taxol. Daunorubicin and methotrexate have been linked to anti-MM46 antibody or anti-EL4 monoclonal antibody for treatment of human melanoma. Most of those TAP therapies show better efficacy in vitro and targeted drug delivery in animal model. It is still very challenging in many aspects of TAP therapy such as rapid clearance in blood, low potency, inefficient internalization of conjugates, poor penetration, and premature release of the drugs. Antibody-Directed Enzyme Prodrug Therapy Antibody-directed enzyme prodrug therapy (ADEPT) is a two step process. In the first step, a drug-activating enzyme is targeted to tumors by a tumor targeting antibody. In the second step, a nontoxic prodrug is administered systemically and converted to the active drug with high local concentration in tumors by the localized antibody-enzyme conjugate. Meanwhile, the

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prodrug remains inactive (without drug-activating enzyme) in normal tissues and thus decreases its nonspecific toxicity. ADEPT provides many advantages: (i) Amplification effect: each localized antibodyenzyme molecule converts a large number of nontoxic prodrugs to potent active drugs and increases the local active drug concentration in tumor. (ii) Bystander effect: the locally activated drug molecules with high lipophilicity diffuse into the cancer cells regardless of the heterogeneous antigen expression. The bystander effect addresses the issues of poor tumor penetration of the antibody-enzyme conjugate. (iii) The antibodyenzyme conjugate does not need to be internalized into each cancer cell for prodrug activation. The enzymes in ADEPT could be a bacterial enzyme without mammalian homologs to minimize nonspecific prodrug activation in normal tissues, such as carboxypeptidase G2, cytosine deaminase, beta-lactaminase, penicillin G amidase. Other bacterial enzymes with mammalian homologs or mammalian enzymes with low expression in normal tissues can also be used, such as beta-glucuronidase, alkaline phosphatase, and alpha-galactosidase. Gene-Directed Enzyme Prodrug Therapy GDEPT is also a two step process. First, a gene that encodes a drug activation enzyme is delivered to tumor cells. The delivered gene will express the drug activation enzyme in the tumor only. In the second step, a nontoxic prodrug is delivered and is converted into active drug in tumor cells by the expressed enzyme. Many enzymes and drugs can be used in this system. For instance, cytosine deaminase for 5-florocytosine prodrug activation, thymidine kinase for ganciclovir, and arabinonucleoside prodrug activation, carboxypeptidase G2 for benzoic acid mustard prodrug activation, carboxypeptidase A for methotrexate-alanine prodrug activation, galactosidase for daunorunbicingalactose prodrug activation, glucuronidase for epirubicinglucuronide prodrug activation, alkaline phosphatase for doxorubicin phosphate prodrug activation, cytochrome P-450 for cyclophosphamide and isofamide prodrug activation. Folate-Targeted Drug Delivery Many cancer cells overexpress folate receptor on the cell surface. For example, 80% of metastatic breast cancer and 90% of ovarian cancer are folate receptor positive. Folate is absorbed by its carrier, and can also be taken up by cells through folate receptor mediated endocytosis. Folate-drug conjugate can bind to folate receptor to achieve targeted drug delivery. Many compounds can be delivered by folate conjugates such as small molecules of chemotherapeutic agents, protein complexes, radioimaging agents, genes, and antisense oligonucleotides.

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Transferrin Targeted Drug Delivery Tumor cells have been reported to have high level of transferrin receptor. Transferrin-drug conjugates can bind to transferrin receptor and be internalized inside the cancer cell. This strategy has advantages for tumor tissue distribution and prolonged half life. Fro instance, transferrin-doxorubicin has the potential to circumvent cardiotoxicity. Transferrin-diphtheria toxin selectively killed brain tumor cells with high level of transferrin receptor, although this conjugates also show neurological side effects due to low level of transferrin receptor in normal brain endothelial cells. Albumin-Drug Conjugate for Targeted Delivery Distinctive characteristics of tumor tissue with lack of lymphatic drainage leads to accumulation of plasma albumin. Thus albumin-drug conjugate can achieve targeted drug delivery. for instance, methotrexate (MTX): albumin (1:1 molar ratio) was accumulated at higher level (14% accumulation) in xenograft tumors compared to MTX alone (0.4% accumulation), and thus showed better efficacy.

Targeted Therapy Definition Therapy directed to a specific molecular target present in the cancer cell, which explains the efficacy of the drug. Targeted therapy has been discovered by exploiting the genetic differences between normal and tumor cells. New agents have been specifically developed to target the gene expression and signaling pathways deregulated in the ▶cancer cells. For example, the treatment of ▶chronic myeloid leukemia has been revolutionized by ▶imatinib, a small molecule inhibitor of tyrosine kinases, including ▶BCR-ABL, PDGFR, and KIT. Recent studies have also confirmed the effectiveness of ▶herceptin in ▶breast cancer. Testing for the presence of ▶HER-2/neu gene mutation to select patients for ▶herceptin treatment is rapidly becoming common practice in the health system that can accommodate the cost of treatment.

References 1. Schrama D, Reisfeld RA, Becker JC (2006) Antibody targeted drugs as cancer therapeutics. Nat Rev (Drug Discov) 5:147–159 2. Springer CJ, Niculescu-Duvaz I (2000) Prodrug-activating systems in suicide gene therapy. J Clin Invest 105:1161–1167 3. Hood JD, Bednarski M, Frausto R et al. (2002) Tumor regression by targeted gene delivery to the neovasculature. Science 296:2404–2407 4. Brannon-Peppas L, Blanchette JO (2004) Nanoparticle and targeted systems for cancer therapy. Adv Drug Deliv Rev 56:1649–1659 5. Rooseboom M, Commandeur JNM, Vermeulen NPE (2004) Enzyme-catalized activation of anticancer prodrugs. Pharmacol Rev 56:53–102

Targeted Drug Design ▶Drug Design

Targeted Radioimmunotherapy ▶Ionizing Radiation Therapy

Targeted Viruses Definition Viruses used in oncolytic virotherapy that have been genetically modified to achieve selective replication in tumor cells. ▶Oncolytic Virotherapy ▶Oncolytic Virus

Targeting, Active Definition Active targeting is one of the mechanisms by which drugs or drug delivery vectors can be preferentially delivered to target cells in vivo. With this method, vectors that possess tissue-specific molecules, such as antibodies, peptides, and sugar chains, actively recognize target cells by molecular interactions. ▶Non-viral Vector for Cancer Therapy ▶Drug Delivery System, in Cancer

TAT Protein of HIV

Tat Definition HIV Transactivator protein product of the HIV tat gene. ▶TAT Protein of HIV

TAT Protein of HIV A DRIANA A LBINI 1 , D OUGLAS N OONAN 2 1 2

IRCCS Multimedica, Milano, Italy University of Insubria, Varese, Italy

Definition Tat is a small viral protein that is encoded by the spliced two-exon tat gene in the HIV genome, responsible for transactivation of the HIV genome.

Characteristics The HIV Tat protein gets its name from its principal activity, Tat stands for Transactivator, which means that it binds to DNA and activates the transcription of DNA into RNA. The Tat protein has an important role in controlling the transcription of the lentivirus HIV genome from its built-in “promoter,” known as the long terminal repeat (which refers to its structure) or ▶LTR, to make the RNA that forms new HIV virus particles. In addition to this major role, Tat has also been implicated in a wide variety of pathologies encountered in persons infected with HIV. How does this small (about 101 amino acids) Tat protein do this? Tat has a capacity to bind a striking number of different proteins, nucleic acids and even polysaccharides. It is this combination of binding to different partners that has linked Tat to numerous events in AIDS and intrigued many researchers, making Tat one of the most extensively studied HIV proteins. Transcriptional Regulation: Control of HIV Replication The HIV LTR acts as a gene promoter. The gene promoter is a portion of DNA that mediates the binding of RNA polymerase (usually through a series of other proteins that bind to the promoter DNA as well) at the beginning of the gene to be transcribed into RNA. This interaction of proteins with DNA controls the transcription of each gene so that it occurs at a certain moment. In the absence of Tat, very little RNA is transcribed from the HIV LTR promoter. When Tat is present, the

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rate of transcription shoots up several hundred fold, making the transcription of the HIV genome efficient. After HIV infects a cell, it is reverse transcribed into DNA, which is then integrated into the genome of the cell (similar to ▶retroviruses). The viral DNA is then packaged along with the rest of the cellular DNA by winding onto the histone proteins. In this state, the HIV LTR promoter is rather inactive. In fact, in order to be transcribed, the promoter region of any gene must be unwound from these histones. The HIV DNA is bound to the histones in a very specific manner, with one histone group just prior to, and another just after, a site which binds DNA binding proteins of the cell known as SP1 and NFκB. The activity of these proteins may be enough to allow the binding of an RNA polymerase, known as RNA polymerase II or RNAP II, to the HIV LTR. The binding of RNAP II to the LTR alone is not enough for efficient transcription. In the absence of Tat it does not appear to be able to advance forward to synthesize RNA beyond the first 44 nucleic acid base pairs. The major role for Tat is to unleash this machinery and send it to work. When Tat is present, it binds to a segment of this short initially polymerized RNA, which forms a peculiar loop known as the TAR (TransActivation Responsive) element. The TAR-bound Tat then brings in a series of other proteins that allow the transcription process to proceed. Tat has been demonstrated to play a key role in the unwinding of HIV DNA from histones, which is one key for allowing RNA transcription. To do this, Tat binds to a group of proteins known collectively as Tatassociated histone acetyltransferase (TAH). The TAH complex can be formed by different cellular proteins such as p300 or is close relative CBP, along with P/CAF and/or TAF250. These TAH proteins have an enzymatic activity that transfers an acetyl group to histones and are known as histone acetyltransferases. The recruitment of the TAH complex to the HIV LTR region by TARbound Tat leads to acetylation of the histones that bind to the LTR, causing changes in their conformation that facilitate RNA transcription. In fact, in cells where p300 and P/CAF are limiting, addition of Tat increases transcription only about sevenfold, while addition of both Tat and p300-P/CAF allows increases transcription about 80-fold. The TAH complex also appears to acetylate the Tat protein itself. This appears to lower the binding of Tat for the TAR, but increases the binding of Tat to another complex of proteins known as the TatAssociated Kinase (TAK) or positive acting transcription elongation factor complex (P-TEFb). This complex of proteins consists of cyclin-dependent kinase 9 (cdk9) and one of the cyclin T isoforms (T1, T2a, T2b). This protein complex can directly bind Tat. More importantly, the kinase activity of the TAK complex phosphorylates (adds a phosphate group to) the RNA polymerase RNAP

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TAT Protein of HIV

II. This phosphorylation appears to alter the activity of the RNAP II, improving its ability to transcribe the HIV genome. Together, the TAH and TAK complexes brought in by Tat unleash the RNAP II to finish the job it started, giving the 100-fold improvement of transcription, and therefore HIV replication, observed when Tat is added to HIV infected cells. Tat also transactivates several cellular genes in addition to the HIV LTR. The activation of these genes is also thought to contribute to the pathogenesis of HIV. The genes include the cytokines IL-6, TNF and IL-1, which are known to be increased in AIDS patients and may have detrimental effects on the overall function of the immune system. AIDS-Associated Pathologies: A Direct Contribution by Tat One of the most striking properties of Tat is its ability to exit from cells, where it is released into the extracellular environment. Several studies have shown that the HIV-1 Tat protein can exit from cells, including HIV infected cells. As the Tat gene does not encode a signal peptide, the release of HIV-Tat has been suggested to occur via an alternative secretion pathway, like that demonstrated for some cytokines. It may also come from cells dying due to HIV. The Tat that is found extracellularly appears to be intact and active, and substantial levels of Tat protein have been observed found in the serum of many HIV patients. Antibodies can be made against Tat in AIDS patients, indicating that it is released, and interestingly an inverse correlation between anti-Tat antibodies and patient survival has been reported in some studies. These data suggest that extracellular Tat may favor HIV replication, and have spawned tests on the possibility of using Tat as part of an AIDS vaccine. A wide range of activities have been attributed to the Tat protein released extracellularly. Several studies have demonstrated that the HIV Tat protein, or peptides based on Tat, are capable of entering cells cultured in vitro. The Tat that enters cells is capable of transactivating the HIV LTR. Tat and Tat peptides have even been used to deliver other proteins into cells, and a peptide based on Tat may find use as a signal to move drugs into cells. In addition to getting into other cells, the Tat that is released also appears to bind to several cell surface proteins, including specific receptors. These activities of Tat have been linked to many of the pathological alterations found in HIV infection. For example, some groups of AIDS patients frequently have ▶Kaposi sarcoma, an otherwise rare, benign vascular tumor. Unlike most Kaposi, the Kaposi sarcoma associated with AIDS is very malignant and could be lifethreatening for many of these patients. Tat was first linked to Kaposi sarcoma when Kaposi-like lesions were found on mice genetically engineered to express

Tat. Soon after, Tat was shown to be a growth factor for Kaposi cells, but the reason for this was not known. Later studies showed that Tat could bind to KDR (VEGFR2), a receptor for the growth factor ▶VEGF, on the surfaces of endothelial and Kaposi sarcoma cells. VEGF (vascular endothelial growth factor) is important in the formation of new blood vessels, as is its receptor KDR. The ability of Tat to bind and activate KDR means that Tat could stimulate the formation of vessels found in Kaposi tumors, as well as the growth of cells of the tumor itself. We now know that Kaposi sarcoma is due to an infection with a herpesvirus, HHV8, that occurs when the immune system is unable to control this virus. All Kaposi cells, whether from aggressive AIDS or benign sporadic of iatrogenic (post-transplant), have KDR, however the Tat stimulation of KDR and perhaps other receptors appears to make AIDS Kaposi potentially lethal. Many Tat proteins have an RGD sequence, three amino acids found in many proteins of the extracellular matrix. Through this RGD sequence Tat can bind to cell surface integrins, proteins that are normally involved in binding to extracellular matrix molecules. Studies have shown that several integrins bind the Tat protein. Tatintegrin binding has been shown to trigger events typical of integrin-extracellular matrix ligand interactions, including activation of p125 Focal Adhesion Kinase. The binding of Tat to these receptors has also been linked to Kaposi sarcoma and other activities of HIV Tat. The immune suppression seen with AIDS appears to affect cells that are not infected with HIV aside from those harboring the virus. Several studies have shown that there is immune suppression of non-HIV infected cells from AIDS patients, and that the number of immune suppressed cells seems to exceed that of the potentially HIV-infected cells. Proteins released from HIV-infected cells are clearly potential candidates for mediating this immune suppression. Tat has been linked to induction of T-cell anergy (lack of activity), T-cell ▶apoptosis (programmed death), but also to a T-cell hyper-activation that appears to prime cells for infection by HIV. These events are probably all closely linked to the same phenomenon. The potential receptor system(s) involved in these activities of Tat include CD26. CD26 is a dipeptidyl peptidase that is known to cleave and alter the activities of chemokines, molecules whose receptors are very important cell surface receptors for HIV and that can regulate HIV infection. Tat has been shown to significantly increase the expression of two key chemokine-HIV receptors, CXCR4 and CCR5, by monocytes and T-lymphocytes, potentially increasing HIV infection. The Tat protein has been reported to act as a growth factor and protect transfected cell lines from apoptosis. Tat has been consistently found to up-regulate the expression of CD95-▶Fas, a protein that signals cells to

Tau

die. The increase in apoptosis is typical for partially activated T-cells, as is entry into anergy resulting from an incomplete stimulation of T-cells. Tat may be capable of partial, but incomplete, T-cell activation. HIV does not readily infect resting T-cells, T-cell activation is a key requisite for HIV infection of these cells. A partial T-cell activation may be sufficient for HIV infection yet detrimental to the host immune response, a potential role that Tat may fulfill. Extracellular HIV Tat has been shown to have wide ranging effects on lymphatic cells such as monocytes, macrophages, dendritic cells and even natural killer cells. Tat has been reported by several groups to be a strong chemoattractant for monocytes. This activity could contribute directly to the recruitment of potentially “infectable” cells toward an HIV-infected cell producing and releasing Tat protein, an activity which may have a direct affect on establishment and spread of HIV infection in the host. Tat has been shown to bind to and activate several chemokine receptors (including CCR3 and CKCR4) and mimic the chemoattractant properties of chemokines. Chronic inflammation is frequently associated with cancer and inflammatory cells can promote tumor growth and tumor angiogenesis, thus Tat may influence the AIDS-associated tumor microenvironment through direct and indirect mechanisms. Tat can even inhibit HIV infection in high doses by binding chemokine receptors, although the physiological relevance of this observation is not yet clear. The Tat protein appears to inhibit dendritic cell phagocytosis and natural killer cell function, apparently by blockage of certain calcium channels. Finally, Tat has been linked to AIDS associated dementia. Tat has been shown to excite neurons, which is associated with neurotoxicity. The molecular identity of the Tat receptor(s) on neural cells is not yet known. However, the neuroexcitory properties of Tat were blocked by lowering extracellular calcium, suggesting that interference with calcium channel function may be involved. Conclusion Tat is known to have a major role in HIV replication through a complex series of interactions with nuclear proteins. In addition, Tat outside the cell appears to be able to stimulate through, or interfere with, several cell surface receptors, sending signals that may be a root cause of many pathologies found in HIV-infected patients.

3. Jeang KT, Xiao H, Rich EA (1999) Multifaceted activities of the HIV-1 transactivator of transcription, Tat. J Biol Chem 274:28837–28840 3. Albini A, Soldi R, Giunciuglio D et al. (1996) The angiogenesis induced by HIV-1 Tat is mediated by the flk-1/KDR receptor on vascular endothelial cells. Nat Med 2:1371–1375 4. Rubartelli A, Poggi A, Sitia R et al. (1998) HIV-I Tat: a polypeptide for all seasons. Immunol Today 19:543–545 5. Gallo RC (1999) Tat as one key to HIV-induced immune pathogenesis and Tat (correction of Pat) toxoid as an important component of a vaccine. Proc Natl Acad Sci USA 96:8324–8326

TATA Box Definition Synonym Goldberg–Hogness box; Is the core promoter sequence (5′-TATAA-3′) of most genes. In eukaryotes, it represents the DNA binding site of the TATA binding protein during the process of gene transcription. Promoters lacking a TATA box were first found in housekeeping genes and have more recently been found in various gene promoters involved in cell-specific transcription.

TATI Definition Tumor-associated trypsin inhibitor (TATI) is a low molecular weight protein used as a tumor marker, e.g. ovarian cancer. ▶Ovarian Cancer

Tau Definition

References 1. Noonan DM, Albini A (2000) From the outside in: extracellular activities of HIV Tat. In: Jeang KT (ed) Advances in pharmacology: HIV: molecular mechanisms and clinical applications. Academic Press, San Diego, CA, pp 229–250

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Is a ▶microtubule-associated protein (MAP) that is functionally modulated by phosphorylation and that is hyperphosphorylated in several neurodegenerative diseases. Tau proteins interact with ▶tubulin to stabilize ▶microtubules and promote tubulin assembly into microtubules.

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Taurodont

Taurodont Definition Unusually large tooth with an unusually large pulp chamber and either multiple roots or a large single root. ▶Dental Pulp Neoplasms

system, the latter composed of four isoprene units with the molecular formula C20H32. Drugs that inhibit microtubule formation resulting in cell cycle arrest and apoptosis, used in cancer chemotherapy. Microtubules are essential to cell division, and taxanes therefore stop this action by freezing the mitosis process. ▶Taxotere ▶Taxol ▶Docetaxel

Tax Taxol Definition

Is a viral oncoprotein encoded by the ▶human T-cell leukemia virus 1 (HTLV-1). HTLV-1 is the causal agent of a human leukemia, adult T-cell leukemia (ATL). During HTLV-1 replication, Tax transcriptionally activates the viral long terminal repeat (LTR). Several major cellular signal transduction pathways including the transcription factors NF-kB, CREB, SRF and AP-1 are induced by Tax. During cell division, Tax binds MAD1 directly to repress its function. ▶Mitotic Arrest-Deficient Protein 1 (MAD1)

Tax Binding Protein-181 ▶Mitotic Arrest-Deficient Protein1

S PYROS D. G EORGATOS , PANAYIOTIS A. T HEODOROPOULOS Department of Basic Sciences, The University of Crete, School of Medicine, Heraklion, Crete, Greece

Definition Taxol (Paclitaxel) was first isolated in 1971 from a crude extract of Taxus brevifolia, a scarce, slow growing yew plant found in the forests of the Pacific Northwest. It is a diterpenoid containing the characteristic taxane ring (Fig. 1). Total chemical synthesis of this compound was achieved in 1994, opening the way for the production of various analogues. Currently, taxol is commercially prepared by hemisynthesis, in which a synthetic side chain is attached to natural products isolated from the needles of Taxus plants. Enzymatic conversion of various taxanes to 10-deacetylbaccatin III (a precursor for taxol hemisynthesis) has been reported.

Characteristics

Tax Helper Protein (GLI2) ▶GLI Proteins

Taxane Definition Are diterpenes synthesized by plants of the genus Taxus (yews). A class of naturally-occurring or synthetic chemicals containing a characteristic 15-member ring

Mode of Action Due to its hydrophobic character, taxol readily crosses the ▶plasma membrane. Once in the cytoplasm, the drug binds with high affinity to the β-subunit of ▶tubulin, modifying and stabilizing ▶microtubules. When modified by taxol, these cytoskeletal structures exhibit decreased dynamic instability and increased rigidity. As a result, the function of the microtubulebased machines (e.g., ▶mitotic spindle) is compromised and the cells cannot divide properly. Taxol also binds to ▶Bcl-2, a protein involved in the process of programmed cell death (▶apoptosis). The cellular effects of taxol vary depending on dose and treatment scheme. In the range of nanomolar concentrations it induces sustained mitotic arrest (Fig. 2), inhibits protein prenylation and triggers apoptosis. At micromolar doses it promotes synthesis and release

Taxol

Taxol. Figure 1 Chemical structure of taxol.

Taxol. Figure 2 Confocal microscopy image, showing human cervical carcinoma cells in mitosis. Cells were treated with 10 nM taxol for 20 h. Immunostaining was done with anti-tubulin antibodies and counter-staining was done with propidium iodide. Chromosomes are shown in red, the mitotic spindle in green.

of cytokines, such as ▶tumor necrosis factor (TNF) and ▶interleukins (IL1 and IL8), increases tyrosine phosphorylation by ▶MAP kinases, induces early response genes and stimulates production of ▶nitric oxide.

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How taxol exerts its cytotoxic action remains elucive. Structure-activity studies differentiate microtubule stabilization from other effects, supporting a direct effect on the genetic and signal transduction machinery. However, other studies suggest that the drug acts by inducing cytoskeletal damage. Recent observations show that taxol activates Raf-1 kinase and induces phosphorylation of Bcl-2. Phosphorylation of the latter may, in turn, lead to dissociation of Bcl-2/▶Bax complexes, unleashing Bax into the cell and thus triggering apoptosis. Although these hypotheses are intuitively attractive, neither of them could fully account for the cell killing action of taxol: programmed cell death is also induced by other microtubule-stabilizing drugs which are free of genomic side-effects (e.g., ▶Taxotere), while apoptosis can still occur independently of Raf-1 phosphorylation, or after Bcl-2 is dephosphorylated by cellular phosphatases. Taxol and other microtubule-acting agents affect dramatically the architecture of the cell nucleus. It is widely known that cells treated with nanomolar amounts of taxanes or ▶vinca alkaloids develop lobulated nuclei or multiple micronuclei and missort key cellular constituents. Also, taxol affects the nuclear envelope. Even at nanomolar concentrations, the drug induces focal unraveling of the nuclear lamina and extensive clustering or ectopic localization of nuclear pore complexes (Fig. 3). Cells that possess a defective nuclear envelope and which are treated with taxol remain alive for at least 24 h after the end of the treatment, but are unable to import karyophilic proteins, such as the transcription factor ▶NFκB, into the nucleus. It has been proposed that inhibition of NFκB import may render the cells prone to programmed cell death. Clinical Pharmacology Antitumor Activity Taxol as a single chemotherapeutic agent has been proven effective against a variety of tumors, including ovarian, breast, head and neck, esophageal, bladder and lung carcinomas. In addition, several schedules of combination therapy have been developed as alternatives for patients with advanced cancer. Pilot studies show that taxol can enhance radiation sensitivity of tumor cells, potentiate tumor response and increase the therapeutic ratio of radiotherapy. Absorption and Excretion The drug is administered as a 3-h or 24-h infusion. It undergoes ▶cytochrome P450-mediated metabolism to 6-OH derivatives and other products. Less than 10% of a dose is excreted intact from the urine. Toxicity The principal toxic effect of taxol is neutropenia. Several other toxic effects, such as myalgias, mucositis,

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Taxotere

Taxol. Figure 3 Nuclear lamina and pore complex lesions after treatment of human endometrial carcinoma cells with 10 nM taxol. Left: indirect immunofluorescence using anti-lamin B antibodies. Right: indirect immunofluorescence using anti-nucleoporin antibodies. The interruption of the nuclear lamina and the formation of large pore clusters is evident in these images.

hypersensitivity reactions, stocking-glove sensory neuropathy and disturbances of the cardiac rhythm, have also been encountered. Structurally and Functionally Related Compounds Structurally Related Compounds . ▶Docetaxel (taxotere), produced by semisynthesis (1986) from 10-deacetyl baccatin III, a taxoid precursor. It is the second member of the taxane class to reach clinical use. . Several chemically synthesized taxoids bearing substitutions or modifications. Nontaxane, Microtubule-Stabilizing Agents . Estramustine, a conjugate of estradiol and nornitrogen mustard. . ▶Epothilones A and B, two macrolides isolated from myxobacterium, Sorangium cellulosum. . A family of marine-derived compounds extracted from sponges (discodermolide and laulimalide) or corals (sarcodictyins A - F and eleutherobin).

References 1. Haldar S, Basu A, Croce CM (1997) Bcl-2 is the guardian of microtubule integrity. Cancer Res 57:229–233 2. Nicolaou KC, Yang Z, Liu JJ et al. (1994) Total synthesis of taxol. Nature 367:630–634 3. Rodi DJ, Janes RW, Sanganee HJ et al. (1999) Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein. J Mol Biol 285:197–203 4. Rowinsky EK, Donehower RS (1995) Paclitaxel (Taxol). N Engl J Med 332:1004–1014 5. Theodoropoulos PA, Polioudaki H, Kostaki O et al. (1999) Taxol affects nuclear lamina and pore complex organization and inhibits import of karyophilic proteins into the cell nucleus. Cancer Res 59:4625–4633

Taxotere E DWARD L. S CHWARTZ Department of Oncology, Albert Einstein College of Medicine, Bronx, NY, USA

Synonyms Docetaxel

Definition

Taxotere is a widely used ▶cancer chemotherapeutic drug with well-documented clinical anti-tumor activity against a range of human cancers, including ▶breast, ▶lung, ▶prostate and ▶ovarian cancers. It is a semisynthetic molecule belonging to the ▶taxane family, and is closely related chemically and pharmacologically to the naturally occurring drug ▶Taxol (▶paclitaxel). Taxotere binds to the β-▶tubulin component of ▶microtubules, a key ▶cytoskeletal protein in cells, and stabilizes the structure of the microtubule polymer, prevents its disassembly, and suppresses microtubule dynamics. These actions interfere with a number of cellular functions in which the microtubules are involved.

Characteristics Chemistry The anti-tumor activity of the taxanes was first observed in preclinical models using a crude extract of the bark of the Pacific yew tree Taxus brevifolia, and Taxol was subsequently (1971) identified as the active constituent. Both Taxotere and Taxol are diterpenoid compounds consisting of a 15-member taxane ring system linked to an unusual 4-member oxetan ring. Limited supplies of bark from the T. brevifolia tree prompted the search for alternative derivatives and sources of starting material.

Taxotere

An inactive compound, 10-deacetylbaccatin III, found in the needles (which are more abundant and also are a renewable source) of several yew species, was used as starting material for the synthesis of Taxotere, which was first reported in 1986. Taxotere and Taxol differ in the nature of the substitutions at the C-10 position of the taxane ring and on the ester side chain attached to C-13, and these differences are the basis for the increased water solubility and greater potency of Taxotere, compared to Taxol (Fig. 1). Mechanisms of Action Microtubules are a component of the cell’s ▶cytoskeleton, and form a well-organized network of hollow tubes in which one end is anchored at the ▶centrosome (the microtubule-organizing center), and the opposite, free end extends out into the cytoplasm. Microtubules are composed of noncovalent ▶heterodimers of α-and β-tubulin, which are arranged head-to-tail to form helical polymers. There is an exchangeable GTP/GDP binding site on β-tubulin which mediates the rapid assembly and disassembly of the microtubules. The free ends of the microtubules undergo frequent periods of slow growth and rapid shortening, a process called dynamic instability, and microtubules also exhibit treadmilling, in which the loss of tubulin at one end is matched by the addition of tubulin at the opposite end. This dynamic behavior is required for the proper functioning of the microtubules. During mitosis, microtubules capture and are responsible for the alignment of the chromosomes and for their subsequent separation to the two daughter cells during anaphase. Properly functioning microtubules are required for the migration of most cell types, and they also play an important role in cell signaling and the intracellular transport of proteins, vesicles, and mitochondria. Much of the early work on the binding and effect of the taxanes on microtubules was done using Taxol.

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Taxol preferentially binds to the microtubule polymer, rather than to unassembled tubulin, at a site that is distinct from the GTP/GDP site and which also differs from the sites at which colchicine, vinblastine and podophyllotoxin bind. Studies suggest that in their interactions with purified microtubules, Taxotere and Taxol are qualitatively indistinguishable. Although Taxotere and Taxol have the same apparent binding site on tubulin, Taxotere binds with a 1.9-fold greater effective affinity. The taxanes promote microtubule assembly, even in the absence of added GTP, by altering the tubulin dissociation constants at both ends of the microtubules. The taxanes reduce the critical concentration of tubulin required for microtubule assembly, with the required concentration 2.1-fold lower in the presence of Taxotere than with Taxol. In addition to promoting both the nucleation and elongation phases of polymerization, they also produce microtubules that are extremely stable and are resistant to depolymerization. The effects on microtubule assembly are maximal at Taxol or Taxotere concentrations that are equimolar to that of tubulin, and evidence indicates that they bind with a stoichiometry of 1 per αβ tubulin dimer. In cells treated with stoichiometric concentrations of taxanes, there is an increase in the microtubule-polymer mass along with the formation of abnormal polymers and a characteristic intracellular “bundling” observed. While these concentrations of the drugs are obtained clinically, it is only for brief periods of time after drug administration. At substoichiometric concentrations, the taxanes effects on microtubule dynamics are more prominent than are their actions on microtubule mass, and the suppression of mitotic spindle dynamics plays a central role in their cytotoxic actions. By interfering with the ▶mitotic spindle, the taxanes block cancer cell proliferation by causing cell cycle arrest at the metaphase/ anaphase boundary. The resultant blocking or slowing of mitosis

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Taxotere. Figure 1 Chemical structure of Taxotere (docetaxel).

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Taxotere

leads to cell death via the induction of the intrinsic mitochondrial pathway of apoptosis. Taxotere is transported out of cells by the ABCtransporter class of membrane transport proteins, which include P-glycoprotein, MDR1 and MDR2. Overexpression of these pumps and the accompanying multi-drug resistance (MDR) phenotype can lead to profound cellular resistance to the taxanes. Microtubule-related determinants may also confer resistance to the taxanes, and these include the levels of expression of microtubuleassociated regulatory proteins, the extent and nature of posttranslational modification of tubulin, and the varying levels of expression of different tubulin isotypes. Interestingly, cancer cells made resistant to Taxol do not necessarily show the same degree of resistance to Taxotere, and this may be due to the observation that Taxotere is retained intracellularly longer than is paclitaxel. In addition to effects on tumor cell proliferation and apoptosis, Taxotere is a potent inhibitor of angiogenesis, and this is likely due to its direct effects on ▶endothelial cells. While it inhibits endothelial cell proliferation at concentrations comparable to those which inhibit cancer cells, it also inhibits endothelial cell motility, invasiveness, and tubule formation in vitro at concentrations substantially (10- to 100-fold) lower than required to cause cell cycle arrest or apoptosis. Based on pharmacokinetic analyses of Taxotere, cancer patients are exposed to these anti-angiogenic concentrations of drug for extended periods of time. Taxotere also has anti-angiogenic activity in several in vivo preclinical models, and these in vivo anti-angiogenic actions were probably not directly due to the antiproliferative or cytotoxic activities of the drug, as other agents that inhibited endothelial cell proliferation in vitro did not affect angiogenesis in vivo. The mechanisms for these actions are not fully understood, and may include effects on signaling pathways, including those mediated by cell surface ▶integrins and cell surface receptors for the angiogenic factor ▶vascular endothelial growth factor (VEGF). The effects of the taxanes on microtubule dynamicity at the low concentrations that inhibit angiogenesis appear to be qualitatively different from those at concentrations that block cell proliferation. Clinical Use Taxotere is a highly effective drug with a spectrum of anti-cancer activity that is virtually identical to that of Taxol. Taxotere initially received regulatory approval for use in patients with metastatic or locally advanced ▶breast cancer who had failed other drug therapies. In addition to increasing survival when used as secondline therapy in patients with advanced disease, subsequent studies showed it was active in the adjuvant treatment (▶adjuvant therapy) of patients with local

breast cancer after definitive local treatment. It has been shown to increase survival in unresectable metastatic ▶non-small cell lung cancer, and is one of the most effective agents in the treatment of ovarian cancer and in hormone-refractory ▶prostate cancer. The wide spectrum of activity that Taxotere was found to have in pre-clinical models has been confirmed in the clinic, and anti-tumor activity has been noted in ▶bladder, ▶endometrial, ▶esophageal, ▶gastric, head and neck, and small cell lung ▶carcinomas, and in lymphoma and ▶melanoma. Taxotere is often used in combination with other chemotherapeutic drugs, including ▶cisplatin, cyclophosphamide, doxorubicin, and prednisone. In addition to being evaluated in a large number of drug combinations, a range of doses and schedules of Taxotere have been studied in clinical trials over the years. The drug is usually administered once a week or once every 3 weeks as short (1 h) intravenous infusions at doses ranging from 60 to 100 mg/m2. ▶Neutropenia is the primary toxicity observed at these doses, with a significantly reduced neutrophil count typically first observed on day 8 after treatment. Complete resolution occurs by days 15–21, and the severity of the neutropenia is related to the dosage of the drug and the extent to which the patients bone marrow function has been compromised by the concomitant or prior use of other myelosuppressive (▶myelosuppression) drugs. The neutropenic effect of Taxotere generally is more severe, but shorter in duration, than that of Taxol. A number of other toxic effects have been associated with Taxotere use. Hypersensitivity reactions (▶dyspnea, bronchospasm, and hypotension) can occur acutely with drug administration, and a unique fluid retention syndrome (peripheral edema, pleural and peritoneal fluid) can occur chronically with multiple courses of therapy. In both instances, the frequency and severity of these toxicities are substantially reduced by premedication with a corticosteroid and H1 and H2 histamine antagonists. Skin toxicities occur frequently, although these are also attenuated by premedication, with the most common manifestation being an ▶erythematous pruritic macropapular rash on the arms, hands and feet. Taxotere causes mild to moderate neurosensory and neuromuscular effects (numbness, ▶paresthesia), and although common (up to 40% of patients), these occur less frequently and are less severe than in patients receiving Taxol. The relative water-insolubility of Taxotere requires it to be formulated for clinical use in the nonionic surfactant polysorbate 80 (Tween 80). Both the hypersensitivity and fluid retention side-effects of the drug have been attributed to the use of this vehicle. A number of different alternatives to the use of Tween 80 have been evaluated in pre-clinical studies, with the objective of developing a less-toxic, better-tolerated formulation, and the

T-Cell Hybrids

identification of ways to better target Taxotere to malignant tissue. These include polyethylene glycol (PEGylated) liposomal Taxotere, immunotargeted-liposomal Taxotere conjugates, Taxotere-fibrinogen-coated olive oil droplets, Taxotere-encapsulated nanoparticle-aptamer bioconjugates, and submicronic dispersion formulations. Some of these formulations have entered early clinical trials.

References 1. Ringel I, Horwitz SB (1990) Studies with RP56976 (Taxotere): a semisynthetic analog of taxol. J Natl Cancer Inst 83:288–291 2. Verweij J, Clavel M, Chevalier B (1994) Paclitaxel (Taxol) and docetaxel (Taxotere): not simply two of a kind. Ann Oncol 5:495–505 3. Hotchkiss KA, Ashton AW, Mahmood R et al. (2002) Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (taxotere): association with impaired repositioning of the microtubule organizing center. Mol Cancer Ther 1:1191–1200 4. Bhalla KN (2003) Microtubule-targeted anticancer agents and apoptosis. Oncogene 22:9075–9086 5. Montero A, Fossella F, Hortobagyi G et al. (2005) Docetaxel for treatment of solid tumours: a systematic review of clinical data. Lancet Oncol 6:229–239

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TCDD ▶Dioxin

2,3,7,8-TCDD ▶Dioxin

T-Cell ALL Definition Arises from the bone marrow precursors of T cell lymphocytes. ▶Childhood Cancer

TBI

T-Cell Antigen Receptor

Definition Total body irradiation. ▶Ionizing Radiation Therapy

T-body ▶Chimeric T Cell Receptors

TBP Definition TATA-box binding protein; an essential protein binding to the TATA-box found in most eukaryotic promoters of genes transcribed by RNA Polymerase II. ▶AAV

Definition

▶T-cell receptor

T-Cell Clone Definition Refers to cells derived from a single progenitor T cell.

T-Cell Hybrids Definition T-cell hybrids are formed by fusing an antigen-specific, activated T cell with a T-cell lymphoma. The hybrid cells bear the receptor of the specific T-cell parent and grow in culture like the lymphoma.

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T-Cell Receptor

T-Cell Receptor Definition TCR; consists of a disulfide-linked heterodimer of the highly variable α and β chains expressed at the cell membrane as a complex with the invariant CD3 chains. T cells carrying this type of receptor are often called α:β T cells. An alternative receptor made up of variable γ and δ chains is expressed with CD3 on a subset of T cells. Both of these receptors are expressed with a disulfide-linked homodimer of ζ chains, which carries out the intracellular signaling function of the receptor.

T-Cell Response P ERIASAMY S ELVARAJ Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA

Definition Expansion of antitumor T cells in response to growth of cancer or to a ▶cancer vaccine or a cancer immunotherapy.

Characteristics In living organisms, the growth and division of cells is a tightly controlled and highly regulated process that maintains the integrity of cellular architecture and survival. Cell growth can occur in our body to replace a dead cell, to heal a wound, to maintain normal homeostasis, or to generate immune cells during an immune response. The number of divisions that cells undergo during these processes is highly controlled and the cells stop dividing when they receive signals either through contact inhibition or other means. Deviations from normal regulatory mechanisms may result in uncontrolled cell growth and the development of various types of cancers in our body. Many genes in the human body play pivotal roles in regulating the normal metabolic and growth patterns of cells. Mutations in these genes lead to the production of altered proteins that may result in the uncontrolled cell growth that is observed in cancer. Role of ▶immunosurveillance in antitumor immunity: Gene mutations occur constantly through the life span of a living organism, resulting in the occasional production of abnormal cells. Most mutant cells with defective metabolism die of a process called apoptosis. The mutant cells that are able to survive with altered

proteins will be recognized by the immune system and destroyed by a process termed immunosurveillance. There is strong evidence for the existence of such immunosurveillance against cancer. Cancers develop in high frequencies in people with immunodeficiency diseases associated with immune system defects such as AIDS, or in animals that have an incomplete immune system, suggesting that cancer specific immunity is a major mechanism for elimination of cancers from the body. This immunity is provided by the cytotoxic activity of tumor specific T cells that recognize tumors and destroy them. However, there are many ways that cancer cells circumvent this immunosurveillance. Some cancers secrete immunosuppressive factors such as TGF-β, which attenuates T cell immune response. Down regulation of MHC class I molecules that stimulate the immune system or upregulation of molecules that induce T cell apoptosis are other mechanisms cancers use to inactivate antitumor T cell immune responses. Molecular requirements of T cell responses. T cells are divided into two major subsets. CD4 antigenexpressing T cells are termed “helper T cells” whereas CD8 antigen-expressing T cells are termed “cytotoxic T cells.” It has been shown that CD4+ T cells provide help by secreting cytokines for the generation of robust CD8+ T cells that can kill tumor cells. Since T cells play a crucial role in the development of antitumor immunity, most cancer immunotherapy is designed to directly or indirectly activate tumor-specific CD4+ and CD8+ T lymphocytes and induce immunological memory against tumors. Studies have shown that both subsets of T cells play an important role in the antitumor immune response. Recent advances in understanding the molecular and cellular requirements for antigenspecific immune responses have led to a number of promising immunotherapeutic strategies for inducing antitumor T cell responses for the treatment of cancer. Many of these strategies include vaccination with ▶dendritic cells (DCs) engineered to express tumor antigens, cytokine transduced tumor cells, peptide vaccines, DNA vaccines, heat shock proteins, hybrid tumor cells, and tumor cells transduced with costimulatory molecules. The rationale for the vaccination strategies mentioned above is that antigen-specific T cells can be stimulated effectively by providing stimulatory signals arising from either tumors themselves (direct priming) or through ▶antigen-presenting cells (APCs) of the host (indirect priming). Normally, T cells are educated in the thymus to react against cells expressing MHC molecules that display peptides derived from foreign or altered proteins. Since tumors express altered proteins, the peptides derived from these altered proteins are associated with MHC molecules and presented to T cells that are specific to the altered protein. For an optimal immune response, antigen specific T cells

T-Cell Response

require at least two specific signals. One of the signals is provided by engagement of the T cell receptor (TCR) with peptide bearing MHC molecules on the APC. The second signal (costimulatory signal) can be delivered by the interaction of various adhesion molecules on the surface of T cells and the APC, one of which is the interaction of CD28 expressed on T cells and B7-1 (CD80) expressed on APCs such as DCs. The absence of a second signal results in T cell clonal anergy, thus preventing the development of tumor specific T cells (Fig. 1). Tumor cells, which lack costimulatory molecules such as B7-1, are poorly immunogenic since they fail to deliver the costimulatory signal necessary for the generation of an anti-tumor T cell immune response. Therefore, one approach to improve the ▶immunogenicity of tumor cells has been to introduce costimulatory adhesion molecules such as B7-1 onto the tumor surface by ▶gene transfection. This B7-1 expression results in the induction of T cell mediated antitumor immunity and subsequent tumor rejection in animals. These studies also demonstrated that ▶costimulation is required only for the initial stimulation and expansion of tumor specific CD8+ cytotoxic T cells (CTLs) and not required for the killing of tumor cells by CTLs. This basic understanding of the role of T cell immunity in eliminating tumors has given rise to many immunotherapeutic approaches focused on expanding tumor specific T cells in a tumor bearing host using vaccination and other immunotherapy approaches.

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Pathways of tumor antigen-specific T cell stimulation in vivo: Tumors transduced with the B7-1 molecule have been suggested to prime T cells directly, whereas DNA vaccines, peptide vaccines, and cytokine transduced tumors may stimulate T cells indirectly through host APC. However, recent studies suggest that tumors transduced with B7-1 molecule use both priming pathways to induce an antitumor immune response. The indirect pathway could occur through host APCs, mainly by DCs taking up the tumor antigens and processing and presenting them to CD8+ T cells through ▶cross-priming. B7-1 gene transfected tumor cell vaccines, once thought to work only by activating CD8+ T cells directly, have now been shown to activate T cells indirectly through cross-priming by professional APCs. The enhancement of cross-presentation by tumors expressing B7-1 has also been attributed to the enhanced recognition of these tumors by ▶natural killer ▶cells. NK cells have been shown to express CD28 and cross-linking of CD28 on NK cells by B7-1 results in the release of factors such as TNF-α and IFN-γ, which subsequently stimulate DCs. Recently it has been shown that activated human NK cells can also directly interact with CD4+ T cells and costimulate TCR-induced proliferation, suggesting a possible crosstalk between CD4+ T cells and NK cells during antigen specific immune response. Many studies have shown that antigen-specific CD8+ CTLs can be generated without CD4+ Tcell help. DCs have

T

T-Cell Response. Figure 1 Tcells require two signals to enpand and become effective cytotoxic Tcells. In the absence of second signal of tumor specific T cells and undergo energy or apoptosis. Thus, tumors by not expressing the second signaling. Molecule can prevent generation omtitumor T cell response.

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T-Cell Response

been shown to play a major role in this CD4+T cell independent expansion of CD8+ CTLs. Antigenspecific CD8+ CTLs developed in the absence of CD4+ T cell help are capable of providing protective antitumor immunity in mice. However, recent studies show that CD4+ T cells play a major role in maintaining the CD8 T cell memory developed during antigen exposure. The CD8+ memory T cells developed in the absence of CD4+ Tcellhelp are defective in responding to antigens in a secondary challenge with antigen, suggesting that for induction of an optimal antitumor immunity, both CD4+ Tcells and CD8+ Tcells are required. These observations demonstrate that interaction of immune cells such as DCs, NK cells and T cells that are activated by direct and indirect pathways cooperate to produce an optimal antitumor T cell immune response during vaccination. Thus, the introduction of ▶immunostimulatory molecules directly into the tumor by gene transfer offers an attractive approach to improve the immunogenicity of tumor cells. Accordingly, the expression of costimulatory molecules such as B7-1 or cytokine molecules by gene transfer results in the induction of tumor immunity capable of inducing wild-type tumor rejection in animals. Apart from these vaccine strategies, it has been shown that adoptive transfer of ex vivo expanded and activated ▶tumor infiltrating T cells reduced tumor burden in animals and humans. Recent studies have also shown that CD4+ CD25+ regulatory T cells (Tregs) (▶T regulatory cells) play an important role in suppressing antitumor immunity in a host. The depletion of Tregs has been shown to increase potency of many antitumor immunotherapeutic methods. Methods to detect antitumor T cell responses: Studying T cell immune responses during vaccination or other type of immunotherapies is valuable in understanding the nature of the immune response. Moreover, identifying a correlation between characteristics of antitumor T cell response and clinical efficacy of a treatment modality will be useful in predicting clinical outcome of a therapy at an early stage. For example, T cell populations carrying longer telomeres will be the dominant population in mice that are treated with the most potent vaccine since it has been shown that the presence of this T cell population is a predictive indicator of robust antitumor immunity. Similarly, better therapeutic efficacy was observed when a T cell population expressing TCR containing specific Vβ was expanded during a vaccine administration. Tumor antigen specific CD8+ T cells can be quantified by CTL assays and intracellular IFN-γ staining of cells obtained from blood in humans or the spleen in the case of mice. Limiting dilution assays or CD8 and IFN-γ staining can be used to determine the frequency of activated CD8+ T cells. Increase in IFN-γ has been shown to occur during antigen-specific CD8 + T cell activation. If the frequency of IFN-γ

staining cells is too low to be detected by the intracellular cytokine staining method, then the ELISPOT assay to determine IFN-γ secreting cells can be carried out. Alternatively, expansion of tumor antigen specific T cells can be monitored using MHC tetramers. Although measuring antigen specific T cells using MHC tetramers is a very sensitive assay, it indicates the mere physical presence of an antigen specific T cell but does not indicate whether the expanded antitumor T cells are functionally active or not. Therefore, MHC tetramer assays are normally combined with functional assays such as CTL assay or intracellular IFN-γ staining to determine the antitumor efficacy of an immunotherapeutic method. Lessons from human immunotherapy: Based on the knowledge obtained from in vitro experiments and animal models of antitumor T cell responses and vaccines, many clinical trials have been conducted. Both cell-based and vaccine-based therapies have been employed. In cell-based therapies, patients were administered ex vivo-expanded tumor infiltrating T cells or DCs loaded with tumor antigens. In vaccine-based therapies, cancer cells or cellular fragments or tumorassociated antigens were modified with adjuvants such as cytokines or other immunostimulatory molecules and administered to induce expansion of tumor antigenspecific T cells. Some of the trials showed moderate success whereas some of them were not as effective as observed in animal models. Interestingly, in many patients, although vaccination produced strong antitumor T cell responses, the regression of the tumor did not occur, suggesting there is a disconnect between the expansion of antitumor specific T cells and efficacy of a cancer vaccine. This could be due to the lack of homing of antitumor T cells to tumor site, or tumors may secrete immnuosuppressive factors that lead to the inactivation of cytotoxic T cells. Also, comparison of the results of T cell response, as measured by MHC tetramer assays and functional assays, showed that not all the tumor antigen specific T cells expanded during a vaccination are functionally active, suggesting aberrations in the development of T cells during vaccination. These results emphasize the need for further studies on the nature and mechanisms of T cell responses during various therapeutic approaches. A careful manipulation of the induction of antitumor T cell immune responses and homing of T cells to tumors will lead to the development of effective therapies to treat various types of cancer.

References 1. Smyth MJ, Dunn GP, Schreiber RD (2006) Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 90:1–50

TCL1

2. Zitvogel L, Tesniere A, Kroemer G (2006) Cancer despite immunosurveillance: immunoselection and immunosubversion. Nat Rev Immunol 6:715–727 3. Peggs KS, Allison JP (2005) Co-stimulatory pathways in lymphocyte regulation: the immunoglobulin superfamily. Br J Haematol 130:809–824 4. Rosenberg SA (2001) Progress in human tumour immunology and immunotherapy. Nature 411:380–384 5. Keilholz U, Martus P, Scheibenbogen C (2006) Immune monitoring of T-cell responses in cancer vaccine development. Clin Cancer Res 12:2346s–2352s

T-Cell Zones Definition In lymphoid tissues are enriched in T cells and are distinct from the B-cell zones and the stromal elements.

T-cells Recognizing Autoantigens ▶Autoimmunity and Prognosis in Cancer

Tcf/LEF Definition T-cell factor/ lymphoid enhancer-binding factor; transcription factors comprising Tcf-1 (TCF7), Tcf-3 (TCF7L1), Tcf-4 (TCF7L2) and LEF-1. Repress transcription when bound to ▶Groucho and activate transcription when bound to β-catenin. ▶Wnt Signaling

TCL1 G IANDOMENICO R USSO Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Roma, Italy

Definition The TCL1 gene is involved in the generation and/or manifestation of mature forms of leukemias, mainly in

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the T-Prolymphocytic leukemia (T-PLL) and in chronic lymphocytic leukemia (B-CLL).

Characteristics Clinical Characteristics of the T-PLL T-PLL is a disease that represents 20% of prolymphocytic leukemias. It occurs at an advanced age of 70–80 years, with a slight male predominance. It is, however, quite frequent in patients with the immunodeficiency syndrome ▶ataxia telangiectasia (AT) (1–5% of these patients develop it). Clinically it is accompanied by splenomegaly (75%), hepatomegaly (42%), lymphoadenopathy (55%), a high blood count (>200 × 109/L), with a very bad survival rate (70%) followed by the N3 position of adenine (9.2%) and the O6 atom of guanine (5%). Figure 2 depicts the chemical structure of the naturally occurring bases in DNA (guanine and adenine) and the TMZ-induced alkylated products (O6-MeG, N7-MeG and N3-MeA).

Temozolomide. Figure 1 Diagram of the chemical structure of temozolomide. Only the methyl group (CH3−) is transferred to DNA. The remainder of the molecule is metabolized in the form of molecular nitrogen (N2), carbon dioxide (CO2) and excreted as a molecule of 5-aminoimadozole-4-carboxamide (AIC) in urine.

Temozolomide and MGMT One of the major cytotoxic DNA lesions or types of DNA damage induced by TMZ treatment is the formation of methyl adducts on the O6 position of guanine (O6-MeG; see Fig. 2). However, the cytotoxic guanine lesion O6-MeG is rapidly repaired by a direct reversal reaction conducted by the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT or more commonly referred to as AGT). This DNA repair protein is encoded by the O6-methylguanine DNA methyltransferase gene, MGMT, located on chromosome 10 at position 10q26. The MGMT gene encodes a single mRNA (NM_002412) that translates to a single protein of 207 amino acids (NP_002403). MGMT functions to remove O6-MeG lesions in DNA via a suicide reaction in which the O6-Me group on guanine is transferred to the cys145 residue in MGMT, rendering MGMT inactive. Once alkylated on amino acid residue

Definition TMZ is a chemotherapeutic DNA alkylating agent of the imidazotetrazine class. IUPAC name = 3-methyl-2-oxo-1,3,4,5,8-pentazabicyclo[4.3.0]nona-4,6,8-triene-7-carbo oxamide CAS number = 85622-93-1 MW = 194.151 g/mol Chemical formula = C6H6N6O2 PubChem Compound information – CID #5394: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD = search&DB = pccompound&term = 5394 Structure – see Fig. 1.

Characteristics

T

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Temozolomide

Temozolomide. Figure 2 Chemical structures of the guanine and adenine bases in DNA and the temozolomideinduced modification to these bases. The guanine base (a) is modified by temozolomide on the O6-atom to yield O6-methylguanine (b) and on the N7-atom to yield N7-methylguanine (c) at a frequency of 5% and >70%, respectively. The adenine base (d) is modified by temozolomide at the N3-position to yield N3-methyladenine (e) at a frequency of 9.2%.

cys145, the MGMT protein is depleted from the cell within a few hours. Depletion of the MGMT protein is facilitated by the ubiquitin-dependent proteosome pathway. The alkylated form of MGMT is targeted for

poly-ubiquitylation; a post-translational modification that initiates proteosome-mediated degradation. Interestingly, MGMT activity is observed to be elevated in many tumors (in relation to the surrounding normal

Temozolomide

tissue) such as those derived from colon, lung, pancreatic and breast cancer, non-Hodgkin’s lymphoma, myeloma and glioma. It is now well established that elevated MGMT expression leads to resistance to clinical alkylating agents due to failure to remove the O6-MeG lesion. To improve TMZ efficacy, several strategies have been developed to limit the repair of the O6-MeG lesion. MGMT activity can be successfully attenuated by the use of free guanine base derivatives, with alkyl groups at the O6 position, which act as a pseudosubstrate and lead to MGMT depletion. Two of the most promising MGMT specific drugs are O6-benzylguanine (BG) and O6-(4-bromothenyl)guanine (Patrin, PaTrin-2, Lomeguatrib). Both of these MGMT-specific small molecule drugs are currently undergoing clinical evaluation to be used in combination with TMZ. In addition, other clinical alkylating agents are found to induce the formation of O6-MeG adducts to DNA, including the platinum drugs cisplatin and carboplatin as well as the alkylating agent dacarbazine. Combination treatments with TMZ are therefore being evaluated as a novel approach to deplete MGMT protein levels and thereby increase TMZ efficacy. Interestingly, the dosing or scheduling of TMZ administration can also be altered to maximize MGMT depletion. For example, the standard 5-day dosing schedule of TMZ (150–250 mg/m2/day) leads to complete depletion of MGMT in peripheral blood cells but MGMT levels will recover within 24 h. The observed TMZ-induced depletion of MGMT suggests that variation in the dosing schedules may improve MGMT depletion and clinical response. Compressed scheduling (five doses every 4 h or once every 8 or 12 h) has shown variation in MGMT depletion as well as elevated myelotoxicity. Alternatively, low (75 mg/m2) extended dosing for 6–7 weeks in cycles of 8 weeks resulted in complete MGMT depletion. Clinical benefit to this or other scheduling paradigms is under intense investigation. The TMZmediated induction of the O6-MeG DNA lesion and the depletion of MGMT has also been used to improve the effectiveness of topoisomerase inhibitors. Topoisomerase inhibitors such as Irinotecan (CPT-11) have been shown to have an enhanced efficacy when delivered in combination with TMZ. The increased level of O6-MeG in DNA acts as a Topoisomerase I (topo-I) trap and an increase in topo-I/DNA covalent complexes. Recently, pre-treatment with interferon-beta was also shown to down-regulate MGMT expression and to improve the response of xenografts to TMZ in pre-clinical testing. Conversely, low or undetectable expression of MGMT is generally considered a strong indicator of potential clinical response to TMZ. Loss of MGMT expression in tumors is generally via elevated methylation and hence inactivation of the MGMT promoter. Although MGMT promoter hypermethylation is associated with

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hypermethylation of many other promoter regions throughout the genome, epigenetic regulation (Epigenetic gene silencing) is not considered a random process in cancer. The development of methylation-specific PCR provides for a highly sensitive and accurate measurement of the methylation status of the MGMT promoter within the tumor tissue. DNA isolated from the tumor can therefore be analyzed for methylation status. Several studies have demonstrated that MGMT methylation status can be used to predict TMZ response. It is possible that MGMT promoter methylation measurements may be used to pre-screen patients for treatment options. However, there are other concerns that define response to TMZ and the cytotoxicity to the O6-MeG DNA lesion. Mismatch DNA Repair and Temozolomide Response The O6-MeG DNA adduct is not inherently cytotoxic by itself. The adduct is stable and genomic DNA containing O6-MeG adducts is efficiently replicated during cell division by human replicative DNA polymerases. However, if not repaired by MGMT, either cytosine or thymine may be inserted opposite the O6-MeG DNA adduct during replication. Insertion of thymine would lead to G to A point mutations in subsequent rounds of cell division and DNA replication, consistent with the elevated level of point mutations in oncogenes and tumor suppressor genes found in tumors with a loss of MGMT expression. The cytotoxicity of the TMZ-induced O6-MeG adduct stems from the replication-dependent formation of the O6-MeG:T mispair and the recognition of this mispair by the post-replication mismatch DNA repair pathway (MMR). The MMR pathway is a multi-protein DNA repair and DNA damage signaling pathway that removes errors of DNA replication and functions in meiotic and mitotic recombination as well as in DNA damage signaling and apoptosis following alkylation damage. Currently, two mechanisms have been proposed for the MMR-dependent cytotoxicity of the TMZ-induced O6-MeG DNA adduct: a “Futile Cycle of Repair” model and a “Direct DNA Damage Signaling” model. In the “Futile Cycle of Repair” model, the MutSα complex (a heterodimeric complex of the two MMR proteins MSH2 and MSH6) recognizes and binds to the O6-MeG:T mispair, recruits the MutLα complex (a heterodimeric complex of the two MMR proteins MLH1 and PMS2) to the mispair and initiates repair of the newly synthesized DNA strand. Since this repair process involves removal and resynthesis of the T-containing DNA strand, the O6-MeG:T mispair is regenerated during each cycle of repair thereby generating the substrate for another cycle. It is proposed that continued rounds of repair may lead to some aborted repair and the formation of double-strand

T

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Temozolomide

breaks and cell death. In the “Direct DNA Damage Signaling” model, MutSα binds to the O6-MeG:T mispair and without repair processing, recruits MutLα and the DNA damage response proteins ATRIP and ATR to initiate the DNA damage checkpoints leading to cell cycle arrest and apoptosis. Critical MMR proteins are therefore required for efficacy of TMZ and hence loss of MMR leads to resistance to TMZ. By a mechanism similar to that described above for MGMT epigenetic gene silencing, MMR can be compromised by tumor-specific promoter methylation of critical MMR genes. Whereas improved prognosis has been reported in tumors with loss of MGMT expression due to promoter methylation, poor prognosis is observed when MMR capacity is compromised by methylation of the promoter for the essential MMR genes MLH1, MSH2 and MSH6. More recently, loss of expression or inactivating mutations in MSH6 (an essential component of the MutSα complex) have been observed in TMZ resistant glioma tumors and in recurrent tumors following TMZ therapy, implicating an essential role for MHS6 in TMZ response. It is likely that all components of the MutSα complex (MSH2 and MSH6) and the MutLα complex (MLH1 and PMS2) may be essential for TMZ efficacy and response to the TMZ-induced O6-MeG DNA adduct. Repair of N7-Methylguanine and N3-Methyladenine DNA Lesions The N7-methylguanine (N7-MeG) and N3-methyladenine (N3-MeA) DNA lesions (Fig. 2) comprise over 80% of the DNA adducts induced by TMZ. Both of these DNA lesions are repaired by the base excision repair (BER) pathway. BER is the predominant DNA damage repair pathway for the processing of small base lesions derived from alkylation damage. BER is normally defined as DNA repair initiated by a lesionspecific DNA glycosylase and completed by either of two sub-pathways: short-patch BER; a mechanism whereby only one nucleotide is replaced or long-patch BER; a mechanism whereby 2–13 nucleotides are replaced. The majority of repair is currently thought to occur via the short-patch pathway. Repair of N7-MeG and N3-MeA is initiated by the methylpurine DNA glycosylase (Mpg; NM_002434). The paradigm for the short-patch BER pathway initiated by Mpg involves base lesion removal and then AP site hydrolysis by AP endonuclease (Ape1; NM_080649), catalyzing the incision of the damaged strand, leaving a 3′OH and a 5′deoxyribose-phosphate moiety (5′dRP) at the margins. DNA polymerase β (Pol β; NM_002690) hydrolyzes the 5′dRP moiety and fills the single nucleotide gap, preparing the strand for ligation by either DNA Ligase I (LigI; NM_000234) or a complex of DNA Ligase IIIα (LigIIIα; NM_013975) and XRCC1

(NM_006297). Each step throughout BER is coordinated via protein-protein interactions with the XRCC1/ DNA Ligase IIIα heterodimer and PARP1 (NM_001618), two important BER scaffold protein complexes. Furthermore, PARP1 has been linked more directly to BER as it interacts both physically and functionally with Pol β and LigIIIα, placing it as a member of the short-patch BER pathway. In addition, PARP1 coordinates with long-patch BER proteins to facilitate the repair of longer stretches of DNA. BER removes >80% of the DNA lesions induced by TMZ. The advent of TMZ has therefore increased interest in the development of BER specific inhibitors since aborted or blocked BER sensitizes most cancer cells to alkylating agents. Blocking almost any step in the BER pathway will improve TMZ efficacy in cell culture assays, suggesting that BER inhibitors (just as observed with inhibitors of MGMT) may prove useful when administered in combination with TMZ. Few small molecule drugs have been developed that are specific for BER proteins. Lucanthone and CRT0044876 have been reported to inhibit Ape1 and will sensitize cells to TMZ. Methoxyamine is another BER inhibitor; however, this compound does not directly inhibit BER enzymes but instead binds to abasic sites in DNA, making them refractory to further repair and the methoxyamine-bound abasic site is highly cytotoxic. Methoxyamine is currently undergoing clinical trials in concert with TMZ. The most common BER target for small molecule drugs (inhibitors) is PARP1 although PARP1 is also involved in the repair of double-strand breaks (DSBs) in DNA via a non-homologous end-joining pathway. PARP1 is the founding member of a large family of ADP-ribosyl transferase proteins including six PARPs (poly-ADP-ribose polymerases), eleven MARTs (mono-ADP-ribosyltransferases), seven SIRTs, four PARGs (poly ADP-ribose glycohydrolases) and PiMARTs. To date, only PARP isoforms 1, 2 and 3 have been found to respond to DNA damage such as that induced by TMZ. Upon binding to a nick (a singlestrand break) or a DSB, PARP is activated to poly-ADPribosylate itself and other target proteins and to synthesize poly-ADP-ribose. Inhibiting PARP activation has been shown to significantly improve TMZinduced tumor cell killing and to improve response to TMZ in pre-clinical xenograft models. Currently, there are at least six PARP inhibitors undergoing clinical trials as anticancer agents, although the specificity of these for PARP1, PARP2, PARP3 or other PARP-family members remains to be determined. Three of these small molecule inhibitors undergoing clinical trials are being evaluated for clinical efficacy in combination with TMZ. These clinical trials (Phase I and II) are for the treatment of solid tumors, metastatic melanoma and glioblastoma multiforme.

Temsirolimus

Although TMZ is effective in the treatment of glioblastoma and is being evaluated for melanoma and other cancers, efficacy can clearly be improved through combination therapy to inhibit one or more DNA repair pathways. Continued development of MGMT and BER inhibitors is expected to have significant impact on response to TMZ in the near future. Further, analysis of these critical DNA repair pathways in tumors can provide valuable biomarkers to anticipate response.

References 1. Almeida KH, Sobol RW (2007) A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification. DNA Repair 6:695–711 2. Gerson SL (2004) MGMT: its role in cancer aetiology and cancer therapeutics. Nat Rev Cancer 4:296–307 3. Jiricny J (2006) The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 7:335–346 4. Newlands ES, Stevens MF, Wedge SR et al. (1997) Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 23:35–61 5. Wang JY, Edelmann W (2006) Mismatch repair proteins as sensors of alkylation DNA damage. Cancer Cell 9:417–418

Temsirolimus J ANET E. DANCEY Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD, USA

Synonyms CCI-779; Torisel™

Definition Temsirolimus is a small molecule inhibitor of mammalian target of rapamycin (mTOR) kinase. Aberrant intracellular signaling through mTOR is associated with the cancer cell proliferation. Inhibition of this pathway by temsirolimus result in anti-proliferative effects that may result in improved survival in patients with cancer.

Characteristics Temsirolimus is a soluble 42-[2,2-bis (hydroxymethyl)]-propionic ester of the macrocyclic lactone rapamcyin (also known as sirolimus). Temsirolimus, sirolimus and other members of this class of agents inhibit the proliferation of normal and malignant cells

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by inhibiting the activity of mTOR, an intracellular serine-threonine kinase within the Phosphoinositide 3-kinase (PI3K)/Akt signal transduction pathway. Temsirolimus, like sirolimus, reacts with the ubiquitous intracellular FK506-binding protein 12 (FKBP12) to inhibit mTOR function. Mechanism of Action Activation of the phosphoinositide 3-kinase (PI3K)/ Akt/mammalian target of rapamycin (mTOR) pathway plays a pivotal role in essential cellular functions such as survival, proliferation, migration differentiation and carbohydrate metabolism, and is important in the molecular mechanisms of diseases such as diabetes and chronic inflammation, as well as cancer. Normal cells such as lymphocytes, ▶endothelial cells and fibroblasts as well as cancer cells are dependent of this signaling pathway. The observed antitumor and immunosuppressive properties of temsirolimus and other agents within this class are due to their ability to disrupt mTOR function. Both activating mutations and amplification of oncogenes and loss of tumor suppressor genes occur within the pathway in human neoplasms with remarkable frequency. Activation mutations of growth factor receptors and PI3K, as well as amplification and or overexpression of PI3K and Akt have been reported in different tumor histologies. Similarly, the loss of tumor suppressor proteins that regulate the PI3K-Akt-mTOR pathway such as tuberous sclerosis proteins 1 or 2 (TSC1/2), phosphatase and tensin homologue deleted on chromosome 10 (PTEN), and serine/threonine kinase 11 (STK11 also known as LKB1) has been linked to the pathobiology of a number of tumor predisposition syndromes, including ▶tuberous sclerosis syndrome (TSC1/2), ▶Peutz-Jeghers syndrome (STK11/ LKB1), and ▶Cowden syndrome (PTEN). In laboratory models, the resultant aberrant activation of the signaling pathway through oncogene stimulation or tumor suppressor gene loss not only leads to a growth advantage during carcinogenesis but also contributes to tumor angiogenesis, metastasis, and resistance to standard cancer therapy. Of interest and relevance to cancer therapeutics development, aberrant pathway activation may also lead to sensitivity to agents that target mTOR. Temsirolimus does not directly interact with mTOR kinase and does not inhibit all mTOR functions. mTOR functions are dependent on its forming complexes with other proteins. Two mTOR-containing complexes have been well characterized: a rapamycin-sensitive complex (also called mTOR complex 1, mTORC1), which includes mTOR accessory protein Raptor along with mammalian ortholog of LST8 (mLST8) (regulatory-associated protein of mTOR); and a rapamycin-insensitive complex (also called mTOR complex 2, mTORC2), which composed of mTOR and Rictor (rapamycin-insensitive

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companion of mTOR). mTORC1, phosphorylates the well-characterized mTOR effectors S6 kinase 1 (S6K1, also known as p70S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). TORC2 controls the actin cytoskeleton as well as Akt. However, mTOR when bound to Rictor can phosphorylate Akt at its hydrophobic motif leading to Akt activation. This positive feed back loop may result in increased phosphorylation of Akt in the presence of temsirolimus inhibition of the Raptor-mTOR complex. Activity in Cancer Models The antiproliferative effects of temsirolimus and other sirolimus derivatives have been evaluated in numerous in vitro and in vivo tumor models. In sensitive cell lines, these agents inhibit tumor and endothelial cell proliferation in picomolar to nanomolar concentrations and may add to the cytotoxicity of other chemotherapeutic agents and radiation. The antiproliferative effects of temsirolimus may be due, at least in part, to its wellcharacterized inhibitory effects on the activation of S6K1 and 4EBP1. Inhibition of these proteins alters the translation of subsets of mRNAs, particularly those that may be involved in regulating cell cycle progression. In a relatively limited number of tumor models, this class of agents may induce cancer cell death apoptosis or autophagy. The molecular mechanisms leading to apoptosis in cancer cells have not yet been fully deciphered. In addition, mTOR inhibitors can target tumor growth indirectly by inhibiting endothelial cells and ▶pericytes proliferation required for tumor angiogenesis. Activity in Clinical Trials Results from cancer clinical trials suggest that temsirolimus is well tolerated and appears to have anti-tumor activity. The most common toxicities seen with the drug are mild to moderate skin reactions, ▶stomatitis, reductions in blood counts, particularly platelets, and metabolic abnormalities such hyperlipidemia and hyperglycemia. These adverse effects are reversible with interruption of dosing or, for hyperlipidemia and hyperglycemia, with specific treatment. Rarely, pneumonitis has been reported in patients that have received temsirolimus. To date, there has been no evidence of clinically significant immunosuppression with intermittent schedules. Effective target inhibition has been shown through pharmacodynamic assays assessing inhibition of either S6K1 or 4E-BP1 phosphorylation in surrogate tissue from patients treated with temsirolimus. More limited data from baseline and on treatment tumor tissue specimens supports that temsirolimus inhibited mTOR and downstream targets. However, optimal dose/schedule for intratumoral target inhibition has not well defined due to limitations in numbers of specimens analyzed.

Results from four phase I studies evaluating increasing doses of temsirolimus on different schedules and with oral and intravenous formulations have been reported. The weekly intravenous schedule is the one that has been most extensively evaluated in phase II and III studies. Phase II studies of single agent temsirolimus evaluating different doses of 25, 75, and/or 250 mg weekly IV have been undertaken in broad range of tumor histologies. The most promising anti-tumor activity has been seen in ▶mantle cell lymphoma and ▶endometrial carcinoma with objective tumor response rates of 30–40%. Moderate activity has been reported in breast and ▶renal cell carcinoma. Minimal single agent activity has been seen in ▶small cell lung carcinoma, ▶melanoma and ▶glioblastoma multiforme. In general, lower doses appear to be as active as higher doses with better tolerability. A phase III trial of temsirolimus, temsirolimus with interferon versus interferon in poor prognosis patients with renal cell carcinoma has been reported. Of the 626 patients, overall survival of patients treated with temsirolimus was significantly prolonged compared to those treated with interferon (median 10.9 months versus 7.3 months, Harzard Ratio for death 0.73, p = 0.0069). The combination of interferon and temsirolimus did not confer greater benefit than interferon alone, possibly due to compromised dose delivery of the agent(s) due to significant toxicity. The identification of tumor types that respond to mTOR inhibitors remains a major issue for the development of temsirolimus. mTOR is ubiquitously expressed and therefore the sensitivity or resistance of specific tissues to temsirolimus cannot be predicted solely on the basis of whether the target protein can be detected in the tumor tissue. Activation status of the PI3K/AKT/mTOR signaling pathway seems to be the most promising strategy to identifying tumor types potentially sensitive to temsirolimus. As a significant number of patients have cancers that are insensitive to temsirolimus, combinations of the mTOR inhibitor with hormonal therapy (▶hormonal treatment), chemotherapy or other targeted therapies, based on the rationale that simultaneous inhibition of multiple signaling pathways are under evaluation. Temsirolimus, which inhibits the downstream kinase mTOR, has demonstrated that it may be a useful cancer therapeutic as it may confer clinical benefit to patients with acceptable toxicity. The proof of principle that temsirolimus can improve cancer patient survival has been recently obtained from a large randomized trial in advanced poor prognostic renal cell carcinoma. The major clinical development challenges will be efficiently identifying the optimal dose, schedule and combination regimens for patients with susceptible

Tenascin-C

malignancies and monitoring and managing toxicities to optimize the therapeutic index. ▶Rapamycin

References 1. Thomas GV (2006) mTOR and cancer: reason for dancing at the crossroads? Curr Opin Genet Dev 16(1):78–84 2. Cully M, You H, Levine AJ et al. (2006) Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6(3):184–192 3. Samuels Y, Ericson K (2006) Oncogenic PI3K and its role in cancer. Curr Opin Oncol 18(1):77–82 4. Faivre S, Kroemer G, Raymond E (2006) Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 5(8):671–688 5. Hudes G, Carducci M, Tomczak P et al. (2006) A phase 3, randomized, 3-arm study of temsirolimus (TEMSR) or interferon-alpha (IFN) or the combination of TEMSR + IFN in the treatment of first-line, poor-risk patients with advanced renal cell carcinoma (adv RCC). J Clin Oncol 24 (June 20 Supplement):Abstract LBA4

Tenascin-C G ERTRAUD O REND Inserm U682 Institute of Development and Pathophysiology of the Intestine and the Pancreas 3, Avenue Moliere 67200, Strasburg, France

Synonyms Hexabrachion; Myotendinous antigen; Glial/mesenchymal extracellular matrix protein; GMEM; Cytotactin; J1 220/200; Neuronectin

Definition Tenascin-C is the founding member of a family of extracellular matrix glycoproteins comprising tenascinX, -R, and -W in addition to tenascin-C. Its name has been created by Ruth Chiquet-Ehrismann 1986 and represents a combination of the Latin verbs “tenere” (to hold) and “nasci” (to grow, develop, to be born), which provided the roots of the English words “tendon” and “nascent,” and reflect the location and developmental expression of the protein observed at that time.

Characteristics

Tenascin-C is part of a ▶tumor-specific stroma of most solid cancers, and plays a role in enhancing proliferation, ▶angiogenesis, and ▶metastasis during tumorigenesis. Moreover, research data support the possibility

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that tenascin-C contributes to cancer formation via interference with genomic stability, blocking the immunosurveillance and by providing a favorable niche for tumor stem cells (▶Cancer stem-like cells, ▶stem cells and cancer). Its high expression correlates with bad prognosis for disease-free survival in patients with glioma, lung, and colon cancer. Molecular Organization The tenascin-C protein, 2′201 amino acids in length giving rise to a 190–300 kDa monomer, is encoded by 6′603 bp, which are organized into at least 28 exons on chromosome 9q33. Tenascin-C is a modular molecule consisting of an N-terminal region containing a chaperone-like sequence forming coiled coil structures and interchain disulfide bonds that are essential for subunit oligomerization into hexamers. Tenascin-C is comprised of 14.5 epidermal growth factor (EGF)like repeats, 30–50 amino acids in length, which contain six cysteine residues involved in intrachain disulfide bonds. In tenascin-C, up to 17 ▶fibronectin type III domains are present that are 90 amino acids in length and that are composed of seven antiparallel β-strands arranged in two sheets. The nature and number of fibronectin type III domains in tenascin-C is generated by alternative splicing that is modulated by the proliferative state of a cell, extracellular pH, and TGFβ1. At least nine different fibronectin type III domains are differentially included or excluded by RNA splicing. This can generate a considerable diversity among different cancers (Fig. 1) and, can cause variable cell responses toward tenascin-C. The C-terminal fibrinogen globular domain resembling the β- and γ-chains of fibrinogen, 210 amino acids in length, forms intrachain disulfide bonds (Fig. 1). Induction and Processing Tenascin-C can be induced in a tumor by various proand antiinflammatory cytokines and growth factors that are mostly secreted by stromal cells. In addition, hypoxia (▶Hypoxia and tumor physiology), ▶reactive oxygen species, and mechanical stress, which are also present in tumor tissue, induce tenascin-C expression. In contrast, glucocorticoids suppress tenascin-C expression. Signaling causing activation of transcription factors such as TCF/LEF, NfkB, c-Jun, Ets, SP1, and Prx-1 is involved in tenascin-C gene transcription. Tenascin-C is cleaved by ▶matrix metalloproteinases and serine proteases, thus potentially releasing cryptic sites within the fibronectin type III domains of tenascin-C. Cell contact with tenascin-C also induces the expression of matrix metalloproteases, thus presenting a positive feedback loop between induction of matrix metalloproteases by tenascin-C and cleavage of tenascin-C by these enzymes.

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Tenascin-C

Tenascin-C. Figure 1 Domain structure, binding partners and expression of tenascin-C in cancer tissue. The N-terminal oligomerization, EGF-like, fibronectin type III and fibrinogen-like domains are schematically depicted as triangle, rhombomeres, boxes and circles, respectively. The alternatively spliced fibronectin type III domains A1-D are shown in black. An electromicrograph of a tenascin-C hexamer is shown at the left corner. Fibronectin type III domains specifically detected in certain cancers are highlighted above the model. EGFR, epidermal growth factor receptor; CALEB, chicken acidic leucine-rich EGF-like domain containing brain protein; RPTPβ, receptor protein tyrosine phosphatase-β/ζ; Nav1.9/NaN, sodium channel subunit β2. Picture was taken from Orend and Chiquet-Ehrismann (2006) Cancer Lett 244:143–163.

Interaction Partners Tenascin-C binds to extracellular matrix molecules such as fibronectin, perlecan, aggrecan, versican, and brevican (Fig. 1), thus potentially forming a tumorspecific extracellular matrix network. Cells can interact with tenascin-C via cell surface receptors including integrins (▶Integrin signaling in cancer) α2β1, α7β1, α9β1, and αvβ3, syndecan, annexin II, and ▶epidermal growth factor receptor (EGFR) among others (Fig. 1). Cell Rounding and Tumor Cell Proliferation Tenascin-C has distinct effects on tumor cells, and tumor-associated cells such as carcinoma-associated fibroblasts, tumor-associated macrophages, and endothelial cells within the tumor stroma based on as yet poorly understood cell type-specific responses toward tenascin-C splice variants. Tenascin-C contains adhesive and antiadhesive sequences that coexist in the native molecule. These opposing activities arise as a consequence of tenascin-C binding to extracellular matrix components and to cell surface receptors. One mechanism that induces cell rounding involves tenascin-C inhibition of cell adhesion to fibronectin. This occurs through competitive binding of tenascin-C to fibronectin, thus masking the binding site for integrin α5β1 coreceptor syndecan-4 (▶Heparanases). This blocks activation of the small GTPase RhoA and focal adhesion kinase. Activation of oncogenic Wnt (▶Wnt signaling), endothelin receptor type A, and ▶MAPK signaling induced by tenascin-C and elimination of G0 and G1 cell cycle (▶Cell cycle targets for

cancer therapy) transition control could contribute to enhanced tumor cell proliferation by tenascin-C. Metastasis Tenascin-C is expressed around invasive carcinoma cells that have undergone epithelial-mesenchymaltransition (EMT) (▶Epithelial to mesenchymal transition). Tenascin-C supports tumor cell migration and invasion by mechanisms that are little understood. Tenascin-C provides a substratum that supports migration of several cell types including glioma and laryngeal carcinoma cells. A mechanism by which tenascin-C supports colon carcinoma cell invasion involves secretion of tenascin-C by carcinoma-associated fibroblasts, activation of EGFR and, expression of hepatocyte growth factor and activation of its receptor c-Met. This triggered downstream activation and inhibition of the small GTPases Rac and RhoA, respectively in the invading carcinoma cells. In addition to an EMTassociated migration, tenascin-C might also promote other forms of migration in cancer cells. Angiogenesis Tenascin-C plays a role during embryonic vascularization and promotes vascular sprouting. It is also expressed during formation of new blood vessels in the adult as, e.g., in granulation tissue of wounds after myocardial infarction, in arthritis, and in neoplastic diseases. In human gliomas, tenascin-C expression correlates with the degree of tumor neovascularization. Tenascin-C may promote angiogenesis by serving as

Tensional Homeostasis

chemoattractant for endothelial cells, by initiating endothelial cell differentiation, survival, and proliferation, events that involve integrin αvβ3 and vascular endothelial growth factor among not yet identified other molecules. Phenotype of Tenascin-C Knockout Mice and Cancer The tenascin-C sequence is highly conserved among species, which suggests that evolutionary forces prevented loss of this gene because of its importance to life. Tenascin-C knockout (▶Gene knockout) mice are hyperactive among other neurological defects, which would make them an easy prey. Abnormal behavior in these mice might be due to its role as ligand for neuronal receptors. Despite an otherwise apparently normal phenotype, which is likely due to compensatory mechanisms, tenascin-C knockout mice show difficulties in regeneration upon disturbance of tissue homeostasis such as during healing of wounds in the eye and in the inflamed kidney. Tenascin-C is expressed in stem cell niches (▶Adult stem cells) such as those of the bone marrow, brain, and skin. Stem cells are required to maintain and restore tissue homeostasis, in particular upon insults to tissues. This may explain the more severe phenotype in injured tenascin-C knockout mice. The first in vivo evidence for a role of tenascin-C in tumor angiogenesis derives from studies with xenografted melanoma cells into mice lacking tenascin-C expression. In these tenascin-C knockout mice, tumor growth and angiogenesis was strongly reduced in comparison to mice exhibiting tenascin-C expression. Clinical Aspects How can we use our knowledge about tenascin-C to combat cancer? Given that expression of tenascin-C correlates with tumorigenesis-enhancing events and with a reduced disease-free survival in patients with some cancers, inhibition of tenascin-C expression at the transcriptional level would be the first choice to block tenascin-C actions in cancer. Unfortunately, this approach is questionable since many factors and conditions that trigger tenascin-C expression are not specific for tenascin-C alone but affect many other genes. Preventing tenascin-C action in a tumor, e.g., by restoration of syndecan-4 function in gliomas offers another approach. However, since tenascin-C has many poorly understood effects at the molecular level on the different cell types within cancer tissue, targeting tenascin-C actions may produce undesirable side effects. The most promising approach today is to target tenascin-C with antitenascin-C-directed antibody fragments that are coupled to cytotoxic reagents in a trojan horse-like strategy, which would trigger destruction of the tumor. Tenascin-C-targeting antibodies are in clinical trials and one needs to await the antitumor response rates in cancer patients.

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References 1. Chiquet-Ehrismann R, Chiquet M (2003) Tenascins: regulation and putative functions during pathological stress. J Pathol 200:488–499 2. Jones PL, Jones FS (2000) Tenascin-C in development and disease: gene regulation and cell function. Matrix Biol 19:581–596 3. Orend G, Chiquet-Ehrismann R (2006) Tenascin-C induced signaling in cancer. Cancer Lett 244:143–163 4. Orend G (2005) Potential oncogenic action of tenascin-C in tumorigenesis. Int J Biochem Cell Biol 37:1066–1083

Tensin2 Definition Is a focal adhesion protein of the tensin family that acts as an important link among extracellular matrix, actin cytoskeleton, and signal transduction. ▶Deleted in Liver Cancer 1

Tensional Homeostasis I NKYUNG K ANG , VALERIE M. W EAVER Department of Surgery, University of California, San Francisco, CA, USA

Definition A mechano-regulatory network that integrates physical and biochemical cues from the tissue ▶microenvironment through mechano—responsive elements such as transmembrane integrins to evoke cytoskeletal re-organization and actomyosin contractility, thereby altering signal transduction and gene expression to modulate cell and tissue phenotype.

Characteristics Cells and tissues experience and respond to externally applied mechanical force through mechano-responsive elements that influence signal transduction and result in the generation of reciprocal intracellular force or contractility. The types of ▶mechanical stress a cell can experience include compressive or tensile stress which is applied perpendicular to the surface of the cell, and shear stress which is applied parallel to the surface of the cell. For example, osteoblasts and chondrocytes within bone and cartilage are subjected to compressive force induced by walking, lung alveolar cells experience

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tensile load resulting from inhalation-induced alveolar sac expansion, and endothelial cells lining the lumens of blood vessels undergo shear force induced by circulating blood flow. Cells integrate external mechanical force on multiple levels. This includes forcedependent changes in the conformation of the plasma membrane lipid bilayers as well as modifications in the orientation and molecular associations of transmembrane proteins. These changes enhance the activity of calcium and potassium ion channels, the extracellular matrix affinity, and cytoskeletal plaque associations of various adhesion molecules including integrins (▶Integrin signaling and cancer). Cells also integrate external force cues to generate reciprocal actomyosin-mediated cell contractility and modulate their ▶mechanical properties through remodeling of the microtubule, intermediate filament and actin cytoskeletal network. Intracellular mechanical force is transduced to the extracellular microenvironment via functional links between transmembrane receptors that bind to extracellular matrix (ECM) proteins and the intracellular cytoskeletal network and ultimately mediate an equilibrium of extracellular and intracellular forces in the cell. This equilibrium or balance between the extracellular forces and the intracellular forces is called tensional homeostasis. When the extracellular mechanical microenvironment is becomes altered, cells and tissues will coordinately respond by adjusting cellgenerated mechanical force or contractility, which in turn elicits changes in cell behavior by modifying the activity and function of signaling pathways and gene expression that determine growth, survival and differentiation. Cells sense and integrate tensional forces by altering the expression and activity of a plethora of putative ▶mechanosensors. Nevertheless, integrins are considered key mechanotransducers by virtue of their external associations with the extracellular matrix and their internal links to various adhesion plaque proteins including vinculin, talin and ▶focal adhesion kinase (FAK), which in turn mediate interactions with the cytoskeleton and activate various signaling cascades. Extracellular mechanical force can alter the conformation of an integrin from a low ligand-binding affinity state to high ligand-binding affinity state, the conformation of extracellular matrix proteins such as ▶fibronectin and collagen I to expose or alter ligand binding sites, and the conformation of vinculin and talin to favor intracellular molecular associations. These mechanically-initiated events promote actin assembly and stabilize adhesion plaque protein assembly and clustering of integrins to convert nascent ▶focal complexes into mature ▶focal adhesions. Forcedependent integrin activation and focal adhesion maturation increase the magnitude and duration of adhesion signaling including ERK ▶MAP kinase and RhoA GTPase (▶Rho family proteins). Elevated and

sustained activity of ERK and RhoA GTPase drive actomyosin-mediated intracellular contractility by altering the function of Rho kinase (ROCK) and phosphorylated myosin light chain. The elevated intracellular tension in turn promotes focal adhesion maturation, creating a feedback loop of biochemical signaling pathways resulting in an elevated intracellular force generated by actomyosin cytoskeleton and inside out remodeling of extracellular matrix proteins (see Fig. 1). When the balance between the external and intracellular stress is altered, the cell and tissue will adapt to the new mechanical microenvironment challenge, which can result in positive outcomes such as an increase in bone and muscle density due to exercise, or in negative outcomes such as atherosclerosis mediated by chronically elevated shear force applied by perturbed blood flow and cardiac hypertrophy due to hypertension. Mechanical compression can also regulate gene expression to influence tissue development as has been documented during embryogenesis. Changes in matrix stiffness determine the lineage commitment of mesenchymal stem cells, such that the cells express neurogenic markers when grown in mechanical environment closer to the stiffness of brain (0.1–1 kPa), myogenic markers at an intermediate stiffness (8–17 kPa), and osteogenic markers at a higher stiffness (25–40 kPa). This lineage commitment is regulated by nonmuscle myosin II and is accompanied by an increase in the size of focal adhesions and in the expression of focal adhesion components including talin and phosphoFAK. These results suggest that a cell dynamically probes its mechanical microenvironment through active engagement of integrin adhesion receptors and generation of actomyosin contractility, and that an increase in focal adhesion maturation and intracellular contractility drives downstream signaling events which determine lineage differentiation. Thus, tensional homeostasis is emerging as a critical determinant in cell fate during normal morphogenesis as well as pathophysiological processes. Solid tumors are characteristically stiffer than normal tissue, which allows detecting tumors by palpation. The elevated stiffness is mediated by increased interstitial tissue pressure and changes in the mechanical properties of malignant cells and the surrounding stroma. The tumor stroma is characterized by an increased deposition and reorganization of matrix proteins including collagen, fibronectin and tenascin, and aberrant ECM cross linking induced by lysyl oxidase, transglutaminase, proteoglycans, and glycation, which contribute to the stiffening of the stroma (▶Extracellular matrix remodeling). In addition, transforming ▶oncogenes such as ▶RAS, ErbB/▶HER2 neu and c-Myc (▶Myc oncogene) can alter the mechano-responsiveness of cells and cooperate with integrin adhesion signaling


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Tensional Homeostasis. Figure 1 Key molecular pathways that mediate tensional homeostasis in cells and tissues. Changes in the mechanical environment of cell, such as an increase in ECM stiffness or elevated extracellular tension, promote integrin clustering to drive the maturation of nascent focal contacts into focal adhesions. The assembly of focal adhesions is associated with increased Rho GTPase activity and elevated and sustained ERK signaling. The combination of enhanced Rho and ERK activity increases actomyosin-mediated intracellular contractility by altering the function of Rho kinase (ROCK) and phosphorylated myosin light chain (MLC-P). Elevated cell-generated force promotes focal adhesion assembly and potentiates growth factor dependent ERK activation in a feed forward vicious cycle. Elevated intra cellular force also alters ECM deposition and organization by orienting and further stiffening ECM. Oncogenes which promote RAS-dependent ERK activation and Rho GTPase activity additionally contribute to cell-generated forces by regulating ROCK and MLCK and myosin II activity.

molecules to enhance cell proliferation, survival and invasion. Indeed, oncogenes such as Ras and ErbB/ HER activate Rho and ERK that induce actomyosin contractility and elevate cell-generated forces to further promote the assembly and maturation of integrin adhesions and enhance growth factor receptor crosstalk. This raises the intriguing possibility that in addition to promoting cell growth and survival by directly modifying the activity of various signaling molecules, some transforming oncogenes might promote malignancy by altering the cells tensional homeostasis. Consistently, Paszek et al demonstrated that increasing ECM stiffness from 140 Pa (approximating the compliance of the normal murine breast) to 1,000–5,000 Pa (similar to the stiffness of a malignant

murine breast) compromised mammary morphogenesis and induced the malignant phenotype of non-malignant mammary epithelial cells in culture, as demonstrated by an increase in cell growth and survival, and the loss of mammary tissue integrity (i.e. disruption of cell-cell junctions and loss of tissue polarity). Stiffening of ECM also significantly increased recruitment and activation of FAK and actin-binding proteins such as vinculin to β1 integrin adhesion, which was accompanied by an increase of larger, mature focal adhesions, contractility, and enhanced growth-factor dependent ERK activation. More intriguingly, malignantly transformed mammary cells with elevated epidermal growth factor receptor (EGFR) signaling that form colonies of disorganized, invasive and continuously growing cancer cells in

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response to a compliant normal matrix also exerted higher cell contractility. Strikingly, inhibiting the activity of Rho GTPase or myosin II or ERK was sufficient to reduce the tumor cells contractility and revert the malignant phenotype of these breast cancer cells toward that of a normal breast acini. Likewise, inhibiting Rho or ERK-dependent myosin activity also normalized the phenotype of non-transformed mammary cells interacting with an abnormally stiffened matrix. Together these findings suggest that breast transformation could arise through the combination of oncogenic mutations that promote cell generated contractility and a progressive stiffening of the ECM which compromises the tensional homeostasis to elevate cell contractility and to increase focal adhesion assembly, which enhance abberant cell growth, survival and invasion. Clinical studies indicate that mammographic density is strongly and reproducibly associated with an increased risk of breast cancer, independent of other risk factors (▶Mammographic breast density and cancer risk). For example breast cancer risk rises to 30% when greater than 50% of the mammography qualifies as dense. Although the high cancer risk linked with breast density could be attributed to decreased detection sensitivity and increased epithelial mass, recent data indicate that elevated collagen and proteoglycan content are also risk factors that contribute to the enhanced transformation frequency associated with this condition. Elevated mammographic density frequently precedes ▶ductal carcinoma in situ (DCIS), and DCIS often occurs predominantly in the mammographically dense areas of the breast. Because higher collagen density and elevated proteoglycan-mediated cross linking correlate with an increase in ECM stiffness, these findings are consistent with the prediction that mammographic density could promote carcinogenesis by perturbing the cells tensional homeostasis. If true, an increase in matrix stiffening would herald an altered tissue tensional homeostasis and constitute a tractable predictor of future tissue transformation. Accordingly, an improved understanding of the parameters that promote matrix stiffening and alter tissue tensional homeostasis would assist in the development of improved detection, prognosis and treatment strategies for solid cancers. To summarize, cells and tissues sense and respond to external force through a process called tensional homeostasis that reciprocally alters the external microenvironment through cell-generated force. Tensional homeostasis is emerging as an important determinant of normal tissue development and adult tissue homeostasis and recent studies indicate that an altered tensional homeostasis likely contributes to the pathogenesis of diseases including cancer and atherosclerosis.

References 1. Keller R, Davidson LA, Shook DR (2003) How we are shaped: the biomechanics of gastrulation. Differentiation 71(3):171–205 2. Paszek MJ, Zahir N, Johnson KR et al. (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241–254 3. Engler AJ, Sen S, Sweeney HL et al. (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689 4. Boyd NF, Rommens JM, Vogt K et al. (2005) Mammographic breast density as an intermediated phenotype for breast cancer. Lancet Oncol 6:798–808 5. Yamaguchi H, Wyckoff J, Condeelis J (2005) Cell migration in tumors. Curr Opin Cell Biol 17:559–564

TEP1 Definition TGF-β-Regulated and Epithelial Cell-Enriched Phosphatase; Rarely used synonym for PTEN. ▶PTEN

Teratocarcinoma Definition

Is an embryonic or ▶germ-cell tumor.

Teratocarcinoma-derived Growth Factor-1 ▶Cripto-1

Teratogenic Definition Refers to substances that cause developmental malformations during gestation. ▶Estrogenic Hormones

Testicular Cancer

Teratoma

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Tertiary Cancer Prevention

Definition

Definition

Plural Teratomata. Term derived from Greek meaning “monstrous tumor”. Is a germ cell tumor derived from pluripotent cells and made up of elements of different types of tissue from one or more of the three germ cell layers. Is a tumor with tissue or organ components resembling normal derivatives of all three germ layers. Rarely, not all three germ layers are identifiable. The tissues of a teratoma, although normal in themselves, may be quite different from surrounding tissues, and may be highly inappropriate, even grotesque (hence the term “monstrous”). Teratomas have been reported to contain hair, teeth, bone and very rarely more complex organs. Usually, however, a teratoma will contain no organs but rather one or more tissues normally found in organs such as the brain, thyroid, liver, or lung.

Prevention of metastatic spread of tumors. A locution used in parallel with ▶primary cancer prevention and ▶secondary cancer prevention. ▶Adjuvant Therapy ▶Immunoprevention of Cancer

Tertiary Structure Definition The three-dimensional structure of a monomeric protein. ▶Structural Biology

N- or C-Terminal Processing Definition Proteolytic attack of a protein substrate can have different outcomes depending on the protease at work and the protein under attack: 1. The protein can be completely degraded into small fragments and amino acids; 2. The protein can undergo limited proteolysis, also referred to as processing or maturation; 3. If the protein is a protease inhibitor, it may form a complex and neutralize the protease; 4. The protein might be neither susceptible to proteolysis nor interact with the protease. When limited proteolysis occurs at either end of a protein it is referred to as N- or C-terminal processing. ▶Cystatins

Terminally Differentiated Cells Definition Cells most distal to the stem cell, being differentiated to perform a specific function, but having permanently lost the ability to divide. ▶Stem Cell Plasticity

Testicular Cancer A XEL H EIDENREICH Division of Oncological Urology, Department of Urology, University of Köln, Köln, Germany

Synonyms Testicular germ cell tumor; Seminomatous germ cell tumor; Nonseminomatous germ cell tumor

Definition About 90% of all testicular tumors are malignant germcell tumors, and the rest comprise benign tumors deriving from Leydig and Sertoli cells and other interstitial components. Testicular germ-cell tumors originate from totipotent primordial germ cells, which undergo neoplastic transformation as a result of a number of endogenous, exogenous, hormonal and genetic, as well as environmental, events. The neoplastic process results in the development of preinvasive carcinoma in situ (CIS) or TIN representing the common precursor for all testicular germ-cell tumors, except spermatocytic seminoma.

Characteristics Testicular cancer represents the most common malignant tumor in young men in the age group of 20–40 years. In 1994, 6,800 new cases of testicular

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Testicular Cancer

cancer were diagnosed in the US. There are striking differences in TC incidences around the world, with the highest incidence of 12–14 per 100,000 person-years in Switzerland and Denmark, and the lowest incidence of less than one per 100,000 person-years among African- Americans and the Chinese populations. Cryptorchidism is the best-known risk factor and, according to case–control studies, the relative risk for TC is 2.5–8.8. Familial and genetic factors have been suggested to be involved in the development of TC with a six- to tenfold higher risk in first degree relatives. Diagnosis The majority of patients present with a painless scrotal mass and the diagnosis is usually established by physical examination of the tumor-bearing and the contralateral testicle, scrotal ultrasonography and determination of the serum tumor markers alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) and lactate dehydrogenase (LDH). Inguinal exploration and scrotal orchiectomy confirms the diagnosis and is the therapy of choice, revealing accurate information with regard to histopathology and pathological-stage classification. Since benign testicular lesions are recognized with increasing frequency, frozen section analysis should be considered. Contralateral testicular biopsy to diagnosis TIN is recommended in high risk patients (testis volume 10.000 ng/ml ß-hCG > 10.000 ng/ml LDH > 10x Norm

Testicular Cancer

delay in decline is predictive for a poor outcome in terms of response to therapy. The prognostic significance of tumor markers at the time of diagnosis becomes evident for advanced disease only, adhering to the International Germ Cell Consensus Classification Group (IGCCCG) classification. The Lugano classification represents the most widely used clinical staging system for testicular cancer (Table 2), and describes the extent of metastatic involvement of the lymph nodes and visceral organs. In recent years the IGCCCG has introduced a new staging system for advanced TC defining three prognostic risk groups with regard to therapeutic outcome. Patients are classified to be at good risk (probability of cure 95%), intermediate risk (probability of cure 70%) or poor risk (probability of cure 50%). The IGCCCG classification gives high prognostic evidence and enables an individualized riskadapted approach in patients with advanced TC. Therapy Once TIN is diagnosed, therapeutic intervention is recommended, since 70% of patients will develop invasive germ-cell tumor within the next 7 years. Local radiation therapy with 18 Gy is the therapy of choice in patients with a contralateral invasive germ-cell tumor. In patients with unilateral TIN and a contralateral normal testis, inguinal orchiectomy appears to be the preferred management, since local radiation bears the risk of damaging the healthy testicle. Non-Seminomatous Germ-Cell Tumors (NSGCT) Clinical stage I NSGCT represents a troublesome entity concerning recommendations for optimal management since about 30% of patients will exhibit microscopic lymph-node disease. Several treatment options such as primary nerve-sparing retroperitoneal lymphadenectomy, primary chemotherapy, and active surveillance have been developed resulting in the same high cure rates of 98%. The European Germ Cell Cancer Consensus Group recommends an individualized, risk

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adapted approach based on the results of prospective randomized trials considering the presence or absence of the risk factors vascular invasion (VI) and percentage of embryonal carcinoma (ECA). VI has been identified as the most powerful clinical predictor of lymph-node metastasis with 48% of NSGCTs with VI developing metastases, compared to 14–22% of tumors without VI. A combination of VI and ECA might be even more powerful. Nowadays, nerve-sparing RPLND – if performed – is regarded as the standard approach. Up to 10% of patients will suffer from pulmonary relapse within the first 2 years, and will be cured by platinumbased chemotherapy. Even in low-volume lymph-node disease such as pathological stage IIA, the nervesparing RPLND can be performed as bilateral radical surgery without compromising the therapeutic outcome. Primary chemotherapy [two cycles of cisplatin, etoposide and bleomycin (PEB)] or surveillance (absence of VI) result in relapse rates of only 7% and 14%, respectively. Low-Stage (IIA/B) NSGCT Low-stage testicular disease comprises clinical stages IIA and IIB associated with a cure rate of 98%. Patients with low volume disease and abnormal tumor marker levels of AFP, ß-hCG, or LDH are treated with 2–3 cycles PEB chemotherapy, patients with negative markers might be offered nerve-sparing RPLND or surveillance Patients with clinical stage IIB TC will undergo primary chemotherapy depending on the serum tumor marker concentrations with three or four cycles of PEB followed by secondary RPLND in about 30% of cases. Clinical Stages IIC and III Inductive chemotherapy represents the therapy of choice, with the number of cycles applied depending on the IGCCCG-based prognostic classification. Patients with “good prognosis” face a long-term survival rate of >90%, and are managed by three cycles of PEB.

Testicular Cancer. Table 2 Clinical Lugano – classification of TC Stage I

Stage IIA Stage IIB Stage IIC Stage IIIA Stage IIIB

Stage IIIC

Tumor markers normalized or decline according to their half-life after orchiectomy No detectable metastases by imaging studies Primary TC confined to the testicle Retroperitoneal metastases 5 cm Supraclavicular or mediastinal metastases Pulmonary metastases Minimal: 2 cm Extrapulmonary visceral metastases

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Patients with “intermediate prognosis” face a survival rate of 70–80% and are managed by four cycles of PEB or cisplatin, etoposide and ifosfamide (PEI). Patients with “poor prognosis” have a survival rate of only about 50%; standard therapy consists of four cycles of PEB or PEI. A major advantage of primary high-dose chemotherapy has not been demonstrated, but this approach is currently being tested in prospective randomized trials. Seminomatous Germ-Cell Tumors Clinical Stage I Seminoma Despite negative CT scans, there is a risk of 12–32% of occult retroperitoneal lymph node metastases depending on the absence or presence negative prognostic markers. The cure rate of clinical stage I seminomatous germ cell cancer is close to 100% and can be achieved by the three different therapeutic options active surveillance, radiation therapy and carboplatin monochemotherapy. Adjuvant retroperitoneal radiation therapy to the paraaortic or paracaval region with 20 Gy or adjuvant chemotherapy with one cycle carboplatin AUC 7 are the standard approach for high risk patients (tumor size > 4 cm, rete testis invasion) and result in a relapse-free long-term survival of 97%. Active surveillance represents the most reasonable approach to patients with good prognostic markers associated with a low recurrence rate of about 12%. Treatment of relapses is more intense with systemic chemotherapy of 3–4 cycles PEB in most cases. Low-Stage (Clinical Stage IIA/B) Seminoma Radiation therapy with 30 Gy (IIA) and 36 Gy (IIB), including the ipsilateral iliac and inguinal lymph nodes, is one standard therapeutic approach for low-stage seminomas. Relapse-free survival is as high as 92.5% in clinical stage IIA/B; relapse rates are about 5% in stage IIA and about 11% in stage IIB seminomas. Primary chemotherapy with two cycles of PEB is an alternative to radiation in clinical stage IIB seminoma. Clinical Stage IIC and III As pointed out for advanced non-seminomatous germcell tumors, therapy should be initiated according to the IGCCCG classification. For patients with good prognosis, three cycles of PEB chemotherapy are the treatment of choice, in patients with intermediate prognosis four cycles of PEB chemotherapy are applied. Residual Tumor Resection (RTR) Following Chemotherapy for Advanced Testicular Cancer. RTR represents an integral part of the multimodality treatment of advanced testicular germ cell tumors. The rationale for RTR is to completely resect mature teratoma and vital cancer which will be found in 30– 40% and 20% of the patients, respectively. Currently, all residual lesions independent on size should be resected

in NSGCT since even small lesions 10% vital cancer cells or those with uncomplete resection might benefit from consolidation chemotherapy with two cycles. Postchemotherapy or postradiotherapy RPLND in seminomas has only to be performed in lesions with a positive PET scan performed about 6 weeks after chemotherapy or radiation therapy in patients with residual lesions >3 cm. Salvage Chemotherapy, High-Dose Chemotherapy In seminomas relapsing after first-line radiation therapy a cure rate of >90% can be achieved by cisplatin-based chemotherapy according to the IGCCCG algorithm with regard to advanced seminomas. About 50% of relapsing seminomas following conventional chemotherapy can be salvaged with another combination chemotherapy consisting of PEI– etopside, ifosfamide and platinol (VIP) or – vinblastine, ifosfamide and platinol (VeIP). Currently, a 10% benefit of high-dose chemotherapy with regard to survival has been demonstrated; therefore, it seems advisable that all relapsing patients should be treated in a tertiary referral center. NSGCT relapsing following conventional chemotherapy, salvage rates are as low as 15–40% using standard salvage protocols such as PEI–VIP or –VeIP. In some institutions the addition of paclitaxel to ifosfamide and cisplatin has been favored due to a high response rate >50%. Conventional-dose cisplatinbased salvage chemotherapy can achieve long-term remission in 15–40% of patients. Early consideration of high-dose chemotherapy seems advisable: trials suggest a benefit for the use of high-dose chemotherapy and autologous bone marrow transfer, with 46% and 50% of the patients being alive and disease-free after a median follow-up of 31 months and 30 months, respectively. Options for third-line chemotherapy are combinations such as paclitaxel and gemcitabine, gemcitabine and oxaliplatin or paclitaxel, gemcitabine and cisplatin, within clinical trials. Genetics With regard to predisposing genetic events, the locus Xq27 predisposes for bilateral TC and bilateral cryptorchidism. Other studies have reported the loci 1p36,

Testosterone

4p14–13, 5q21–21, 14q13–q24.3 and 18q21.1–21.3 to be highly associated with TC. Recently, it has been demonstrated that somatic mutations of exons 10, 11 and 17 of KIT occur significantly more often in patients with bilateral TC as compared to patients with unilateral disease. The results indicate that KIT might be involved in the development of familial and a minority of sporadic germ cell tumors and that KIT mutations primarily take place during embryogenesis such that primordial germ cells with KIT mutations are distributed to both testes. Currently, all molecular markers such as p53, Ki-67, bcl-2, cathepsin D and E-cadherin have not been proven to be clinically useful prognosticators; only reverse transcriptase-polymerase chain reaction for AFP, hCG and germ cell alkaline phosphate (GCAP) mRNA for the detection of circulating tumor cells appears to be an interesting approach, with 60% of clinical stage I testicular cancer patients exhibiting positive signals that turn into negative signals following adjuvant chemotherapy.

Future Directions in TC Based on the excellent therapeutic outcome, there appear to be only a few developments possible that will have further impact on the survival of testicular cancer patients. However, there might be many options to improve quality of life either due to reduction of acute toxicity or due to the development of treatment regimes associated with a significantly reduced long-term toxicity. It has been that the risk of cardiovascular disease is significantly increased after standard chemotherapy with 3–4 cycles PEB and/or salvage treatment (RR = 2.59). The increased risk is not due to an increase in classical cardiac risk factors but directly dependent on first line therapy. For the future, attempts to minimize treatment should be undertaken especially in patients with good prognosis in whom this type of long-term toxicity might be a greater risk to long-term survival than testicular cancer itself. Elucidation of those mechanisms involved in the development of intrinsic and extrinsic chemorefractoriness in testicular cancer will be a major issue in the future, to apply effective chemotherapeutic protocols and to save even more lives. There are some promising approaches using modern molecular techniques such as gene expression profiling to explore the role of mismatch repair genes, multidrug resistance genes and potentially unknown genes. Despite the high cure rates, it will be necessary for testicular cancer to be treated by clinicians and institutions with sufficient experience in diagnosis and management of germ-cell tumors. Specific problems such as extended tumor masses, relapsing tumors or poor prognosis at initial diagnosis must be referred to

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tertiary centers having the ability of an interdisciplinary approach.

References 1. Cavalli F, Manfardini S, Pizzocaro G (1980) Report on the international workshop on staging and treatment of testicular cancer. Eur J Cancer 6:1367–1372 2. Heidenreich A, Srivastava S, Moul JW et al. (2000) Molecular genetic parameters in pathogenesis and prognosis of testicular germ cell tumors. Eur Urol 37:121–135 3. International Germ Cell Consensus Classification Group (1997) A prognostic factor-based staging system for metastatic germ cell cancers. J Clin Oncol 15:594–603 4. Schmoll HJ, Souchon R, Krege S et al. (2004) European consensus on diagnosis and treatment of germ cell cancer: a report of the European Germ Cell Cancer Consensus Group (EGCCCG). Ann Oncol 15:1377–1399 5. Skakkebaek NE, Bertlesen JG Giwercman A et al. (1987) Carcinoma in situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytic seminoma. Int J Androl 10:19–28

Testicular Feminization TFM ▶Androgen Receptor

Testicular Germ Cell Tumor ▶Testicular Cancer

Testicular Tumors ▶Germ Cell Tumors

Testosterone Definition Testosterone is a steroid hormone from the androgen group. In mammals, testosterone is primarily secreted in

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2,3,7,8-Tetrachlorodibenzo-p-dioxin

the testes of males and the ovaries of females, although small amounts are also secreted by the adrenal glands. It is the principal male sex hormone. ▶Sex Hormone Dependent Cancers ▶Hormonal Carcinogenesis

2,3,7,8-Tetrachlorodibenzo-p-dioxin ▶Dioxin

TFF ▶Trefoil Factors

TFIIH Definition Is a protein complex that plays a key role in both transcription and DNA repair.

Tetracopeptide Repeat TPR Domains Definition A structural motif that mediates protein: protein interactions. Each TPR motif consists of repeats of a 34-amino acid sequence. It is present in a wide range of proteins; in particular many of the co-chaperones involved in the heat shock protein 70 and 90 folding systems. ▶Molecular Chaperones

Tetraploidization

TG2 Definition An enzyme (EC 2.3.2.13) of the transglutaminase family. Like other ▶transglutaminases, it crosslinks proteins between an N of a lysine residue and a glutamine residue in two protein chains, creating a bond (isopeptide) that is highly resistant to proteolysis. ▶Transglutaminase-2

Definition Process whereby the entire genome is duplicated.

TGc Tetraspanin

▶Transglutaminase-2

▶Metastasis Suppressor KAI1/CD82

TF

TGF

Definition

Definition

Tissue factor.

Transforming Growth Factor.

▶Proteinase-Activated Receptors

▶Transforming Growth Factor Beta

Th1 Immune Response

TGFa

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TGF-β Superfamily

Definition

Definition

Transforming growth factor alpha.

Is a group of structurally related multifunctional peptides that control proliferation, morphogenesis, differentiation, migration and a variety of other functions in many cell types. Members of this family include the transforming growth factor βs (▶TGF-β), ▶bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activins/inhibins, mullerian inhibitory substance (MIS), glial cell derived neurotrophic factors (GDNFs) and macrophage inhibitory cytokine-1 (▶MIC-1).

▶ADAM17

TGF-b Definition

▶Transforming growth factor β; Is a multifunctional dimeric 25 kDa polypeptide growth factor, whose main functions are growth inhibition, immunosuppression and regulation of extracellular matrix formation and turnover. There are three different mammalian gene products. Activation renders the biologically latent TGF-β into its active form, which can bind to the cell surface receptors and initiate signaling. The TGFβ superfamily contains at least forty cytokines currently divided into two classes: the bone morphogenetic proteins (BMP) and the TGFβ/activin. These cytokines regulate cell fate (proliferation, differentiation and apoptosis) and extracellular matrix deposition.

Th1 Definition CD4+ helper T cells providing cytokines related to cellmediated immune reactions. ▶Allergy

▶Smad Proteins in TGFβ Signaling

Th2 Definition

TGF-β Activation Definition

CD4+ helper T cells providing cytokines related to humoral immune reactions. ▶Allergy

An event that renders the biologically latent ▶TGF-β into its active form, which can bind to the cell surface receptors and initiate signaling.

T Th1 Immune Response

TGF-β-Regulated and Epithelial Cell-Enriched Phosphatase Definition

▶TEP1; Alternative, rarely used name for ▶PTEN.

Definition Two types of effector CD4 + T helper cell responses can be induced by a professional antigen presenting cell (APC), designated Th1 and Th2, each designed to eliminate different types of pathogens. The Th1 response is characterized by the production of interferongamma, which induces B-cells to make opsonizing

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Th2 Immune Response

(coating) antibodies, and leads to cell mediated antitumor immunity. ▶DNA Vaccination

Th2 Immune Response Definition

Is characterized by the release of ▶interleukin 4, which results in the activation of B-cells to make neutralizing (killing) antibodies, leading to humoral immunity. ▶DNA Vaccination

Thalidomide and its Analogues R ICKY A. S HARMA 1 , A DRIAN J. B LOOR 2 , A NGUS G. DALGLEISH 3 1

Radiation Oncology and Biology, University of Oxford, Churchill Hospital, Oxford, UK 2 The Christie NHS Trust, Manchester, UK 3 St. George’s Hospital Medical School, London, UK

Synonyms Thalidomide = (ph)thal(ic acid) + (im)id(e) + (i)mide, C13H10N2O4; Immunomodulatory drugs (IMiDs) = CC-4047, Lenalidomide (CC-5013 or Revlimid)

Definition First synthesized in 1954 in Germany from the glutamic acid derivative α-phthaloylisoglutamine, thalidomide was used as a sedative and treatment for morning sickness in the 1950s until its teratogenic effects became apparent. Phocomelia is the most well known toxicity of thalidomide, making it absolutely contraindicated in pregnancy. The IMiDs are immunomodulatory derivatives of thalidomide, rationally designed to be more potent at inhibiting ▶cytokine production. The chemical structures of thalidomide and two IMiDs are shown in Fig. 1.

Characteristics Thalidomide is commonly used in the treatment of moderate to severe erythema nodosum leprosum and less frequently in the treatment of a wide range of

non-malignant clinical conditions refractory to standard therapies, such as rheumatoid arthritis, the inflammatory and wasting effects of chronic tuberculosis, Behcet’s disease, Crohn’s disease, aphthous ulcers, and cachexia associated with HIV infection. The growth and survival of myeloma cells is critically dependent on the interaction with the bone marrow microenvironment. Thalidomide (Thal) and immunomodulatory drugs (IMiDs) act via mechanisms to disrupt this interaction and inhibit myeloma cell growth and survival. For abbreviations, see main text. Mechanisms of Action Thalidomide influences the production of and cellular effects of inflammatory cytokines, particularly those involved in ▶angiogenesis. It has also been shown to activate cytotoxic T-lymphocytes, a process involving the release of interleukin-2 (IL-2) and ▶interferon (IFN)-γ. Thalidomide enhances IL-4 and IL-5 production and down-regulates IL-12 production, effecting a shift in cytokine profile which is generally considered favorable in the treatment of cancer. Thalidomide may also have direct effects on the growth and survival of malignant cells, e.g. it inhibits the activity of ▶cyclooxygenase-2 via inhibition of ▶nuclear factor κ-B (NF-κB) activity. IMiDs demonstrate a similar range of biological activities to thalidomide, with significantly greater potency in certain activities. For example, CC-4047 exhibits a 20,000-fold higher potency than thalidomide at inhibiting ▶tumor necrosis factor (TNF)-α production. Several mechanisms of action are shown in Fig. 2, which lead to alterations in the interplay between ▶multiple myeloma (MM) cells and the bone marrow environment.

Multiple Myeloma Early studies using thalidomide in patients with relapsed multiple myeloma described responses in around half of the patients treated, although typically with a duration of less than 12 months. Initial attempts to achieve high target doses (up to 800 mg/day) resulted in frequent dose limiting toxicity. The reported side effects were dependent on dose and patient age. Somnolence and constipation were common, although manageable in most cases. Peripheral neuropathy occurred in up to a third of patients, particularly with long term treatment, and it was disabling and irreversible in some cases. Particular concerns were raised concerning patients with multiple myeloma who may develop neuropathies for other reasons, thus increasing the severity of the symptom, e.g. concomitant drugs, amyloid deposition or paraproteinaemia. There was also a significant rate of venous thromboembolism (VTE) complicating treatment with thalidomide.

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Thalidomide and its Analogues. Figure 1 Mechanism of action of thalidomide and IMiDs in multiple myeloma.

Thalidomide and its Analogues. Figure 2 Chemical structures of thalidomide, lenalidomide and actimid (CC-4047).

More recent clinical studies have suggested that high doses of thalidomide cause unnecessary toxicity since its efficacy in treating multiple myeloma is equivalent at lower doses (100–400 mg/day). A number of groups have investigated the combination (TD) of thalidomide and dexamethasone in patients with relapsed multiple myeloma. Collectively, the results suggest that the TD combination is superior to thalidomide monotherapy for treating relapsed disease and, although there is no published data to directly confirm this advantage, TD combination treatment is widely used in this context. Although an overall survival benefit has not yet been demonstrated, the response rate may be further improved with the addition of cytotoxic agents (Table 1), albeit at the cost of increased toxicity. TD is effective in 30% of patients resistant to thalidomide when it is used as a single agent.

Based on its activity in relapsed patients, TD has also been tested in patients newly diagnosed with multiple myeloma. The response rate is superior to that obtained using standard infusional chemotherapy with Vincristine, ▶Adriamycin and Dexamethasone (VAD) and offers the benefits of an oral regime versus one requiring the insertion of a central venous catheter. These advantages have led to TD being widely adopted as standard first line therapy in younger patients prior to autologous stem cell transplant (ASCT), although whether the improved response rates will translate into an overall survival advantage is not yet known. In patients not suitable for ASCT, two recent studies have demonstrated that the addition of thalidomide to standard chemotherapy (Melphalan and Prednisolone) results in improvements in both response rate and survival (Table 1).

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Thalidomide and its Analogues. Table 1

Summary of the activity of thalidomide in the treatment of multiple myeloma

Disease status Relapsed disease

Newly diagnosed disease (Pre autologous stem cell transplant (ASCT)

Regimen Thalidomide: Monotherapy Thalidomide + Dexamethasone (TD) Thalidomide + Steroids + Chemotherapy Thalidomide + Novel agents Thalidomide + Dexamethasone

Thalidomide + Dexamethasone + Chemotherapy Newly diagnosed disease (Not candidates for ASCT)

Melphalan + Prednisolone + Thalidomide (MPT)

Maintenance post ASCT

Thalidomide + Bisphosphonates

Response rate 25–48% Complete response rare Response rate 41–55% Synergistic in-vitro and in-vivo Effective in relapse post ASCT Response rate 36–79% Many combinations tested Increased toxicity with intensive regimens Ongoing phase I/II trials Bortezomib, IMiDs, investigational drugs Response rate 65–75% CR in 7–15% Survival benefit over standard induction chemotherapy plus ASCT unproven Response rate up to 90% Many regimens, increased toxicity over TD Survival benefit over standard induction chemotherapy/TD plus ASCT unproven Response rate 75–80% CR in up to 15% Response rate and event free survival superior to MP but increased non-hematological toxicity Event free survival and overall survival superior to no maintenance in single study Survival advantage most marked if 100 K) of ▶small molecule libraries to identify a set of compounds which can be developed into drugs. TR-FRET assay technology allows for the generation of a simple automation friendly assay that is amenable to HTS and can be readily adapted

to families of target proteins implicated in disease. Members of the family of protein ▶kinases have been tied to cancer and TR-FRET is a valuable technology suited to studying kinases. The processes by which normal healthy cells are transformed into cancer cells are being elucidated. While there are several mechanisms that drive the transitions from normal to cancer cells, a common feature of all cancer cells is the loss of ability of a given cancer cell to control its growth or ▶cell cycle. Regulation of cell growth is a complex process where the cell communicates with its external environment and responds with a series of carefully coordinated internal events. This process is termed ▶cell signaling. Cell signaling allows a cell to respond to changes in its external environment and exert its own influence in a localized manner. Understanding and compensating for an aberrant mechanism of cell signaling in cancer cells is an underlying quest in drug discovery and development. Cell signaling within a cell is mediated by various proteins. Typically, cell signaling proteins are either modifiers (▶enzymes) of other proteins (▶substrates) or subunits of larger complexes that are formed or disbanded for a particular function. For example, kinases are a family of enzymes that modify their substrates by adding a phosphate group to specific amino acid residues. In turn, the phosphorylated protein may now interact with another protein forming a complex such as a ▶transcription factor that is able to enter the nucleus and initiate the ▶transcription of specific genes. Thus, the activities of several proteins are coordinated together to obtain a change in the cell such as the production of new proteins. In normal cells, cell signaling is carefully regulated. In cancer cells, events in cell signaling such as the activity of kinases are misregulated leading to inappropriate activation of cellular processes, such as cell growth. A central activity in identifying new therapeutics to cancer is the identification of small molecule compounds that inhibit improper kinase mediated cell signaling. Kinases are a large family of proteins involved in cell regulation and make up 2% of the human genome; 518 kinases have been identified. Kinases are intracellular proteins and by modifying their substrates through ▶phosphorylation they cause changes in protein localization and/or protein complex formation. Most kinases fall into two categories based on their ability to phosphorylate specific amino acid residues on their substrates; typically tyrosine and serine/threonine. Kinases also typically have high homology in their catalytic domains due to the binding of ▶ATP and divalent metal ions (Mg2+ or Mn2+) to facilitate transfer of γ-phosphate from ATP to substrate. Substrate to kinase specificity is based on cellular localization or the presence of specialized protein domains which facilitate

Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery

protein:protein binding interactions. Kinases may be their own substrates; known as ▶autophosphorylation. A complex cascade of protein:protein interactions and enzymatic activity is mediated by kinases and their substrates. Phosphorylation of a protein can result in either activation of signaling (▶EGFR kinase) or inhibit further signaling from occurring (▶src kinase). Misregulation of kinase signaling as in the case of EGFR results in increased cell growth and ▶oncogenesis. Thus, specific kinases and the larger protein family have been targeted as a point of intervention for identifying novel anti-cancer drug candidates. Kinase activity can be inhibited by small molecules which are able to interfere with ATP binding and thus, prevent phosphorylation of substrate. Anti-cancer drug therapies have been identified which take advantage of a small molecules ability to enter cells, locate and block a target kinase. Examples are: ▶Dasatinib, ▶Erlotinib, ▶Gefitinib, ▶Imatinib, ▶Lapatinib, ▶Nilotinib, ▶Sorafenib, ▶Sunitinib, and ▶Vandetanib. Gifitnib (also called Iressa) targets EGFR kinase which is misregulated due to overexpression in some cancers, including incidences of breast cancer. The inappropriate activity of EGFR kinase in cancer cells leads to the activation of the ▶Ras cell signaling pathway. The Ras signaling pathway is comprised of a series of distinct kinases which are involved in either further perpetuating or shutting down the pathway. The inappropriate activation of the Ras pathway leads to changes in the cells which result in increased and misregulated cell growth. A mutation in the ATP binding site of the kinase domain of EGFR renders the cells susceptible to the effect of Iressa. The identification of novel small molecules for additional kinases implicated in cell growth misregulation has key implications for cancer therapies. Since kinases play a key role in cancer and small molecules have been identified as kinase targeted cancer therapies, HTS has emerged as a useful approach for identifying novel anti-cancer small molecule drug candidates. A key component of a kinase based HTS is a biological assay which can be readily adapted to robotics and measure a test molecule’s ability to prevent phosphorylation of substrate by the target kinase. Historically, radioactivity based assays were used to measure kinase activity. The cost of radioactive assays is prohibitive for HTS where large numbers of samples are being processed and tested. Radioactive assays have the additional disadvantage of having multiple steps which increases the challenges of automation. Radioactive assays were preferred to conventional fluorescence based measurements which were relatively insensitive due to their limited assay dynamic range. HTS assays generally have a ▶homogenous assay format which is automation friendly. Homogenous assays combine the biological event and assay readout (detection) into a single measurement, minimizing the

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number of steps needed to perform a given measurement. Radioactive assays are often heterogeneous assay formats, where multiple steps are required to separate the desired biological signal from the total signal generated by the radioactive molecule. TR-FRET technology eliminated these hurdles to HTS by providing (i) a non radioactive assay format (ii) reasonable dynamic range of assay and (iii) homogenous assay format for ease of automation. TR-FRET is based on ▶Time Resolved Fluorescence (TRF) and Fluorescence Resonance Energy Transfer (FRET) technologies. Assay sensitivity can be a significant limitation when using conventional ▶fluorophores due to the naturally fluorescent properties of many compounds and proteins. In time-resolved detection technology, there is a time delay between the ▶excitation of the fluorophore and the ▶emission detection of the fluorophore. When TRF detection is coupled with long lasting fluorophores, sensitivity issues can be overcome for biological assays. FRET technology enabled homogenous assay formats using TRF technology by incorporating two distinct fluorophores into the assay. The two fluorophores are defined as a donor molecule and an acceptor molecule. The excitation of the donor fluorophore causes an emission that in turn is able to excite the acceptor fluorophore and cause a specific measurable emission. The ability of the donor fluorophore to excite the acceptor fluorophore is based on the proximity of the two molecules to each other and is the basis of FRET. FRET enables homogenous assay design based on the proximity of two fluorophores which can be used to measure biologically relevant protein to protein binding interactions. Thus, TR-FRET can be applied to measuring the activity of proteins (such as kinases) and adapted for drug discovery with HTS. TR-FRET assays for drug discovery are designed to use fluorophores specific for TRF and amenable to FRET. TRF uses specific fluorophores from the family of elements known as the ▶lanthanides which have a long emission profile and a large ▶Stokes shift. The Stokes shift is defined as the difference between the excitation and emission wavelengths. Lanthanides have Stokes shifts of 300 nm which is three times greater than conventional fluorophores. The large Stokes shift enables detection of emission signal distinct from excitation energy allowing for greater dynamic range in assay measurements. Fluorescence decay of lanthanides is long-lived (microseconds) relative to conventional fluorophores (nanoseconds). This spectral property allows for measurements removing most background fluorescence noise and further improving the assay sensitivity. The lanthanides are coupled to molecules which are able to capture and transfer light to the lanthanide to generate fluorescence upon excitation. Fluorophores modified for TRF are lanthanide

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Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery. Table 1 Commonly used TR-FRET pairs for drug discovery Lanthanide

a

Stokes shift (nm)

Europium 300 (Eu) Terbium (Tb) 200 Samariumb 225 (Sm) a b

Fluorescence Lifetime (μsec)

Excitation (nm)

Emission (nm)

FRET Acceptor

Excitation (nm)

Emission (nm)

730

340

615

APC

615

665

1050 50

340 340

495 642

Fluorescein 495

520

Variations will occur based on specific fluorophore-chelate or fluorophore-cryprate and acceptor molecules. Samarium has TRF properties and is not routinely used for TR-FRET.

▶chelates or lanthanide ▶cryptates. TRF fluorophores have been identified which can serve as donors for a FRET pair. A FRET pair for biological measurements is based on the distance between two fluorophores to activate the acceptor fluorophore. Typically, FRET occurs within 70–90 Å which allows for measuring interactions between proteins. TR-FRET pairs must meet the following criteria to be useful for biological measurements as in HTS; (i) emission spectra of each fluorophore must have a non-overlapping region to allow measurement of each molecules’ fluorescence (ii) high energy transfer (high ▶quantum yield) to enable sufficient signal detection, and (iii) fluorescence emission must fall outside of spectrum of proteins to avoid high background. Several key properties for fluorophores for TRF and the FRET pairs commonly used in kinase assay design are shown in Table 1. TR-FRET assay format is applied towards kinase assays for the identification of small molecules which are able to inhibit the enzymatic activity of the target kinase. A sample assay design is presented in Fig. 1 which can be generally applied to kinase family members. Advances in fluorescence technology enabling TR-FRET measurements have led to the availability of a variety of assay reagents which can be applied in kinase assays. Many kinases and their specific or a generic artificial substrate are commercially available. Specific antibodies which detect the presence of a phosphorylated substrate in solution can be coupled with a member of the TR-FRET pair. The second member of the TR-FRET pair can be coupled with the substrate protein. An active kinase will phosphorylate the substrate; which is tagged with the acceptor fluorophore of the TR-FRET pair. The phosphorylated substrate becomes bound to the anti-P-antibody which is tagged with donor fluorophore. This binding event can only occur if the kinase is active on the substrate protein. The binding event causes TR-FRET by bringing the donor and acceptor fluorophores into proximity of each other enabling FRET. The TR-FRET signal measured is only generated by the phosphorylated substrate, minimizing background signal from

Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery. Figure 1

each individual fluorophore. If a small molecule inhibits the kinase activity, FRET does not occur as there is no phosphorylation event to cause the anti-P-Ab to bind to the substrate. Test molecules can have their own fluorescent properties causing artifactual results in TR-FRET. A ratio of emission/excitation wavelengths is routinely used to minimize fluorescence changes within an assay. The general assay design shown in Fig. 1 is frequently used in HTS. The specific conditions of the assay are customized for each kinase and substrate pair and each donor and acceptor TR-FRET detection pair. A commonly used TR-FRET pair is Eu3+ as the donor with ▶allophycocyanin (APC) as the acceptor molecule (Fig. 1). Excitation of Eu-chelate or Eu-cryptate at 337 nm causes emission at 613 nm which in turn causes excitation of APC which emits at 665 nm. A ratio of 665 nm/613 nm is typically used to monitor specific assay signal.

Tissue Engineering

TR-FRET technology is not limited to small molecule screening or kinases in drug discovery. Macromolecules (antibody base protein therapies, enzyme replacement therapies) are used treat an array of diseases; most frequently by targeting proteins on the surface of cells. Some examples of the use of TR-FRET technology in drug discovery beyond the kinase family are: (i) ▶cytokines in the extracellular environment (ii) key intracellular molecules involved in cell signaling such as ▶cAMP and (iii) ▶protease family of enzymes where misregulation has serious implications in disease. TR-FRET can be applied as long as an appropriate fluorophore pair can be identified such that the proximity of the two fluorophores leads to a sufficient assay signal. In general, TR-FRET has been applied in large scale to kinases as a protein family due to the range of commercially available reagents and the direct link between kinases and their role in cellular regulation. TR-FRET for anti-cancer drug discovery by HTS has the advantage of a homogenous assay format with sufficient sensitivity to identify putative kinase inhibiting small molecules. The high throughput nature of TR-FRET kinase assays has made it a useful assay format for characterizing the specificity of kinases. Since there is high homology between kinase domains of different kinases and small molecule drugs interfere with the ATP binding site of the kinase domain, small molecules drugs are known to inhibit kinases beyond the original target kinase. In some cases, this activity can be favorable in cancer treatment. Imatinib targets tyrosine kinase ▶BCR/ABL and the tyrosine kinase ▶c-kit. Another example is Sorafenib which can inhibit both Raf (serine/threonine kinase) and VEGFR(tyrosine kinase), key players in cell signaling. The selectivity profile of small molecule kinase inhibitors is emerging and could provide key information in determining which small molecules to pursue as a drug. Alternatively, selectivity profile information could determine which small molecules to abandon due to their activity as pan-kinase inhibitors or their ability to inactivate a key kinase necessary for cell viability and thus behave as a toxin. TR-FRET is a key assay technology for identifying and characterizing anti-cancer small molecule drugs; especially against the protein kinase family which has been implicated in cellular misregulation leading to cancer. TR-FRET combines TRF and FRET technologies to provide a sensitive and high throughput assay format for characterizing the activity of small molecules against kinases. TR-FRET is an approach to rapidly screen large collections of putative drug candidates to identify a subset of molecules able to inhibit kinase activity. Application of TR-FRET extends beyond small molecule and kinase drug discovery. TR-FRET is utilized for characterizing macromolecules and a

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variety of protein families and signaling events. The central role of kinases in cancer and the ease of kinase assay design with TR-FRET has made TR-FRET technology a key component in today’s technology rich anti-cancer drug discovery research.

References 1. Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic/Plenum Publishers, New York, NY 2. Manning G, Plowman GD, Hunter T et al. (2002) Evolution of protein kinase signaling from yeast to man. Trends Biochem Sc 27(10):514–520 3. Noble MEM, Endicott JA, Johnson L (2004) Protein kinase inhibitors: Insights into drug design from protein structure. Science 303:1800–1805 4. Zaman GJR, Garritsen A, Boer T de et al. (2003) Fluorescence assays for high throughput screening of protein kinases. Comb Chem High Throughput Screen 6(4):313–320 5. Manning G, Whyte DB, Martinez R et al. (2002) The protein kinase complement of the human genome. Science 298:1912–1934

Time-to-Event Analysis ▶Kaplan-Meier Survival Analysis

TIMP ▶Tissue inhibitor of metalloproteinases

T Tissue Engineering Definition Process of growing functional tissues in three dimensions using supporting structures and culture methods that stimulate appropriate growth, morphogenesis and differentiation. ▶Three-Dimensional Tissue Cultures

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Tissue Factor

Tissue Factor Definition TF; Cell surface receptor which is present in subendothelial tissue, platelets, and leukocytes necessary for the initiation of thrombin formation from its zymogen. Its best known function in blood coagulation. An integral membrane protein which is found on the outside of blood vessel cells and on monocytes during ▶inflammation. Upon vessel injury, tissue factor is exposed to the blood stream and binds circulating factor VII. TF can also be produced by tumor cells, triggering their coagulation. Moreover, TF possibly mediates the ▶adhesion of cancer cells to exposed subendothelial ▶extracellular matrix components at permeabilized vessel sites. ▶Proteinase-Activated Receptors ▶Coagulopathy ▶Protease Activated Receptor Family

Tissue Inhibitors of Metalloproteinases L ILIANA G UEDEZ 1 , E NRIQUE Z UDAIRE 2 , W ILLIAM G. S TETLER-S TEVENSON 1 1

Extracellular Matrix Pathology Section, Cell and Cancer Biology Branch, National Cancer Institute, Bethesda, MD, USA 2 National Cancer Institute, National Institutes of Health, Advanced Technology Center, Gaithersburg, MD, USA

Synonyms TIMPs

Definition The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the ▶matrix metalloproteinases (MMPs), proteases that play a central role in the degradation of extracellular matrix components in diseases such as cancer. Four TIMPs (TIMP1–4) which share high sequence homology have been identified. Tumor cells express MMPs that are important for cell ▶invasion and metastasis. Because of their implication in tumor progression, TIMPs have been regarded as a potential therapeutic target in the management of cancer.

Characteristics

▶Extracellular matrices (ECM) play a central role in the structure and functions of tissues. The ECM consists of

a network of fibrous proteins that include collagens, ▶laminins, ▶fibronectin and proteoglycans that provides a unique structural scaffold for normal tissue function. Alterations in the ECM protein scaffold always develop in pathological conditions such as cancer. In fact, the tumor cells penetrating the subepithelial basement membrane define a malignant tumor. Thus, degradation and remodeling of the ECM are pivotal for tumor growth. These functions are carried out by several enzymatic-cascade systems that control the tumor ▶microenvironment and its vascularization. Matrix metalloproteinases, MMPs, are among the major class of enzymes responsible for tumor-associated matrix degradation. This is supported not only by the nature of their association with tumor progression but also by the observations that in animal models MMP inhibitors are effective anti-tumor and anti-metastatic agents. MMPs are a large family of proteases with more than 20 members. Collectively, the MMP family members are capable of degrading essentially all extracellular matrix components. The expression of MMPs by the tumor cells and/or tumor stromal cells determines not only the behavior of malignant cells, but enables them to carry out pivotal functions during the metastatic process such as ▶migration and invasion. It is widely known that the endogenous inhibitors of the MMP family are the tissue inhibitors of metalloproteinases or TIMPs. These proteins regulate the proteolytic activities of the MMPs in the remodeling tissue or tumor microenvironment. TIMPs are a highly conserved, small family of proteins, with sequence homologies present through out evolution including worms, chicken, and Drosophila. In contrast to the large number of members in the MMP family, only four timp genes are present in the human genome. These genes are located in different chromosomes, timp-1 on the chromosome X, timp-2 in chromosome 17, timp-3 on Chromosome 22, and timp-4 on chromosome 3. Also, there is an association of the TIMP gene loci with the synapsin gene loci, with the exception of TIMP-2. In both human and mouse, the genes for TIMP-1, 3, and 4 are located in the introns of synapsins I, III and II respectively, but the transcriptional orientation of the two gene families is in opposite direction. TIMPs are small proteins with molecular weight of the core proteins in the range of 21 kDa; TIMP-3 and TIMP-1 are N-glycosylated, while TIMP-2 and TIMP-4 are not. However, they all contain 12 cysteine residues that must correctly pair into six disulfide bonds to give the correct secondary/tertiary structure required for MMP inhibitory activity. The selectivity of TIMPs for inhibiting MMP activity resides in the N-terminal domain. The TIMP family is also unusual in that the N-terminal amino acid is a cysteine residue. X-ray diffraction studies show that it is the amino group of this N-terminal cysteine that actually coordinates with the

Tissue Spectroscopy

Zn atom in the MMP active site, which results in inhibition of the proteolytic activity. Also, TIMP-2 selectively binds to zymogen pro-MMP-2 via the carboxyl terminal domain, and is important for the cell surface activation of pro-MMP-2 by MT-1-MMP. Interestingly, TIMPs -2 and -4 can interact with the catalytic domains in all activated MMPs, but TIMP-1 is a poor inhibitor of MT-1-MMP. TIMP family members have a distinctive pattern of tissue expression. TIMP-2 is unique in that expression of this inhibitor is constitutive in most tissues, whereas TIMP-1, TIMP-3 and TIMP-4 proteins expression is inducible, and TIMP-3 is the only member that is specifically bound to the ECM. Given that TIMPs regulate MMPs activation, their role in tumor development has been the subject of an intense research. In experimental systems, gene knockout either in cell lines in vitro or in vivo models reveals that the absence of some TIMPs accelerate tumor growth, as well as tumor-associated ▶angiogenesis. In clinical investigations, tumor specimens consistently demonstrate alterations in the balance of MMP/TIMPs. Based on these findings, there has been an interest in these proteins as potential ▶biomarkers for prognosis and diagnosis, as well as targets for cancer therapy. In response, synthetic inhibitors of MMPs were developed and tested in clinical trials, but these compounds failed to demonstrate meaningful anti-tumor effects. However, re-examination of TIMP expression in tumor specimens shows no clear pattern when analyzed as an independent variable. For instance, in an increasing number of clinical reports, TIMP-1 has been found to be overexpressed in human tumors and correlated with poor prognosis. These observations lead to the idea that the role of TIMPs in cancer is more complex than one first thought, in that although TIMPs may have MMP inhibition as a main function, these proteins can also regulate steps throughout the malignant process independent of their MMP inhibitory role. Thus, TIMP-1 has been shown as an autocrine growth factor and anti-apoptotic protein even independent of its MMP inhibitory ability. TIMP-3 can directly inhibit tumor growth in vivo by restricting angiogenesis. Recently, TIMP-2 is shown in vitro and in animal models to be an effective anti-tumor and anti-angiogenic factor. These TIMP-2 effects are also direct on cells and independent of its MMP inhibitory activity, mediated by binding to α3β1 integrin. Despite of their structural similarities, it is clear that TIMPs exhibit a wide spectrum of effects during the process of tumorogenesis. These conflicting TIMP effects in tumor growth are new challenges for the field of MMPs and TIMPs in cancer. Clarifying this will be important for the design of novel anti-cancer therapies. A current approach is being investigated for TIMP-2 and TIMP-3 that involves either over expression of these TIMPs by retroviral-gene transfers into tumor tissues or

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adenovirus systemic-gene delivery. These are being implemented either as monotherapies or as adjuvants to directly inhibit tumor growth or angiogenesis.

References 1. Finan KM, Hodge G, Reynolds AM et al. (2006) In vitro susceptibility to the pro-apoptotic effects of TIMP-3 gene delivery translates greater in vivo efficacy versus gene delivery for TIMPs -1 and -2. Lung Cancer 53:273–284 2. Guedez L, Stetler-Stevenson WG (2007) The role of metalloproteases and their endogenous inhibitors in regulation of tumor-associated angiogenesis. In: Figg W, Folkman J (eds) Angiogenesis: an integrative approach from science to medicine. Springer, Inc. New York, NY (in press) 3. Jiang Y, Goldberg ID, Shi Ye (2002) Complex roles of tissue inhibitors of metalloproteases in cancer. Oncogene 21:2245–2252 4. Visse R, Nagase H (2003) Matrix metalloproteases and tissue inhibitors of metalloproteases: structure, function, and biochemistry. Circ Res 92:827–839

Tissue Kallikreins ▶Kallikreins and Cancer

Tissue Proliferation ▶Regeneration

Tissue Regeneration Definition Is a regular maintenance cycle in which tissue cells constantly undergo remodeling and restoration.

Tissue Spectroscopy ▶Fluorescence Diagnostics

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Tissue Stem Cells

Tissue Stem Cells ▶Adult Stem Cells

Tissue-transglutaminase ▶Transglutaminase-2

Tissue-Type Plasminogen Activator tPA; Serine proteinase that converts plasminogen to enzymatically active plasmin, an anticoagulant.

TLR4 Definition

▶Toll like Receptor 4 is a membrane protein and is described as a pattern recognition receptor. It cooperates with CD14 in the binding of endotoxins and transmits intra-cellular signals through MyD88 and initiates a cascade that activates nuclear factor κ-B (NFκB). This results in the production of cytokines and other biologically active molecules. ▶Kupffer Cells

TMPRSS1 Definition Hepsin.

▶Proteinase-Activated Receptors ▶Plasminogen-Activating System

TMPRSS10 Tissue Typing

Definition Corin.

Definition

▶Histocompatibility testing.

TMPRSS2/ERG Fusions TJP-1 ▶Zonula Occludens Protein-1

TKO

J IANGHUA WANG , Y I C AI , M ICHAEL I TTMANN Department of Pathology, Baylor College of Medicine, Houston, TX, USA

Definition TMPRSS2/ERG fusions are fusions of the promoter and 5′ portion of the androgen-regulated TMPRSS2 gene and coding portions of the ERG gene that occur in the majority of prostate cancers.

Characteristics Definition Technical Knockout.

Chromosomal rearrangements resulting in gene fusions with expression of functional proteins are common in leukemias, lymphomas, and sarcomas. The presence of

TMPRSS2/ERG Fusions

such ▶fusion genes is often linked to specific tumor phenotypes, for example, the ▶BCR–ABL1 fusion encoded by the Philadelphia chromosome in chronic myelogenous leukemia. In contrast, gene fusions with expression of a functional protein have generally been considered to be rare events in common epithelial malignancies such as lung, colon, and breast cancer. This idea has been brought into question by the finding of recurrent fusion of the androgen-regulated TMPRSS2 gene to the ▶ETS transcription factors, particularly the ERG gene, in the majority of prostate cancers (▶Prostate cancer). Overall, 50–80% of clinically detected prostate cancers contain TMPRSS2/ERG fusion genes, with a much smaller percentage containing fusions with genes for other ETS transcription factors such as ETV1 and ETV4. The existence of a recurrent chromosomal rearrangement in prostate cancer is a paradigm shift in the study of epithelial malignancies and other epithelial tumors may have their own recurrent chromosomal rearrangements that are yet to be identified. The TMPRSS2/ERG gene fusion involves fusion of the promoter region of the TMPRSS2 gene as well as some 5′ portions of this gene with coding sequence of the ERG gene. The TMPRSS2 gene encodes a transmembrane-bound serine protease that is localized to prostatic epithelium and is overexpressed in neoplastic prostate epithelium. It is well known that normal and neoplastic prostatic epithelium contain the ▶androgen receptor and express numerous genes whose expression is regulated by androgens. The TMPRSS2 promoter contains a 15 bp sequence that is homologous to the consensus androgen response element, which is in accordance with androgen-inducible expression of the gene. Experiments in cells with TMPRSS2/ERG fusions indicate that the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of ETS family members in prostate cancer. Fusion proteins involving ETS transcription factors are characteristic of ▶Ewing sarcoma and occur at lower rates in some leukemias. Based on this finding and the known biology of these transcription factors and fusion proteins, these genes are considered as oncogenes that promote neoplastic progression. Thus, the TMPRSS2/ERG gene fusion results in constitutive expression of an oncogenic transcription factor in the neoplastic prostatic epithelium. It is unknown whether decreased TMPRSS2 expression as a result of loss of one TMPRSS2 allele due to this rearrangement plays a role in prostate cancer initiation or progression. The ERG gene is located on chromosome 21q22.2 while the TMPRSS2 gene is 2.8 Mbp telomeric to ERG at 21q22.3. Initial characterization of the mechanism by which the fusion gene arises reveals that about half of the cases there is a large deletion of the genomic DNA between these two genes, while in other cases there appears to be more complex chromosomal

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translocations. More detailed studies of the mechanism by which the gene fusion arises and the exact regions fused are needed. The fusion gene initiates transcription from the TMPRSS2 promoter. All reports to date indicate that there is significant heterogeneity in the structure of the mRNA transcripts of the fusion gene, both among different cancers and within a single cancer. Thus some cancers express a single mRNA type, while others express multiple isoforms of the fusion gene. These different isoforms presumably arise via alternative splicing of the initial fusion transcript. In all cases, the fusion mRNA includes the TMPRSS2 exon 1 and often exon 2 as well. Transcripts with TMPRSS2 exons 3–5 have also been reported but appear to be uncommon. In some cases, the native ERG translation initiation codon in ERG exon 3 is maintained, fused to the untranslated TMPRSS2 exon 1. The most common transcript contains the TMPRSS2 exon 1 fused to ERG exon 4, such that translation would have to arise from an internal ATG codon and give rise to a slightly truncated protein. Of particular interest is an isoform in which TMPRSS2 exon 2 is fused with ERG exon 4. In this case, translation can be initiated from the TMPRSS2 translation initiation codon and results in a true fusion protein containing the first five amino acids of the TMPRSS2 protein fused to a slightly truncated ERG protein. Expression of this isoform is associated with early biochemical recurrence, characterized by detectable serum ▶prostate specific antigen (PSA), following radical prostatectomy. Such early recurrence has been shown to be a hallmark of aggressive disease and is associated with cancer progression and death from disease. Thus there is significant heterogeneity in both the structure of TMPRSS2/ERG fusion genes and the isoforms of transcripts arising from the fusion gene (Fig. 1). TMPRSS2/ERG fusion can be detected in high grade prostatic intraepithelial neoplasia, a noninvasive lesion that is generally believed to be the precursor of invasive prostate carcinoma, and thus is the gene fusion may be an early event in prostate cancer initiation. In all series studied to date, 50–80% of clinically detected prostate carcinomas contain the TMPRSS2/ERG fusion gene. These findings argue strongly that the fusion gene plays a critical role in prostate carcinogenesis but the exact biological role of the TMPRSS2/ERG fusion gene in prostate cancer has not been determined. Analysis of expression microarray databases reveals that ERG expression is associated with increased expression of histone deacetylase 1 (▶Histone deacetylases), activation of the ▶Wnt signaling pathway and downregulation of apoptotic pathways (▶Apoptosis). These observations need to be validated, but indicate that the TMPRSS2/ ERG fusion gene may have significant biological activities in vivo that promote carcinogenesis. Further studies are needed to characterize the biological

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TMPRSS2/ERG Fusions

TMPRSS2/ERG Fusions. Figure 1 The TMPRSS2 and ERG genes are located at chromosome 21q22.2–22.3 and undergo interstitial deletions or other genomic rearrangements to yield a fusion gene containing the androgen-regulated TMPRSS2 promoter and 5′ portion of the TMPRSS2 gene fused to coding regions of the ERG gene in 50–80% of clinically detected prostate cancers. The transcript from the fusion gene undergoes alternative splicing to yield various isoforms in prostate cancer cells. The resulting proteins may have a variety of biological activities that promote prostate cancer initiation and progression.

activities of the TMPRSS2/ERG fusion gene in human prostate cancer cells. A number of studies have examined whether the presence of the TMPRSS2/ERG fusion gene is associated with more aggressive clinical behavior in prostate cancer. Examination of outcome in patients treated by watchful waiting showed a strong association with death from disease. Retrospective studies have shown an association of the presence of the fusion gene with higher Gleason grade (link Gleason grading), extracapsular penetration, or biochemical recurrence in radical prostatectomy cohorts, although other study did not see these associations. Other factors may also come into play including the isoform(s) of fusion transcript expressed, the total expression level of fusion transcripts and the presence of large interstitial deletions. As described above, certain fusion transcript isoforms are associated with more aggressive disease, possible due to differences in translation initiation efficiency. Increased androgen receptor activity has been associated with prostate cancer initiation and progression and certainly the TMPRSS2/ERG fusion gene may be an important androgen receptor target in prostate cancer. Small studies have indicated that when cancers with similar isoforms are compared, that higher transcript expression may be associated with poorer prognosis. Finally, large interstitial deletion of the region between TMPRSS2 and ERG on chromosome 21 may inactivate potential tumor suppressor genes located between these two genes on chromosome 21. Comprehensive studies are needed to understand the

impact of the TMPRSS2/ERG fusion on prostate cancer progression and clinical outcome. The presence of a recurrent gene fusion in any malignancy raises the possibility of specific diagnostic tests, such as the use of the Philadelphia chromosome as specific diagnostic test for chronic myelogenous leukemia. A diagnostic test to detect the TMPRSS2/ ERG fusion transcripts in cancer cells in blood should be specific for prostate cancer and detection of specific isoforms may have prognostic utility to assist in treatment planning. Further studies and development of appropriate technologies are clearly indicated.

References 1. Iljin K, Wolf M, Edgren H et al. (2006) TMPRSS2 fusions with oncogenic ETS factors in prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming. Cancer Res 66:10242–10246 2. Perner S, Demichelis F, Beroukhim R et al. (2006) TMPRSS2:ERG fusion-associated deletions provide insight into the heterogeneity of prostate cancer. Cancer Res 66:8337–8341 3. Rubin MA, Chinnaiyan AM (2006) Bioinformatics approach leads to the discovery of the TMPRSS2:ETS gene fusion in prostate cancer. Lab Invest 86:1099–1102 4. Tomlins SA, Rhodes DR, Perner S et al. (2005) Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310:644–648 5. Wang J, Cai Y, Ren C et al. (2006) Expression of variant TMPRSS2/ERG fusion messenger RNAs is associated with aggressive prostate cancer. Cancer Res 66:8347–8351

TNF-a in HIV Infection

TNF Definition

▶Tumor necrosis factor. ▶Neutrophil Elastase

TNF-a Definition

▶Tumor necrosis factor α; Is a potent paracrine and endocrine mediator of inflammatory and immune functions. It regulates growth and differentiation of a wide variety of cell types and acts in combination with other cytokines.

TNF-a in HIV Infection PASCAL C LAYETTE SPI-BIO, Service de Neurovirologie, CEA, CRSSA, EPHE, Fontenay aux Roses Cedex, France

Definition In the 1950s–1960s, it was reported that several patients with malignant tumors had a spontaneous regression of their tumors after bacterial infections. In the 1970s, a bacterially-induced circulating host factor was associated with this anti-tumor activity and was designated “▶tumor necrosis factor” (TNF). Subsequently, TNF was isolated, cloned and found to be the leader of the “TNF superfamily” and a pleiotropic cytokine representing a major mediator of the inflammatory and immune responses. The human TNF gene is located on chromosome 6p23–6q12 between class I HLA region for HLAB gene and the gene encoding the complement factor c; it contains four exons in a total length of 3.6 kb. Human TNF-α is a non-glycosylated protein of 157 amino acids with a molecular weight of 17 kD. A 233 amino acid precursor (26 kD) is synthesized, expressed at cell surface and cleaved by the 85 kD TNF-α converting enzyme (TACE). This TNF precursor is biologically active and considered as the “membrane TNF” form. The 17 kD peptides interact together and form circulating homotrimers. The level of circulating TNF ranges

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between 10 and 80 pg/mL in non-pathological conditions. TNF is secreted by various cell populations such as cells of the macrophage lineage (monocytes, macrophages, microglial cells), CD4+ and CD8+ T cells, dendritic cells, neutrophils, adipocytes, keratinocytes, astrocytes, neurons and pancreatic cells. Two distinct membrane receptors for TNF have been identified and cloned: . A 55 kD receptor (p55) or TNF-R1, newly designated as CD120a and referred as TNF receptor superfamily member 1A (TNF-RSF1A) . A 75 kD receptor (p75) or TNF-R2, newly designated as CD120b and referred as TNF receptor superfamily member 1B (TNF-RSF1B) TNF-R1 and -R2 are glycoproteins of 455 and 461 amino acids, respectively. Soluble forms of TNF-R1 (at least two molecular weights 32 and 48 kD) and -R2 (42 kD) are generated by proteolytic cleavage. Cells such as monocytes/macrophages, endothelial cells express both TNF-R1 and -R2. It was first suggested that TNF-α was bound by TNF-R2 and transferred to TNF-R1, which then is activated; in fact, TNF-R1 may be the main receptor for soluble TNF-α, whereas membrane TNF preferentially interacts with TNF-R2.

Characteristics Physiopathological Role of TNF-a in HIV Infection Human immunodeficiency virus (HIV) is a ▶retrovirus that infects preferentially T CD4+ lymphocytes and ▶macrophages, a major source of TNF-α. As a consequence, close relationships exist between HIV and TNF-α; an implication of TNF-α has been reported in HIV replication, neuroAIDS, ▶cachexia and in the development of opportunistic infections and tumors. Relationships Between HIV Replication and TNF-a TNF-α is a pro-inflammatory cytokine and thus has been considered as a cytokine that could increase HIV replication. In fact, the effects of TNF-α on HIV replication is dual and opposite: . Inhibition of HIV entry . Increase of expression of proviral genome and production of HIV particles The inhibitory effects on HIV entry is due to the capacity of TNF-α to favor the synthesis of β-chemokines e.g. regulated upon activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α and MIP-1β, the natural ligands that compete with HIV particles to bind their receptor, which is also the co-receptor of HIV. The transcription factor ▶NFκB participates, in part, to the deleterious effects of TNF-α on HIV production; this transcription factor is activated by TNF-α, and interacts with domains regulating the

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expression of HIV (long terminal repeat or LTR), in which two binding sites has been identified (Fig. 1). TNF-α is a cytokine that is secreted by cells of the immune system in the area of microbial infections e.g. viral infections. In HIV infection, immune cells are HIV targets, and dysregulations of TNF-α synthesis induced by the HIV infection were explored in macrophages. Several authors demonstrated that the infection of macrophages by HIV is not sufficient to induce the synthesis of TNF-α in the absence of other environmental factors. In contrast, TNF-α synthesis may be increased in the presence of soluble factors or cells; only few contaminating lymphocytes favor, for example, the macrophagic TNF-α synthesis in response of HIV infection.

TNF-α in HIV Infection. Figure 1 Structure of TNF receptor superfamily members. TNF-R1 (p55, CD120a, TNF-RSF1A), TNF-R2 (p75, CD120b, TNF-RSF1Bg).

Role of TNF-a in NeuroAIDS TNF-α is also a neurotoxic factor that is implicated in the neuronal death in HIV disease. TNF-α can be synthesized in central nervous system (CNS), and the passage of blood brain barrier is not required for its presence in situ. The mechanisms of this neurotoxicity involve the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and the N-methyl-Daspartate (NMDA) receptor. TNF-α inhibits the glutamate uptake by astrocytes and favors the synthesis or release of the phospholipid mediator platelet-activating factor (PAF) which enhances excitatory transmission. Moreover, TNF-α also participates in the pathogenesis of neuroAIDS by regulating NFκB activation and HIV replication levels in perivascular macrophages and microglial cells. Indeed, viral factors such as the envelope glycoprotein gp120 and the transactivator protein ▶Tat are known to be neurotoxic factors, and Tat can induce TNF-α synthesis. Both TNF receptors are present in the CNS, and on neurons in particular. The role of TNF-R2 in the CNS remains largely unknown. TNF-R1 can promote apoptosis of neurons via the release of silencer of death domains (SODD) permitting the association with intracellular proteins such as TNF receptor-associated death domain (TRADD), FAS-associated death domain (FADD) and caspase-8 (FLICE/CASP8). The activation of acid sphingomyelinase by caspase-8 contributes to the cleavage of sphingomyelin to phosphocholine and ceramide, which activates various enzymes e.g. phosphatases, protein kinases involved in apoptosis (Fig. 2). A new neurodegeneration process has recently emerged; the silencing of survival signal (SOSS). TNF-α, at picogram concentrations, inhibits the survival signaling mediated by insulin-like growth factor 1 (IGF 1) in neurons; TNF-α inhibits tyrosine phosphorylation of insulin receptor substrate 2 (IRS2) and the activation of the survival enzyme, phosphatidylinositol 3′ kinase (▶PI3K) and the

TNF-α in HIV Infection. Figure 2 Different regulatory elements in the HIV LTR domain (LTR: long terminal repeat).


formation of phosphatidylinositol(3,4,5)P3 (▶luositoe lipids), known to play a pivotal role in the regulation of cell proliferation and survival. However, as the effects of TNF-α on HIV replication, the effects of TNF-α in apoptosis may be opposite. TNF-α may be also a neuroprotective factor, for example, in excitotoxic conditions. Members of the family of TNF receptor-associated factors (▶TRAF), particularly TRAF2 (Fig. 3), may play a major role in this process; it increases the TNF-α-induced ▶JNK and NFκB activation known to favor cell proliferation, and a dominantnegative mutant enhances apoptosis.

Role of TNF-a in Cachexia, Opportunistic Infections and Tumors TNF-α plays a major role in cachexia in HIV-infected patients but also in the development of opportunistic infections e.g. Mycobacterium avium, and tumors

TNF-α in HIV Infection. Figure 3 Different signaling pathways mediating biological effects of TNF-α. Tumor necrosis factor-a triggers the release of SODD and the DD domain of TNF-R1, that recruits TRADD via an interaction with the DD domain of this cell factor. Then, TRADD interacts with FADD, TNF-R-associated factor 2 (TRAF2) or kinase receptor-interacting protein (RIP). FADD is involved in the apoptotic signal cascade via caspase-8, TRAF2 in JNK activation, and RIP in NFκB activation.

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e.g. malignant lymphoma and ▶Kaposi sarcoma. The immune activation and TNF-α synthesis induced by the Mycobacterium infection amplifies the infection of proximal cells by HIV and their level of viral replication. This viral replication accentuates the TNF-α secretion, and harmful connections between th1e replication of HIV or opportunistic pathogens, and immune activation or inflammation, are created. In Kaposi sarcoma, TNF-α favors the acquisition of the phenotype and functional features of AIDSrelated KS spindle cells by endothelial cells. Moreover, TNF-α could be also responsible for deleterious effects of compounds delivered as treatment of opportunistic infections e.g. amphotericin B and cryptococcal meningitis. Clinical Relevance of Molecules Inhibiting TNF-a Synthesis In order to reduce the TNF-α -dependent HIV replication and the chronic inflammation that is associated with HIV infection, several inhibitors of TNF synthesis and TNF-receptor antagonists were evaluated in vitro or in vivo as therapeutical agent (e.g. RP-55778, pentoxifylline, chimeric humanized monoclonal antibody cA2). These molecules decreased in vitro TNF-α synthesis and HIV-1 replication. In vivo, pentoxifylline and cA2 were tested but no beneficial effects were observed on HIV viral load; only TNF-α synthesis was decreased. These data confirm that an inhibitor of TNF synthesis could not be used as pharmacological agent in monotherapy of HIV disease. Systemic ▶inflammation is decreased by highly active antiretroviral therapy (HAART); therefore, TNF-α and TNF-R2 may constitute reliable predictive markers of efficiency of HAART, or its failure. In tissues, particularly in CNS, HAART is less efficient to decrease HIV replication and chronic inflammation. As a consequence, anti-TNF-α molecules could be theoretically a good pharmacological strategy as adjuvant therapy. Nevertheless, caution is also required because it is important to preserve antimicrobial effects promoted by TNF-α against a variety of infectious agents, and particularly HIV.

References 1. Arch RH, Gedrich RW, Thompson CB (1998) Tumor necrosis factor receptor-associated factors (TRAFs)- a family of adaptor proteins that regulates life and death. Genes Dev 12:2821–2830 2. Venters HD, Dantzer R, Kelley KW (2000) A new concept in neurodegeneration: TNF-α is a silencer of survival signals. Trends Neurosci 23:175–180

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TNF-Related Apoptosis Inducing Ligand

TNF-Related Apoptosis Inducing Ligand A MRIK J. S INGH , R OYA K HOSRAVI -FAR Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA, USA

Synonyms APO2 ligand; APO2L; TRAIL

Definition TRAIL is primarily a membrane-bound cytokine that is one of several members of the ▶tumor necrosis factor (TNF) gene superfamily. Members of this family induce apoptosis through engagement of cell surface ▶death receptors (DRs).

Characteristics Introduction ▶Apoptosis, or programmed cell death, is a genetically conserved physiological event. Apoptosis plays a major role in the elimination of injured or unwanted cells in many physiological and pathological conditions such as normal development, defense against viral infection, dysregulated immune disease and uncontrolled cell growth. Thus, deregulated apoptosis might be related to serious human pathologies including neoplasia, degenerative disorders, and autoimmune diseases. Induction of apoptosis is an important mechanism for ▶chemotherapeutic agents or ▶radiation to kill cancer cells. Many endogenous cytokines also kill target cells via induction of apoptosis. Among these cytokines, several members of the TNF family have been extensively characterized: TNF-α, Fas ligand (FasL/APO1L/ CD95L) and TRAIL. TRAIL has a unique selectivity for triggering apoptosis in cancer cells, but not normal cells. In agreement with these observations, TRAIL knockout mice were more susceptible to experimental and spontaneous tumorigenesis and ▶metastasis. Thus, TRAIL has been considered an important cellular factor both in natural defense mechanisms and as a potential therapeutic agent to treat human cancers. TRAIL and Its Cell Surface Receptors TRAIL was first identified in the mid 1990s by a homology search of an expressed sequence tag database with a highly conserved sequence motif characteristic for the TNF family members. The open reading frame for human TRAIL encodes a protein of 281 amino acids long. TRAIL is primarily expressed as a type II transmembrane protein in which the carboxyl terminus of the

receptor-binding domain protrudes extracellularly. Similar to TNF-α and FasL, TRAIL can also be cleaved from the cell membrane by ▶metalloproteases to yield a soluble and biologically active form. Structural studies have demonstrated that biologically active TRAIL protein forms a homotrimer. A critical cysteine residue that coordinates with a divalent zinc ion stabilizes this homotrimeric structure. TRAIL has been shown to bind five different cell surface ▶TRAIL receptor molecules: TRAIL-R1 (DR4), TRAIL-R2 (DR5/TRICK2/KILLER), TRAIL-R3 (DcR1/TRID/LIT), TRAIL-R4 (DcR2/TRUNDD), and osteoprotegrin (OPG). These receptor molecules, members of the TNF receptor (TNF-R) family, are type I transmembrane polypeptides. TRAIL-R1 and -R2 containing a cytoplasmic ▶death domain (DD) that is essential for death signaling are able to transmit apoptosis-inducing activity of TRAIL across the cell membrane. By contrast, the other three receptors lack a functional death domain. Thus, they may act as ▶decoy receptors, probably by competing with DR4 or DR5 for TRAIL. Unlike other TRAIL receptors, OPG is a soluble protein, originally identified to regulate osteoclastogenesis. Although OPG has been shown to interact with TRAIL, it has a much weaker affinity for TRAIL than other TRAIL receptors; therefore, it is unclear whether or not OPG can efficiently act as a decoy receptor for TRAIL under physiological conditions. Expression of TRAIL receptors closely parallels that of TRAIL, suggesting that most tissues and cell types are putative targets for TRAIL. Organization of TRAIL-Induced Apoptotic Signaling Biologically active trimeric TRAIL protein activates TRAIL receptors via inducing their oligomerization. Similar to other TNF family members, stimulation of the death domain-containing TRAIL receptors DR4 or DR5 recruits the cellular adaptor protein Fas-associated death domain (FADD/MORT1) through interaction of the death domains on each molecule. FADD, interacting with activated DR4 or DR5, then recruits initiator ▶caspases (cysteinyl aspartases) such as procaspase-8 and/or procaspase-10, to form a ▶death-induced signaling complex (DISC). Procaspase-8 or procaspase-10 molecules undergo autoproteolytic activation induced by proximity of these procaspases leading to the formation of active ▶caspase-8 or caspase-10. Although procaspase-10 has been identified as a DISC component, the involvement of caspase-10 in initial death signaling activated by stimulated TRAIL receptors is controversial. Once activated, caspase-8 initiates proapoptoticsignaling cascades leading to the cleavage of cellular factors. Active caspase-8 transmits its proapoptotic activity to executioner caspases, such as caspase-3 and caspase-7, through two main pathways. One proapoptotic signal pathway, termed the extrinsic or


mitochondria-independent pathway, directly activates executioner caspases. The other, termed the intrinsic or mitochondria-dependent pathway, involves mitochondrial events that activate the executioner caspases. The mitochondria-dependent pathway can be initiated, in part, by Bid (BH3 interacting death domain agonist), a ▶Bcl-2 (B-cell lymphoma/leukemia-2) family member or through the uncontrolled production of ▶reactive oxygen species (ROS). In the case of the former, caspase-8-cleaved Bid (tBid, truncated Bid) translocates to the mitochondria and induces cytochrome c release into the cytoplasm. The cytoplasmic cytochrome c binds to ▶Apaf-1 (apoptotic protease activating factor-1) and participates in activation of another initiator caspase, caspase-9. This complex is referred to as the ▶apoptosome. The activated caspase-9 is then able to activate executioner caspases. Thus, activation of executioner caspases represents the point at which the mitochondria-dependent and -independent proapoptotic signaling pathways, having diverged at caspase-8, meet again. In most cell types, despite the existence of a mitochondria-independent TRAIL-induced apoptotic signal pathway, the engagement of the mitochondriadependent pathway is required, as well, for efficient apoptosis. Mitochondrial events also include the release of SMAC (second mitochondria-derived activator of caspases)/DIABLO, apoptosis-inducing factor (AIF) and endonuclease G from the mitochondria. The release of SMAC/DIABLO from the mitochondria appears to be induced by t-Bid and occurs simultaneously with the release of other mitochondrial factors including cytochrome c. Once activated, executioner caspases liberate a DNase termed CAD (caspase-activated DNase) by cleaving an inhibitor of CAD (ICAD/DFF-45). CAD activation leads to DNA fragmentation, a hallmark of apoptosis. Activation of executioner caspases also leads to the cleavage of numerous cytosolic, cytoskeletal and nuclear proteins. Mechanisms to Attenuate TRAIL-Induced Apoptosis Regulation of TRAIL-induced apoptosis occurs at multiple points and involves an intricate network of molecules and signals. The expression levels and subcellular localization of the factors in the TRAILinduced ▶apoptosis signaling cascade influence TRAIL signal strength and cellular susceptibility to TRAIL-induced apoptosis. Anti-apoptotic molecules and diverse extracellular cell survival signals influence TRAIL susceptibility of cells by attenuating cellular factors involved in TRAIL signaling. Expression and mutations of the death domaincontaining TRAIL receptors DR4 and DR5 can influence TRAIL susceptibility. ▶DNA damaging agents, such as chemotherapeutic agents and ionizing

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radiation, have been shown to upregulate DR5 expression. These agents, however, also upregulate the TRAIL decoy receptors DcR1 and DcR2 that, consequently, may abrogate the augmented susceptibility due to upregulated DR4 and DR5. Since numerous DNA damaging agents have been shown to sensitize cells to TRAIL, whether or not TRAIL sensitization by such agents requires upregulation of DR5 is unclear. Early studies suggested that expression levels of DcR1 and DcR2 might be associated with selective induction of apoptosis in tumor cells because the levels of these decoy receptors were higher in normal cells than in tumor cells. Subsequent studies, however, did not show a solid correlation between the expression levels of the decoy receptors and TRAIL susceptibility. This suggests that physiological levels of the decoy receptors might not be sufficient to inhibit TRAILinduced apoptosis. Nevertheless, these decoy receptors may contribute to TRAIL resistance under certain physiological or pathological conditions, which may regulate the expression levels and subcellular localization of these decoy receptors. In addition to receptor molecules, many cytosolic factors also modulate TRAIL-induced apoptosis. Caspase-8 plays a critical role in TRAIL death signaling. Accordingly, caspase-8-deficient Jurkat T cells were shown to be resistant to TRAIL-induced apoptosis. In childhood ▶neuroblastoma, the gene for caspase-8 was found to be frequently silenced through ▶DNA methylation and gene deletion. Furthermore, cell lines derived from these neuroblastoma tissues were resistant to TRAIL-induced apoptosis. Cellular FLICE ((FADD-like interleukin-1β-converting enzyme)-like inhibitory peptide (c-FLIP)) is structurally related to procaspase-8 but lacks an active site for proteolytic action. This protein inhibits TRAIL-induced apoptosis by competing with procaspase-8 for FADD, preventing formation of a functional DISC. High expression levels of c-FLIP has been observed in many cancer cells that are TRAIL-resistant. Anti-apoptotic ▶Bcl-2 family members such as Bcl-2 and Bcl-XL have been shown to inhibit apoptosis mediated by various death receptors. Overexpression of these proteins prevents the release of mitochondrial factors by interacting with proapoptotic Bax (Bcl-2associated X protein) and Bad (Bcl-xL/Bcl-2-associated death promoter) and attenuating their apoptotic function. Overexpression of Bcl-2 and/or Bcl-XL protects various TRAIL-sensitive cells from TRAIL-induced apoptosis. Bcl-2 and Bcl-XL are also upregulated by many extracellular cell survival stimuli, including growth factors and ▶hypoxia. The deficiency of Bax results in a significant reduction of apoptosis induced by TRAIL. Treatment of Bax-deficient cells with TRAIL induces the formation of a functional DISC, caspase-8 activation and Bid

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cleavage. Mitochondrial events, however, involving the release of mitochondrial factors, such as cytochrome c and SMAC/DIABLO, were impaired in Bax-deficient cells. The impaired mitochondrial events prevent post-mitochondrial proapoptotic signaling, including caspase-9 activation. Recently, a line of evidence showed that Bax requires Bak (Bcl-2 antagonist killer 1), another Bcl-2 family member, to act as a gateway for the tBidinduced release of mitochondrial factors. Cells lacking both Bak and Bax, but not cells lacking only one of these proapoptotic components, are completely resistant to tBid-induced cytochrome c release and apoptosis. Whether or not Bax-deficient cells express Bak remains to be determined. Inhibitor of apoptosis protein (IAP) family members, including c-IAP1, c-IAP2, X chromosome-linked IAP (XIAP) and ▶survivin, are potent inhibitors of endogenous caspases, which results in blockade of TRAIL-induced apoptosis. Specifically, IAPs exert their inhibitory activity by interacting with caspase-9, -3, and -7, but not caspase-8. SMAC/DIABLO interacts with IAP family members, antagonizing their inhibitory activity and releasing the bound IAPs from caspases. Caspases free of IAPs are more susceptible to proteolytic cleavage and activation. The protein levels of IAPs are regulated by diverse signals and mechanisms, including autoregulation by intrinsic ▶ubiquitin protein ligase activity. This intrinsic E3 ligase activity has also been shown to regulate the levels of other proteins such as caspase-3 and SMAC/DIABLO. Downregulation or inactivation of IAPs induces spontaneous TRAIL-induced apoptosis. Numerous growth factors and cytokines attenuate TRAIL-induced apoptosis. These survival signals regulate many factors involved in proapoptotic and antiapoptotic signaling. ▶Receptor tyrosine kinase (RTK)mediated activation of the ▶phosphatidylinositol-3 kinase (PI-3K)/protein kinase B (PKB/Akt) signaling axis leads to the direct phosphorylation of numerous downstream targets including proapoptotic factors Bad and caspase-9 as well as class O ▶Forkhead boxbinding family transcription factors (FOXOs). Phosphorylation of Bad and caspase-9 attenuates their proapoptotic activity, ablating the propagation of downstream proapoptotic signaling cascades. Phosphorylation of FOXOs prevents their translocation to the nucleus and results in the transcriptional blockade of genes implicated in promoting apoptosis and cell cycle arrest. A human ▶prostate cancer cell line with constitutive activation of Akt is almost completely resistant to TRAIL, indicating that Akt plays a critical role in tumorigenesis and apoptosis. Augmentation of TRAIL-Induced Apoptosis Although TRAIL is a potent apoptosis inducer, some cells are resistant to TRAIL. Resistance to conventional

therapies such as chemotherapy or radiation poses a major problem in cancer treatment. Subtoxic levels of chemotherapeutic agents facilitate TRAIL-induced apoptosis in vitro and in vitro, suggesting that this combination of therapy may be superior to TRAIL alone. Actinomycin D (Act D) sensitizes many TRAILresistant cells to TRAIL-induced apoptosis. The combination TRAIL and Act D effectively killed TRAIL-resistant prostate cancer cell lines. In these cells, Act D downregulated XIAP. Act D also promoted TRAIL-induced apoptosis in TRAIL-resistant ▶pancreatic cancer cells by decreasing the expression of c-FLIP. Etoposide increased expression of DR4 and DR5 and significantly promoted TRAIL-induced apoptosis in mammary epithelial cells. CPT-11 (Irinotecan/ Camptosar®) and taxol were also observed to enhance TRAIL-induced apoptosis in cells from ▶colon, ▶breast, ▶liver, and ▶ovarian cancers. Adriamycin treatment also resulted in synergistic cytotoxicity and apoptosis for multiple myeloma that are resistant to TRAIL alone. Although the potential of a TRAIL combination therapy with chemotherapeutic agents is evident, in many cases the molecular basis of the synergistic action of chemotherapeutic agents for TRAIL is poorly understood. Clinical Potential for TRAIL Therapy TRAIL is a potent inducer of apoptosis in a wide variety of tumor cell lines in vitro and cancer xenograft animal models in vivo. Additionally, TRAIL did not show any detectable toxic side effects in safety tests using animals such as mice and chimpanzees. From a clinical point of view, one of the most important issues in drug development is safety. Numerous studies have demonstrated stringent selectivity of TRAIL to tumor cells but not to normal or non-transformed cells. Recent reports, however, challenge this established apoptotic selectivity of TRAIL to tumors, demonstrating effective induction of apoptosis in cultured normal hepatocytes and neurons. These observations suggest the potential for damage to normal tissues and organs in clinical trials. Reevaluation of these results, however, reveals that the toxicity observed in cultured normal human hepatocytes is associated with the preparation method of the recombinant TRAIL protein. The non-histidine-tagged form of TRAIL versus the histidine-tagged variant is believed to be safer and more appropriate in clinical trials. Recently, an agonistic ▶TRAIL receptor antibody specifically targeting DR5 was also shown to be a safe and effective cancer therapy similar to non-histidine-tagged TRAIL. Perspectives In the past, most TRAIL research has focused on its proapoptotic activity. Thus, the normal physiological functions of TRAIL remain poorly understood. Recent studies using TRAIL knockout mice and an animal model for the chronic blockade of TRAIL function have

Tobacco Carcinogenesis

shed light on the role of TRAIL in normal physiology and have demonstrated a key role for TRAIL in immune surveillance of tumor and infected cells. A better understanding of the physiological role of TRAIL will no doubt broaden the possible therapeutic applications of this molecule. Still, little is known about TRAIL-triggered death signaling. In particular, the modulation of TRAIL death signaling, under the settings of a continuous challenge with extracellular cell survival stimuli, is poorly understood. Many of these stimuli have been shown to attenuate apoptosis. Identification of signaling pathways and cellular factors involved in cell survival will suggest mechanisms whereby potential targets can be specifically blocked, thus enhancing TRAIL activity in therapeutic applications. Additionally, understanding the mechanisms by which chemotherapeutic agents act and sensitize cells to TRAIL-induced apoptosis will provide various new combination therapies for TRAIL. Although numerous chemotherapeutic agents have been shown to augment TRAIL-induced apoptosis in vitro and in vivo, toxicity tests of the combinations in cultured normal human cells have not been intensively investigated. Such tests would reduce the concerns raised in clinical trials and provide better combination therapies that have higher efficacy and less toxicity than individual therapies. TRAIL has great potential to be further developed as a promising new drug for cancers and autoimmune diseases. Even though there is concern for toxic side effects, there remains great interest to determine whether clinical trials will produce similar results for human cancers as in animal models.

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forms although the most common is wrapped in the form of cigarettes. It is consumed worldwide and causes many types of cancers and other diseases due to mainly its content of different carcinogens. The nicotine is the substance causing addiction. ▶Tobacco-Related Cancers ▶Tobacco Carcinogenesis

Tobacco Addiction ▶Nicotine Addiction

Tobacco Carcinogenesis S TEPHEN S. H ECHT The Cancer Center, University of Minnesota, Minneapolis, MN, USA

Definition Refers to cancer induction by tobacco products and their constituents in laboratory animals and humans.

Characteristics References 1. LeBlanc HN, Ashkenazi A (2003) Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10:66–75 2. Wang S, El-Deiry WS (2003) TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 22:8628–8633 3. Jin Z, El-Deiry WS (2005) Overview of cell death signaling pathways. Cancer Biol Ther 4:139–163 4. Almasan A, Ashkenazi A (2003) Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy. Cytokine Growth Factor Rev 14:337–348 5. Ashkenazi A (2002) Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2:420–429

Tobacco Definition It is the common name of Nicotiana species, particularly of Nicotiana tabacum. It can be consumed in many

Tobacco Products and Human Cancer Worldwide tobacco use continues to be immense and pervasive. According to estimates by the World Health Organization, there are about 1.3 billion smokers in the world and millions of smokeless tobacco users. Cigarette smoking causes well over one million cancer deaths annually worldwide, and accounts for 26% of all cancer mortality in developed countries. In addition to lung cancer, cigarette smoking causes the following types of cancer: oral cavity, pharynx, larynx, esophagus, pancreas, bladder, nasal cavity, stomach, liver, kidney, ureter, cervix, and myeloid leukemia. ▶Secondhand tobacco smoke causes lung cancer in non-smokers, although the risk is less than in smokers. Smokeless tobacco products, such as moist snuff used orally, are accepted causes of oral cavity cancer and are implicated as causes of pancreatic cancer. Tumor Induction in Laboratory Animals Experimental studies demonstrate that cigarette smoke as well as its condensate cause cancer in laboratory animals. Inhalation studies have conclusively produced

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cancer in the respiratory tracts of hamsters, rats, and mice. Cigarette smoke condensate has been extensively tested on mouse skin, where it consistently induces benign and malignant skin tumors. Smokeless tobacco has been shown to induce oral cavity cancer in rats. Chemistry of Tobacco Smoke When cigarette tobacco is burned, mainstream and sidestream smoke are generated. Mainstream smoke is the material drawn from the mouth end of a cigarette during puffing. Sidestream smoke is the material released into the air from the burning tip of the cigarette plus the material which diffuses through the paper. Secondhand tobacco smoke is a composite of sidestream smoke and exhaled smoke. Mainstream smoke is an aerosol containing about 1 × 1010 particles per milliliter. About 95% of the smoke is made of gases, mainly nitrogen, oxygen, and carbon dioxide. The particulate phase of mainstream smoke contains more than 4,000 compounds. Many components are present in higher concentrations in sidestream than in mainstream smoke, but a person’s exposure to sidestream smoke is far less than to mainstream smoke because of dilution with room air. There are over 60 ▶carcinogens in cigarette smoke that have been evaluated by the International Agency for Research on Cancer, and for which there is sufficient evidence for carcinogenicity in laboratory animals or humans, and 15 are rated as carcinogenic to humans. Carcinogens in cigarette smoke include ▶polycyclic aromatic hydrocarbons (PAH) and their nitrogen containing analogues, ▶N-nitrosamines, ▶aromatic amines, heterocyclic aromatic amines, aldehydes, low molecular weight organic compounds such as benzene and 1,3butadiene,andinorganic compounds. In addition, cigarette smoke contains tumor promoters, co-carcinogens, and toxicants such as acrolein and nitrogen oxides. The most important compounds with respect to human lung cancer appear to be the PAH, typified by benzo[a]pyrene (BaP),

and the tobacco-specific N-nitrosamine ▶4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), both considered as human carcinogens. Other compounds that may play a role as causes of lung cancer include 1,3-butadiene, benzene, aldehydes, and metals. Whereas PAH occur in all products of incomplete combustion, ▶tobaccospecific N-nitrosamines are found only in tobacco products because they are derived from ▶nicotine and related compounds. Tobacco-specific N-nitrosamines are the most prevalent strong carcinogens in unburned tobacco and are believed to play a significant role in the induction of oral cavity cancer by these products. Mechanisms of Tumor Induction Fig. 1 presents a conceptual framework for understanding mechanisms of tobacco carcinogenesis. While this scheme focuses on smokers, similar considerations apply to smokeless tobacco users. The central track of Fig. 1, involving exposure to carcinogens, the formation of covalent bonds between the carcinogens and DNA (▶DNA adduct formation), and the resulting permanent mutations in critical genes of somatic cells is the major established pathway of cancer causation by cigarette smoke. While nicotine, the main known addictive agent in cigarette smoke, is not carcinogenic, each puff of each cigarette contains a mixture of carcinogens. Extensive data using specific ▶biomarkers, including carcinogen metabolites in urine, DNA adducts, and protein adducts, demonstrate the uptake of these carcinogens by smokers and confirm the expected higher levels of their metabolites in urine of smokers than non-smokers. Most cigarette smoke carcinogens require a ▶metabolic activation process, generally catalyzed by ▶cytochrome P450 enzymes (P450s), to convert them to forms that can covalently bind to DNA, forming DNA adducts. P450s 1A1 and 1B1, which are inducible by cigarette smoke via interactions with the aryl hydrocarbon receptor, are particularly important in the metabolic

Tobacco Carcinogenesis. Figure 1 Conceptual framework for understanding tobacco carcinogenesis.


activation of PAH. The inducibility of these P450s may be a critical aspect of cancer susceptibility in smokers. P450s 1A2, 2A13, 2E1, and 3A4 are also important in the activation of cigarette smoke carcinogens. Competing with the activation process is ▶metabolic detoxification, which results in excretion of carcinogen metabolites in generally harmless forms, and is catalyzed by a variety of enzymes including ▶glutathione-S-transferases and UDP-glucuronosyl transferases. The balance between ▶carcinogen metabolic activation and detoxification varies among individuals and is likely to affect cancer susceptibility with those having higher activation and lower detoxification capacity being at highest risk. This is supported by considerable evidence from molecular epidemiologic studies of polymorphisms, or variants, in these enzymes. The metabolic activation of carcinogens results in the formation of DNA adducts which are absolutely central to the carcinogenic process. Starting in the mid 1980s, extensive studies have demonstrated the presence of DNA adducts in human tissues. There is massive evidence, particularly from studies which use relatively non-specific adduct measurement methods, that adduct levels in the lung and other tissues are higher in smokers than in non-smokers, and some epidemiologic data link higher adduct levels with a higher probability of cancer development. Cellular DNA repair systems remove DNA adducts and return the structure of DNA to normal. These systems include direct base repair by alkyltransferases, excision of DNA damage by base and ▶nucleotide excision repair, mismatch repair, and double strand repair. If these repair enzymes are overwhelmed by DNA damage or for other reasons cannot efficiently perform their function, then adducts may persist, leading to higher probability of cancer development. There are polymorphisms in some DNA repair enzymes and the resulting deficient DNA repair can lead to a higher probability of cancer development, according to some molecular epidemiologic studies. Persistent DNA adducts can cause miscoding during replication when DNA polymerase enzymes process them incorrectly. There is considerable specificity in the relationship between specific DNA adducts caused by cigarette smoke carcinogens and the types of mutations which they cause. G to T and G to A mutations are often found. Mutations have been frequently observed in the KRAS ▶oncogene in lung cancer and in the TP53 ▶tumor suppressor gene in a variety of cigarette smokeinduced cancers. The cancer causing role of mutations in these genes has been firmly established in animal studies. The KRAS and ▶TP53 mutations observed in lung cancer in smokers reflect DNA damage by cigarette smoke carcinogens. In addition, numerous cytogenetic changes have been observed in lung cancer, and chromosome damage throughout the aerodigestive

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tract is strongly linked with cigarette smoke exposure. ▶Apoptosis can remove cells with DNA damage, and serves as a protective counterbalance to these mutational events. The balance between mechanisms leading to apoptosis and those suppressing apoptosis will have a major impact on tumor growth. While the central track of Fig. 1, proceeding through genetic damage, is clearly established as a major pathway by which cigarette smoke carcinogens cause cancer, epigenetic pathways also contribute, as indicated in the top and bottom tracks of Fig. 1. Nicotine and tobacco-specific nitrosamines have been shown to bind to nicotinic and other cellular receptors leading to activation of Akt (also known as protein kinase B), protein kinase A, and other changes, resulting in decreased apoptosis, increased ▶angiogenesis, and increased transformation. Furthermore, the occurrence of co-carcinogens and tumor promoters in cigarette smoke is well-established. These compounds, while not carcinogenic themselves, clearly enhance the carcinogenicity of cigarette smoke carcinogens. This occurs through mechanisms which to date are poorly defined. Another important epigenetic pathway is enzymatic ▶methylation of promoter regions of genes, which can result in gene silencing. If this occurs in tumor suppressor genes, the result can be unregulated proliferation. The chronic exposure of smokers and smokeless tobacco users to the DNA damaging intermediates formed from tobacco carcinogens is consistent with our present understanding of cancer induction as a process which requires multiple genetic changes. Thus, it is completely plausible that the continual barrage of DNA damage produced by tobacco carcinogens causes the multiple genetic changes that are associated with cancer, and lead to a series of well established ▶signal transduction mechanisms resulting in uncontrolled cell growth. While each dose of carcinogen from a tobacco product is small, the cumulative damage produced in years of usage is substantial.

References 1. Hecht SS (1999) Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 91:1194–1210 2. Hecht SS (2003) Tobacco carcinogens, their biomarkers, and tobacco-induced cancer. Nat Rev Cancer 3:733–744 3. International Agency for Research on Cancer (2004) Tobacco Smoke and involuntary smoking. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol 83. IARC, Lyon, FR, pp 33–1452 4. Hecht SS (2005) Carcinogenicity studies of inhaled cigarette smoke in laboratory animals: old and new. Carcinogenesis 26:1488–1492 5. International Agency for Research on Cancer (2007) Smokeless tobacco and tobacco-specific nitrosamines. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol 89. IARC, Lyon, FR, in press

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Tobacco Dependence

Tobacco Dependence ▶Nicotine Addiction

Tobacco-Related Cancers A LBERTO R UANO -R AVINA , M O´ NICA P E´ REZ -R I´ OS , J UAN M IGUEL B ARROS -D IOS Área de Medicina Preventiva y Salud Pública, Facultad de Medicina y Odontología, Universidad de Santiago de Compostela, C/San Francisco, s/n CP 15782

Definition

Refers to all those cancers where ▶tobacco has an etiologic role either in active or in passive form. Due to the risk for health associated with tobacco consumption and also to its widespread use, tobacco-related cancers have been sometimes studied as a whole.

When inhaled, tobacco smoke goes through the mouth, pharynx, larynx, main bronchus and then diffuses on the lungs, where its components entry in the circulatory system and are distributed into the body. These components are eliminated mainly on the urine. As can be deducted, all the organism of a smoker is exposed to the harm caused by tobacco, although not all the organs have the same susceptibility for developing a cancer caused by it. Although it is not exactly known how the tobacco causes cancer on different anatomic locations, one of the most important mechanisms of action is the following: carcinogenic substances contained in tobacco smoke are activated in different cells into more reactive ones. These activated substances can bind to the DNA forming the so-called ▶DNA adducts. These DNA-adducts have a covalent union among the activated carcinogenic substance (usually a PAH) and the double-strand of the DNA. If the adduct is not repaired by the replication machinery, it can cause a mutation when the cell replicates that can ultimately lead to cancer. It has also been observed a specific mutation in the ▶p53 gene due to tobacco exposure, especially in ▶lung cancer. This mutation appears on the second position on codon 249 and is a guanine to thymine transversion.

Characteristics Tobacco as a Carcinogen In 2000, 1.47 million deaths from cancer were attributable to smoking, accounting for 32% of all smoking attributable deaths. Tobacco is estimated to cause about 1 of each 5 cancer deaths worldwide, with a higher ▶attributable fraction of 29% in high-income countries and 18% in low and middle-income regions. Although the most known tobacco product is cigaret, there are other types of tobacco products, such as pipes, cigars, bidis, or betel quids, among others. The last two ones are especially frequent in India. Scientific evidence has shown that all tobacco forms cause cancer. Tobacco smoking was declared a human carcinogen in 1986 by the International Agency for Research on Cancer (IARC). Tobacco contains more than 4,000 different chemical substances and about more than 60 have been classified as human carcinogens or probable or possible carcinogens. These substances can be classified mainly in ▶polycyclic aromatic hydrocarbons (PAHs) and ▶tobacco-specific nitrosamines. The most characteristic substance of the first group is benzo[a]pyrene and of the second is 4-(methylnitrosamino)-1-(3-pyrydil)-1-(butanone), also known as NNK. ▶Nicotine, which accounts for 0.5–4% by weight of tobacco leaves, is readily absorbed after tobacco use and is responsible for the addiction. Around the world, there are 1.1 billions of smokers and a quite high percentage of them will develop a tobacco-related cancer.

Cancers Related with Tobacco In general, the risk of developing a tobacco-related cancer depends mainly on the duration on smoking and on the number of cigarettes per day. Cumulative exposure increases with time, so the probability of developing a cancer increases with age. The risk of cancer diminishes from the time since quitting and, for some cancers, it can be the same of a never-smoker’s risk after some years of abstinence. This is an important message to keep in mind, it is never too late to abandon the tobacco habit and the human health will be benefited with this decision at any age. Lung Cancer. This is the best known cancer related to tobacco consumption and is the one which causes more deaths. About the 80% of lung cancer deaths are attributed to tobacco in males and the 48% in women worldwide. When comparing a smoker with a nonsmoker, the former has 20-fold more possibilities of developing lung cancer. In the appearance of the disease, the number of cigarettes per day along with smoking duration determines the risk, although the last factor is more important than the first. Some discrepancies exist on the time since quitting for the risk of lung cancer of a former smoker to be the same to the risk of a never smoker. There also exist interactions between tobacco smoking and other risk factors of lung cancer including occupation or radon exposure in the risk of developing the disease.

Tobacco-Related Cancers

Oral Cavity and Pharyngeal Cancers. Tobacco is a risk factor for both cancers. Men smokers pose a risk tenfold higher versus never smokers while the average risk in women is fivefold higher. There is also an interaction with alcohol consumption. Laryngeal Cancer. Tobacco causes laryngeal cancer, there is a strong dose–response relationship between tobacco and the risk of developing this cancer and also an interaction with alcohol intake. Esophageal Cancer. Tobacco is a risk factor for this cancer; the average risk is about seven to eight times higher than the risk of lifetime nonsmokers. The risk of esophageal cancer due to tobacco increases with heavy alcohol consumption. Pancreatic Cancer. Tobacco increases the risk of having a pancreatic cancer. For heavy smokers, the risk of this cancer is about three to five times higher than for never-smokers. Bladder and Kidney Cancers. Smoking increases the risk of bladder, kidney, and renal pelvis cancers. An interesting finding was that the urine of smokers is more mutagenic than the urine of never smokers. Cervical Cancer. Smoking women have a risk of cervical cancer two to three times higher than never smoking women. Although it is not clear if there is an interaction with human papillomavirus (HPV), it seems to exist a higher risk for smokers that are HPV positive. Stomach Cancer. Tobacco causes stomach cancer. The risks of smokers versus nonsmokers are around 1.5–2 times higher. Although not confirmed, there seems to be an interaction with Helicobacter Pylori. This infection is harder to be eradicated in smokers compared with nonsmokers. Acute Myeloid Leukemia. There exists a risk of acute myeloid leukemia for smokers versus nonsmokers and that is 1.5–2 times higher in smokers.

Evidence for Other Cancers Some cancers have not been related to tobacco. Although the studies performed on them cannot allow us to conclude a causal relationship, we should not disregard a possible association, since there are methodological aspects in the design of such studies that make difficult to assess a possible relationship. In any case, it is true that these cancers would not pose the association (in terms of relative risk) of those cancers with a demonstrated causal association nowadays. For ovarian cancer and endometrial cancer, tobacco does not seem to pose a risk for the former but seems to reduce the risk of the latter. For colorectal cancer, although the published literature seems to indicate a higher risk for smokers, this is not conclusive. For liver cancer, although it seems to be a positive association among smoking and liver cancer, the studies are

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inconclusive due to the lack of adjustment for other risk factors of this cancer. For prostate, brain, and breast cancers, it seems that tobacco is not associated with the incidence of these cancers. Passive Smoking We refer to ▶passive smoking exposure when we breath (or are exposed to, i.e., the case of the fetus) tobacco products produced by others. This is also called involuntary smoking. Passive smokers inhale the mixture of the mainstream smoke exhaled by the smoker and also the sidestream smoke produced by the smoldering cigarette. This complex mixture has essentially the same composition that the one to which smokers are exposed and the main difference with smokers is that this mixture is usually diluted on the air, although its concentration depends on many factors. It is called ETS (Environmental tobacco smoke) or more recently ▶secondhand smoke. In 1992, the US Environmental Protection Agency (EPA) declared the ETS as a human carcinogen. In 2006, the US Surgeon General has published an extensive report on the health consequences of involuntary smoking (see references for more information). It states that there is no risk-free level of exposure to secondhand smoke. Some of the results affecting cancer incidence related with this exposure are the following: ETS causes lung cancer, with exposed lifetime nonsmokers having a 20–30% more risk than nonexposed. The evidence is suggestive but not sufficient for nasal sinus cancer. Respecting prenatal and postnatal exposure to ETS the literature suggests that there could be some relationship with ▶childhood leukemias, ▶lymphomas, and ▶brain tumors. These last relationships have to be demonstrated in more rigorous studies. Conclusions Tobacco use is nowadays one of the most, if not the most, hazardous agents for human health and also in the cancer appearance. This modifiable exposure causes many different types of cancer (along with cardiovascular and respiratory diseases among others) such as lung, oral cavity, pharynx, larynx, esophagus, bladder, kidney, pancreas, cervix, stomach, and acute myeloid leukemia. The best way for prevention is not to smoke and a clear message has to be given to current smokers that their risk of cancer diminishes immediately from the moment they decide to abandon the habit. A great effort has to be made in order to avoid tobacco use in the new generations. Passive smoking also entails a risk for health, for both adults and for infants. In adults, it causes lung cancer and in infants it could cause childhood cancer (to be further studied), along with other diseases and alterations different than cancer. Exposure to ETS should also be avoided at any concentration in every place.

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References 1. International Agency for Research on Cancer (2004) IARC Monographs on the evaluation of the carcinogenic risks to humans: tobacco smoke and involuntary smoking, vol 83. International Agency for Research on Cancer, Lyon (France), pp 1452; ISBN 92 832 1283 5 2. National Cancer Institute (1999) Health effects of exposure to environmental tobacco smoke. The report of the Environmental Protection Agency. Smoking and Tobacco Control Monograph no. 10. US Department of Health and Human Services, National Institutes of Health, National Cancer Institute, Bethesda, MD, NIH Pub no. 99–4645 3. Ruano-Ravina A, Figueiras A, Montes-Martínez A et al. (2003) Dose-response relationship between tobacco and lung cancer: new findings. Eur J Cancer Prev 12(4): 257–263 4. US Department of Health and Human Services (2006) The health consequences of involuntary exposure to tobacco smoke: a report of the Surgeon General. US Department of Health and Human Services, Centers for Disease Control and Prevention, Coordinating Center for Health Promotion, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, Atlanta, GA 5. US Department of Health and Human Services (2004) The health consequences of smoking: a report of the Surgeon General. US Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, Atlanta, GA

Tolbutamide Definition Is a potassium channel blocker; may be used in the management of type II diabetes if diet alone is not effective. Tolbutamide stimulates the secretion of insulin by the pancreas. Since the pancreas must synthesize insulin in order for this drug to work, it is not effective in the management of type I diabetes, where the beta-cells of islets of Langerhans are unable to produce insulin, for instance as the result of cell degeneration.

Tolbutamide Intravenous Test Definition A test to detect insulin-producing tumors (insulinoma). After a 1-g intravenous dose of tolbutamide, plasma insulin and glucose are measured at intervals up to 3 h; higher insulin responses and lower glucose values characterize patients with such tumors.

Tobacco-Related Lung Cancer Definition Cancer that develops in the lungs those who smoke and/ or use of smokeless tobacco for long-term period. ▶Toxicological Carcinogenesis

Tolerance Definition State in which the immune system shows a lack of reactivity toward certain antigens, notably those that are expressed by normal cells and tissues.

Tobacco-Specific N-Nitrosamines Tolfenamic Acid Definition A group of carcinogens structurally related to nicotine which occur only in tobacco products. ▶Tobacco Carcinogenesis

Definition

A ▶non-steroid anti-inflammatory drug (NSAID) that also induces Sp protein degradation.

Toll-Like Receptors

Toll-Like Receptors K ASPER H OEBE Cincinnati Children’s Hospital Research Foundation, Divsion of Molecular Immunology Cincinnati, OH, USA

Definition Toll-like receptors (TLRs) represent a family of proteins that are involved in the innate sensing of conserved microbial components. They are composed of an extracellular domain containing leucine-rich repeats and a cytoplasmic Toll/Interleukin-1 receptor/Resistance (TIR) domain that is responsible for mediating downstream signaling cascades and subsequent inflammatory response, ultimately leading to the eradication of infectious agents.

Characteristics Toll-like receptors were originally described as proteins with homology to the protein Toll, which was first identified in Drosophila melanogaster. In flies, Toll served a regulatory function in the embryonic dorsoventral polarity, and was later shown to be an essential component of the Drosophila ▶innate immune system. Based on the fact that Toll serves an immune function in Drosophila, it was proposed that TLRs might “activate ▶adaptive immunity” in mammals, but the precise function of TLRs was revealed by forward genetics. It was long recognized that the inflammatory responses induced by pathogens could be mimicked by specific molecules of microbial origin initially referred to as “endotoxin” and later shown to be lipopolysaccharide (LPS). It was then shown that LPS was capable of augmenting the adaptive immune response to a protein antigen, and in 1975, it became clear that the immuno-▶adjuvant effect of LPS depended upon the integrity of a single locus known as Lps. It was not until 1998, that positional cloning revealed TLR4 as the membrane spanning component of the LPS receptor and this discovery accelerated our understanding of how the innate recognition of microbes occurs. Further genomic analysis revealed additional TLRs and to date a total of ten human TLRs (1–10) and 12 mouse TLRs (1–9; 11–13) have been described. Each of these recognizes a limited repertoire of broadly conserved molecules of microbial origin. For example, unmethylated DNA (common to prokaryotic genomes and DNA viruses) is recognized by TLR9; gram-positive bacterial lipopeptides are recognized by TLR2, acting in conjunction with TLRs 1 and 6, whereas TLR7 and TLR8 recognize single stranded viral RNAs or guanosine related analogues such as loxoribine and imidazoquinoline. All TLRs

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have a common cytoplasmic TIR motif that is believed to bind to intracellular adaptor molecules containing TIR motifs as well. Besides TLRs, the TIR motif is shared by the IL-1 receptor and IL-1 receptor-related family of signaling adaptor molecules. To date a total of five TIR domain-containing intracellular proteins have been identified that include MyD88, Mal (also known Tirap), Trif (also known as TICAM-1), Tram (also known as TICAM-2) and Sarm. Although most TLRs activate a common signaling pathway involving the adaptor molecule MyD88, other adaptor molecules involving Tram and/or Trif are able to activate a MyD88-independent pathway specifically downstream of TLR3 and/or TLR4. To date, many of the kinases involved in the TLR signaling pathways have been described and include for instance IRAK-1, IRAK-4 and Transforming Growth factor-β-activated kinase-1 (TAK1) (Fig. 1). Although TLRs are primarily expressed on cells of the innate immune system, including monocytes macrophages and dendritic cells, their expression is not limited to these cells and can also be found in lymphoid or non-hematopoietic cells. Whereas most TLRs are expressed on the surface of the cells, TLRs that recognize nucleotide structures (TLRs 3/7/8/9) are expressed in intracellular compartments such as endosomes and/or endosplasmic reticulum. TLRs and Cancer Treatment Although TLRs have limited involvement in the recognition, development or initiation of tumor growth, their immunostimulatory function has been utilized in cancer treatment in a variety of ways. Particularly, TLR7/8 agonists such as ▶imiquimod, have long been used as topical immunomodulators, indicated for the treatment of external genital and perianal warts. This drug has also been approved for the treatment of superficial basal cell carcinoma and actinic keratoses, and also has activity against cutaneous metastases of malignant melanoma and vascular tumors. Many of the therapeutic effects of TLR activation can be attributed to the production of proinflammatory cytokines including tumor necrosis factor-alpha (▶TNFα), type I ▶interferons and interleukin 12. These cytokines have diverse functions that range from direct anti-tumor or pro-apoptotic effects (e.g. TNF), priming of natural killer (NK) cells (e.g. type I IFN) and the maturation of DCs leading to cytolytic T cell activation (e.g. type I IFN and IL-12). Although many cells of the immune system are involved in tumor surveillance and tumor recognition, the induction of anti-tumor immune responses capable to suppress or eradicate tumors is generally poor. CD8+ cytolytic T cells are the main cells involved in tumor eradication. As the antigens on tumor cells originate from self, most CD8+ T cells have low avidity or are tolerant to these antigens and require a potent

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Toll-Like Receptors. Figure 1 Toll-like receptors are key proteins in the innate recognition of microbial components. Upon recognition, TLRs initiate a signaling cascade that involves the TIR domain containing adaptor molecules MyD88, MAL/TIRAP, Trif and/or Tram, ultimately leading to NFκB activation resulting in proinflammatory cytokine production and/or translocation of IRF3/7 leading to type I interferon production (see text for further explanation).

stimulatory environment to become activated and acquire and execute their effector functions. Tumors create a suppressive environment by secretion of antiinflammatory molecules, prevention of antigen expression and recruiting and inducing regulatory T cells on the site of tumor growth. The design of experimental therapies is currently geared to implement TLR agonists to act on the above mentioned ▶immunosuppressive pathways, thereby enhancing the induction of tumor specific cytolytic T cells. Specifically, vaccines based on the administration of dendritic cells containing tumor antigens or the administration of irradiated autologous or allogeneic tumor cells, have shown improved efficacy when coadministered with TLR-agonists. In both settings, the addition of TLR-agonists is thought to target DC function, leading to proinflammatory cytokine production and the subsequent upregulation of costimulatory molecules. In the event tumor cells were used as a vaccine therapy, only cell associated but not

soluble TLR-agonists were shown to improve the adaptive immune response, suggesting targeting of TLRs with antigen in the same DC is crucial for efficacy. In addition, consistent TLR activation also leads to a reversal of T regulatory cell-mediated immune suppression and as such break tolerance towards self antigens. Together, these effects lead to an enhanced tumor-specific CD8+ and CD4+ T cell response ultimately leading to a regression and/or eradication of tumor growth. ▶Inflammation

References 1. Beutler B (2004) Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430:257–263 2. IshiiK J, Coban C, Akira S (2005) Manifold mechanisms of toll-like receptor-ligand recognition. J Clin Immunol 25:511–521

Tongue Cancer

3. O’NeillL A (2006) How Toll-like receptors signal: what we know and what we don’t know. Curr Opin Immunol 18:3–9

Tongue Cancer A NTHONY P O -W ING Y UEN Division of Otorhinolaryngology, Department of Surgery, The University of Hong Kong, Hong Kong, SAR, China

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Clinical Features The stepwise development of tongue cancer sometimes appears clinically in its premalignant stage as leukoplakia (white discoloration) or erythroplakia (red discoloration). These lesions histologically have various degrees of dysplasia. With further accumulation of genetic abnormalities involved in cancer development, these dysplastic epithelial cells become cancer cells which can invade and metastasize. The risk of malignant change of these dysplastic lesions is in the range of 5–20%. Carcinoma of tongue appears as nonhealing painful ulceration mostly at the lateral border of oral tongue. A typical ulcerative carcinoma of the lateral border of oral tongue with surrounding leukoplakia is shown in Fig. 1.

Synonyms Lingual cancer

Definition Tongue cancers are malignant neoplasms of the tongue affecting oral tongue in the oral cavity or tongue base in the oropharynx.

Characteristics Epidemiology The world age standardized incidence is about 1.6 per 100,000 persons. The age standardized incidences of tongue cancer increase with age from about 0.1 per 100,000 persons in age group 15–19 years to about 7.5 per 100,000 persons in age group 65–75 years. The male to female ratio is about 1.5. The common predisposing factors of tongue carcinoma are smoking and drinking. Betel leaf chewing is the main reason for high incidences of tongue carcinoma in a few Asian countries including India and Taiwan.

Pathology and Genetic Aberration The commonest cancer affecting the tongue is squamous cell carcinoma (▶oral cancer) arising from the mucosal epithelial cells. Other pathology including minor salivary gland malignancies and sarcomas are rare. The development of cancer is believed to be a multi-step process with accumulation of many genetic abnormalities. A common genetic abnormality in tongue carcinoma is p53 (▶p53 protein, biological and clinical aspects) mutation and its overexpression. The aberrantly underexpressed and overexpressed genes of tongue carcinoma are summarized in Table 1. These aberrantly expressed genes are involved in the control of cell cycle (▶cell cycle target for cancer therapy), cell proliferation, ▶apoptosis, cell ▶adhesion, cell ▶motility, and ▶angiogenesis.

Subclinical Nodal Metastasis Tongue carcinoma has high propensity of nodal metastasis even in its early stage. The nodal recurrence rate is 30–50% in untreated clinically N0 neck of T1-2 carcinomas. The commonest site of nodal metastasis is ipsilateral level II, and 95% metastatic nodes are found in the ipsilateral levels I, II, and III. Contralateral nodal metastasis and contralateral nodal recurrence are found only in less than 5% patients. Most subclinical nodal metastases contain only tiny focal micrometastasis of less than 2 mm diameter within the lymph nodes or on the capsular lymphatic vessels. It has been reported that these cancer cells occupied a median of only 6% of nodal cross-sectional area of a lymph node and only 1% nodes in the neck contained micrometastasis. The small size of the micrometastasis amidst the large number of normal nodes in the neck would cause tremendous difficulty in the clinical detection. Although preoperative radiologic screening including CT, MRI, PET, or ultrasound scans are useful screening methods, they are not sensitive in the detection of most micrometastasis inside the small nonpalpable nodes. The risk of nodal recurrence of observed N0 neck remains high after these radiologic imaging screening. TNM Staging and Prognostic Factors The current TNM staging system for carcinoma of oral tongue is shown in Appendix 1. The largest tumor diameter has been used for many years in staging T1-3 oral tongue carcinoma. The largest diameter of oral tongue carcinoma is however either the tumor length or width. Tumor thickness is almost never the largest diameter of oral tongue carcinoma and therefore has no contribution to the current staging system. It has been shown by many studies that tumor thickness, but not the largest dimension, is a significant independent prognostic factor in predicting subclinical nodal metastasis, local recurrence, and survival of oral

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Tongue Cancer. Table 1

Aberrantly expressed genes of tongue carcinoma

Underexpressed genes

▶E-cadherin (83–85%) α-Catenin (94%) β-Catenin (89%) γ-Catenin (83%) ▶Maspin (49%) P14 (21%) P15 (47%) P16 (54–74%) ▶P21 (40–73%) P27 (72%) ▶PTEN (29%) RARβ (▶retinoid receptor crosstalk in cancer) (78%)

Overexpressed genes Akt (▶AKT signal transduction pathway in oncogenesis) (46%) AMFR (42%)

▶bcl-2 (9–26%) Cerb-B2 (17–50%) C-myc (75%) c-Met (42%) cyclin B1 (37%) ▶cycin D1 (53–68%) EGFR (▶epidermal growth factor receptor ligands) (32–34%) EpCam (63%) Ets-1 (▶ETS transcription factor)(42%)

▶Furin (76%)

MMP-2 (▶matrix metalloproteinases) (49–53%) MMP-9 (▶matrix metalloproteinases) (78%) MT1-MMP (▶matrix metalloproteinases) (35%) ▶Osteopontin p34cdc2 (66%) p53 (▶p53 protein, biological and clinical aspects) (27–62%) PCNA (32%) ▶Podoplanin (60%) Rab1A (98%) RARα (▶retinoid receptor crosstalk in cancer) (65%) S100A2 (29%) Syndecan-1 (58%) TIMP-1 (▶matrix metalloproteinases) (65%) TIMP-2 (▶matrix metalloproteinases) (43%) VEGF-A (▶vascular endothelial growth factor) (67%) VEGF-C (▶vascular endothelial growth factor) (49–100%) VEGFR-1 (▶vascular endothelial growth factor) (89%) VEGFR-3 (▶vascular endothelial growth factor) (74%)

tongue carcinoma. The thicker the tumor, the higher would be the risk of local recurrence, subclinical nodal metastasis and treatment failure. It is however still unresolved yet on the best cutoff thickness value for staging purpose. The proposed cutoff thickness values for prognosis or staging by various studies vary between 3 and 9 mm. The other important prognostic factor is histologic feature of perineural spread which is a significant risk factor of local recurrence after surgical treatment. Pretreatment Assessment Preoperative endoscopy and biopsy should be done to confirm the diagnosis and evaluate the extent of local tumor infiltration. Small neck nodes less than 1 cm

along the jugular chain may not be palpable with fingers. Ultrasonography of the neck and ultrasound guided fine needle aspiration for cytology should be done to screen for presence of small nodal metastasis that may not be palpable. Tumor thickness is an important factor for prognosis evaluation and treatment planning. Preoperative assessment of tumor thickness cannot be done with palpation. Intraoral ultrasonography using 7.5 MHz probe can be used to document the tumor thickness. MRI can also be used to assess the tumor thickness for preoperative evaluation of prognosis. Both T1 and T2 weighted MRI images can show the tumor thickness satisfactorily. MRI images in three-dimensional planes can also help the surgeon in the planning of surgical resection.

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Appendix I TNM stage of oral tongue carcinoma, AJCC/ UICC 2002

Tongue Cancer. Figure 1 Ulcerative carcinoma over the lateral border of tongue with surrounding leukoplakia.

Treatment Options The treatment of oral tongue carcinoma remains controversial. Brachytherapy alone or surgery alone are each commonly used as primary treatment for early carcinomas by radiation oncologists and surgeons. The curative results of brachytherapy and surgery are similar for early stage carcinoma with over 90% local control. Either surgery or brachytherapy alone are not effective for stage III and IV carcinomas, combined surgery and postoperative chemoradiotherapy are recommended for advanced stage carcinomas. Other less commonly practiced alternative treatment options include laser surgery or photodynamic therapy for early stage carcinomas. Concurrent intra-arterial regional chemoperfusion of high dose of cisplatin and radiotherapy may be considered as alternative treatment option for advanced T4 stage carcinoma. Controversy of Elective Neck Dissection Versus Observation of N0 Neck Since there is high risk of nodal recurrence of observed N0 neck of early tongue carcinoma, elective neck dissection is commonly practiced in many cancer centers worldwide. In retrospective studies of elective selective neck dissection versus observation of N0 neck, the regional recurrence rates could be reduced from 30–50% of observation to 10–15% after for elective selective I,II,III neck dissection. The regional recurrence related mortality rates could be reduced from 20–25% for observation to 4–10% for elective selective I,II,III neck dissection group. From the results of retrospective studies, elective neck dissection can reduce both initial regional recurrence rate and regional recurrence related mortality, and the reduction of noderelated mortality contributes to long-term survival benefit.

Primary tumor (T) Tx Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ T1 Tumor ≤2 cm in greatest dimension T2 Tumor >2 cm but ≤ 4 cm in greatest dimension T3 Tumor >4 cm in greatest dimension T4 Tumor invades adjacent structures (e.g., through cortical bone, into deep extrinsic muscle of tongue, maxillary sinus, skin; superficial erosion of bone/ tooth socket by gingival primary is not sufficient to classify as T4) Regional lymph nodes (N) Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in single ipsilateral node, ≤3 cm in greatest dimension N2a Metastasis in single ipsilateral node, >3 cm but ≤6 cm N2b Metastasis in multiple ipsilateral nodes, all ≤6 cm N2c Metastasis in bilateral or contralateral nodes, all ≤6 cm N3 Metastasis in lymph node >6 cm in greatest dimension Distant metastasis (M) Mx Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis Stage 0 Tis N0 M0 I T1 N0 M0 II T2 N0 M0 III T3 N0 M0, T1-2 N1 M0, T3 N1 M0 IV T4 N0-1 M0, anyT N2-3 M0, anyT anyN M1

There is however no prospective randomized study comparing the long-term benefit of elective neck dissection compared with observation in the treatment of N0 neck of early stage I and stage II oral tongue carcinoma. There is risk of mortality and morbidity of elective neck dissection. Both elective neck dissection and observation have their proponents in different cancer centers. Instead of performing elective neck dissection for all patients with early tongue carcinoma, patients should be informed of the possible choice of both treatment options of either observation or elective neck dissection of N0 neck. Observation is particularly suitable for patients with thin carcinomas of less than 3–4 mm. The risk of nodal recurrence of patients with thin tumors of 3–4 mm is in the range of

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10–15%. Patients choosing observation treatment of N0 neck should be advised to have regular follow-up after primary treatment for early detection of nodal recurrence. Early nodal recurrence can be salvaged with modified or radical neck dissection successfully. Of those patients who cannot be closely followed up, elective neck dissection is a more suitable treatment of choice. Outcome of Treatment Local or regional lymph node recurrences account for over 90% of recurrences. Majority of local recurrences cannot be salvaged. Over 90% nodal recurrences of closely observed neck can be successfully salvaged with neck dissection. Of those patients with elective selective neck dissection of cN0 neck, the nodal recurrence rate of pN0 neck is less than 5% and is 30–40% for pN+ neck. Radiotherapy of pN+ neck is therefore advised. The overall 5-year disease free survival rates are in order of 90–100% for stage I, 60–80% for stage II, 30–50% for stage III and less than 20% for stage IV.

DNA, processes an unbroken double-strand DNA and then reanneals the broken strand. It is a major target for antineoplastic agents, such as anthracycline antibiotics, and ▶etoposide. ▶Adriamycin ▶Topoisomerases

Topoisomerase II-DNA Cleavage Complexes Definition Transient intermediates in the catalytic cycle of DNA ▶topoisomerase II. ▶Replication Factories and Foci ▶Topoisomerases

References 1. Tsantoulis PK, Kastrinakis NG, Tourvas AD et al. (2007) Advances of the biology of oral cancer. Oral Oncol 43(6):523–34 2. Yuen APW, Wei WI, Wong YM et al. (1997) Elective neck dissection versus observation in the surgical treatment of early oral tongue carcinoma. Head Neck 19:583–588 3. Yuen APW (2004) Cancer of the tongue: Operative techniques in otorhinolaryngology. Head Neck Surg 15:234–238

Tonsils and Adenoids

Topoisomerase III Definition Topoisomerase III is an enzyme that changes the degree of supercoiling of DNA by cutting one strand of DNA and passing an intact strand through the break. ▶Topoisomerases ▶Poly(ADP-Ribosyl)ation

Definition Prominent oval masses of lymphoid tissues on either side of the throat.

Topoisomerases Topoisomerase II Definition Is a nuclear isomerase enzyme that alters the topology of DNA. This enzyme transiently cleaves double-stranded

Definition Enzymes that produce a reversible covalent complex with DNA through which another DNA strand or duplex is passed. Passage of strands or duplexes changes DNA topology introducing or removing supercoils. Type I and III topoisomerases pass single

Toxic Epidermal Necrolysis

strands and change DNA supercoiling in units of one. Type II topoisomerases pass duplex DNA and change DNA supercoiling in units of two. ▶Topoisomerase II nuclear enzymes induce transient breaks in both strands of the DNA helix simultaneously. The clinically used DNA topoisomerase II inhibitor ▶etoposide is known to induce DNA double strand breaks. ▶Celastrol ▶Decatenation G2 Checkpoint ▶Genistein

TOR Definition The phosphatidylinositol (PI) kinase homologue TOR (“target of ▶rapamycin”) is the cellular target of the complex of FK506 binding protein (FKBP) with the immunosuppressant rapamycin. TOR is part of the general mitotic signaling pathway involving a tyrosine kinase, a phosphatidylinositol 3-kinase (PI3K), Akt/ PKB kinase, p70S6 kinase (p70S6k) and 4E-BP1 (PHAS-I). TOR is conserved from yeast to mammals and is also known as FRAP, RAFT, RAPT ▶mTOR refers to mamalian TOR. ▶Autophagy

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limiting or degenerate into ventricular fibrillation and result in death. Drugs that prolong action potential duration (as a result of a reduction in repolarizing current), induce early after depolarizations, which can generate ectopic beats and increase the spatial dispersion of repolarization, which in turn increases the possibility for reentry, have the potential to cause this tachyarrythmia. ▶Lead Optimization

Totipotent Definition Able to give rise to all cell types. In mammals, only the fertilized egg and early cleavage stage blastomeres are truly totipotent. Cells of the inner cell mass and ▶embryonal stem (ES) cells are unable to differentiate into cells of the trophectoderm lineage. ▶Adult Stem Cells

Totipotent Stem Cells Definition

Torisel Definition An inherited condition characterized by benign tissue masses that form on the skin, hair follicles, and gums during infancy, and in the breasts following puberty, and are associated with a higher risk for developing cancers. ▶Temsirolimus

Are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also ▶totipotent. These cells can differentiate into embryonic and extraembryonic cell types. ▶Adult Stem Cells

T Toxic Epidermal Necrolysis Definition

Definition

A life-threatening, exfoliative mucocutaneous disease in which much of the skin becomes intensely red, blisters, and peels off in the manner of a second-degree burn. It is thought to be an idiosyncratic reaction to a drug or other chemical agent.

A peculiar ventricular tachyarrythmia, with characteristic ECG (lengthened QT interval), that can be self

▶Rituximab

Torsade de Pointes

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Toxicity

Toxicity Definition Refers to unwanted biological activity. ▶ADMET Screen

Toxicity Testing ▶Preclinical Testing

Toxicokinetics Definition Refers to study of the fate of administered drug and metabolites in the animals used in toxicity studies. The term usually refers to those studies of drug disposition that form part of toxicity study rather than specialist studies of adsorption, distribution, metabolism and excretion conducted as separate studies. ▶Preclinical Testing

Toxicological Carcinogenesis TAKUJI TANAKA Deaprtment of Oncologic Pathology, Kanazawa Medical University, Kanazawa, Japan

Synonyms Chemical carcinogenesis; Experimental carcinogenesis

Definition

In the broadest possible sense, ▶carcinogenesis is a process of generation of benign and malignant neoplasm. Agents such as viruses, radiation, and chemicals are able to induce ▶cancer in humans and experimental animals. However, the importance of chemicals as a cause of cancer has long been recognized in basic and clinical studies, and is emphasized by the epidemic of ▶tobacco-related lung cancer in the twentieth century. Carcinogenesis may be considered

as a form of toxicity in which cells achieve a different steady state from the normal and do not respond normally to homeostatic mechanisms. Carcinogenesis induced by chemicals is called “toxicological (chemical) carcinogenesis.” Basic and clinical research in the field of toxicological carcinogenesis has led to many major advances, ranging from the fields of epidemiology and international human studies to laboratory research on mechanisms involved in the complex processes that are associated with the initiation and development of malignant disease (cancer). Many chemical carcinogens have been identified, and their effects documented in experiments in which animals exposed to the agents at the maximum tolerated dose develop neoplasm. Toxicological carcinogenesis and ▶human cancer epidemiology studies have clearly identified specific chemicals that can act as human carcinogens in both occupational and environmental settings. The main groups of relevance to human disease include ▶polycyclic aromatic hydrocarbons, aromatic amines, nitrosamines, ▶alkylating agents, and heterocyclic amines. Cancer resulting from exposure to chemicals in the environment has taken on new importance. Knowledge about the mechanisms and natural history of cancer development from toxicological carcinogenesis as well as epidemiology of human cancer is critical in the control and prevention of human neoplastic disease.

Characteristics Mutagens are agents that can permanently alter the genetic constitution of a cell. The most widely used screening test, the Ames test, uses the appearance of mutants in a culture of bacteria of the Salmonella species. Approximately 90% of known carcinogens are mutagenic in this system. Moreover, most, but not all, mutagens are carcinogenic. This close correlation between carcinogenicity and mutagenicity presumably occurs because both reflect ▶DNA damage. The in vitro mutagenicity assay is a valuable tool in screening for the carcinogenic potential of chemicals. Cultured human cells are also being increasingly used for assays of mutagenicity. Chemical carcinogens may cause development of neoplasm either directly or indirectly. They can be grouped into two main classes according to the mechanism by which they stimulate development of neoplasm: (i) ▶Genotoxic carcinogen causes direct damage to DNA by forming chemical:▶DNA adducts. The abnormal areas of DNA are prone to damage in replication and some adducts are resistant to normal ▶DNA repair mechanisms. (ii) ▶Non-genotoxic carcinogen is a carcinogen for which there is no evidence of direct interaction with cellular DNA. This type of carcinogen can be divided into two subgroups. ▶Mitogenic carcinogen binds to receptors on or in cells and stimulates cell division without causing

Toxicological Carcinogenesis

direct DNA damage. In experimental skin carcinogenesis such agents have been shown to bind to and activate ▶protein kinase C, causing sustained epidermal hyperplasia. ▶Cytotoxic carcinogen produces tissue damage and leads to hyperplasia with cycles of tissue regeneration and damage. In some cases it is believed that cytokines generated in response to tissue damage act as mitogenic factors. Chemical carcinogens can be further divided into two groups: (i) ▶Direct-acting carcinogen: the agent is capable of directly causing neoplasia. (ii) ▶Procarcinogen: the agent requires conversion to an active carcinogen. This conversion takes place through normal metabolic pathways. In procarcinogens the ▶cytochrome P450 (CYP) monooxygenase system plays an important role in conversion in many instances. ▶Detoxification reactions also occur, the accumulation of carcinogen being determined by a balance between: (i) dose of procarcinogen; rate of detoxification and elimination; and (ii) rate of conversion to the active form. Three stages have been defined in toxicological carcinogenesis (Fig. 1). Studies of toxicological carcinogenesis among experimental animals have shed light on the individual stages in the progression of normal cells to cancer. From these studies, one can

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define three stages (▶multistep development) of toxicological carcinogenesis: 1. Initiation is the first stage and likely represents mutations in a single cell. The nature of the initial changes in cells is still uncertain. In experimental toxicological carcinogenesis in skin, the Harvey ▶ras gene has been identified as being frequently mutated. This gene is involved in epidermal proliferation and when it becomes abnormal epidermal cells are less responsive to signals that normally cause terminal differentiation. Only relatively few genes have been identified as being mutated in other animal models of toxicological carcinogenesis. 2. Promotion follows initiation and is characterized by clonal expansion of the initiated cell. Induction of cell proliferation takes place at this stage. The altered cells do not exhibit autonomous growth, but remain dependent on the continued presence of the promoting stimulus, including an exogenous chemical or physical agent or an endogenous mechanism, such as hormonal stimulation. In this phase of carcinogenesis a promoting agent brings about increased cell proliferation. Promotion is initially reversible if the promoting agent is withdrawn.

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Toxicological Carcinogenesis. Figure 1 Toxicological carcinogenesis as a multi-step process.

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Toxicological Pathologist

3. Progression is the third stage, in which growth becomes autonomous and is independent of the carcinogen or promoter. At this stage, additional genomic changes presumably endow cells with a relative growth advantage that, in turn, results in their further clonal expansion. Cancer is the end result of the entire sequence and is established when the cells acquire the capacity to invade and metastasize. If there is persistent cell proliferation, initiated cells acquire secondary genetic abnormalities in oncogenes, which first lead to dysregulation and eventually to autonomous cell growth. The ultimate end-point of progression is development of an invasive neoplasm. The various tests that have been applied to identifying agents with carcinogenic potential may be classified into several general areas on the basis of the time involved in the assay: short, medium, and long. These include short-term tests for mutagenicity (e.g., the Ames test), gene mutation assays in vivo (e.g., The LacZ mouse, the LacI mouse, the LacI rat), assay for chromosomal alterations (e.g., ▶micronucleus assay, sister chromatid exchange), measurement of primary DNA damage in vitro and in vivo, and chronic bioassays for carcinogenicity (e.g., chronic 2-year bioassay, medium-term bioassays-Ito model, multi-stage models of neoplastic development, transgenic and knockout mice as models of carcinogenesis). History It is widely recognized that exposure to chemicals in the workplace and the environment can contribute to human cancer risk. This was first indicated in 1775 by Dr. Pott, who attributed scrotal skin cancers to prolonged exposure to soot in London chimney sweeps. In 1914, Dr. Boveri first hypothesized that cancer was a genetic disease, prior to the discovery of the genetic material. In 1915, Dr. Yamagiwa and co-workers successfully induced skin cancer in rabbits by painting their ears continuously with benzene solutions of tar. In the 1930s Dr. Kenneway and co-workers demonstrated that pure chemicals isolated from coal tar could also produce tumors in animals. In the 1950s there were parallel discoveries of the structure of the DNA double helix and its establishment as the hereditary material and mutagenic potential of ionizing radiation and certain chemical carcinogens in humans and experimental systems, and extensive investigations into the relationship between chemically induced mutations and human cancer. The 1980s saw the elucidation of the first oncogenes that appeared to be responsible for the initiation of cancer as first predicted by Dr. Boveri. This era also saw the development of the Ames Salmonella bacterial mutagenesis assay (the Ames test) and similar genetic toxicology assays. These

developments firmly established the basic paradigm for the field of toxicological carcinogenesis: chemicals capable of induction of mutations are presumed to be carcinogens. It was predicted that any chemical or physical agent that can covalently damage DNA could also cause mutations through its DNA-damaging mechanism, and hence can be a carcinogen. The data that followed in the 1990s appeared to strongly support this central assumption, as numerous chemicals that were initially tested for DNA damage or mutations were also carcinogens in experimental animals. Since then, our understanding of the molecular basis of cancer has improved substantially. In addition, investigations into the molecular basis of toxicological carcinogenesis, as well as more extensive human cancer epidemiology studies using modem molecular tools, have greatly expanded our knowledge in this area.

References 1. Clayson DB (2001) Toxicological Carcinogenesis. CRC Press LLC, Boca Raton, FL 2. Tanaka T (1997) Effect of diet on human carcinogenesis. Crit Rev Oncol Hematol 25:73–95 3. Sugimura T, Ushijima T (2000) Genetic and epigenetic alterations in carcinogenesis. Mutat Res 462:235–246 4. Williams GM, Iatropoulos MJ, Weisburger JH (1996) Chemical carcinogen mechanisms of action and implications for testing methodology. Exp Toxicol Pathol 48:101–111 5. Ito N, Tamano S, Shirai T (2003) A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 94:3–8

Toxicological Pathologist Definition Is usually a veterinary or medical graduate with experience in the pathological changes that can be induced by chemicals agents including drugs. ▶Preclinical Testing

Toxicology Definition Study of the nature, effects, and detection of poisons in living organisms. The basic assumption of toxicology is

TP53

that there is a relationship among the dose (amount), the concentration at the affected site, and the resulting effects. ▶Biomonitoring

TP ▶Thymidine Phosphorylase

TP53

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The main function of the p53 protein is to act as an “emergency brake” to prevent the proliferation of cells with damaged genetic material, caused by exposure to genotoxic agents (Fig. 1). In a broader context, the protein acts as an integrator of multiple exogenous and intracellular signals to regulate cell proliferation during replicative ▶senescence, differentiation and development. Inactivation of TP53 in mice resulted in accelerated development of multiple tumors, while a fraction of p53-deficient embryos displayed a lethal defect in neural tubule closure, resulting in exencephaly.

Characteristics The TP53 gene spans 20 kb and contains 11 exons, the first one being non-coding. The coding sequence contains five regions showing a high degree of conservation in vertebrates, located in 2, 5, 6, 7 and 8. An ▶orthologue has recently been described in

P IERRE H AINAUT International Agency for Research on Cancer (IARC), Lyon, France

Synonyms p53

Definition

The TP53 ▶tumor suppressor gene is located on chromosome 17p13.1 and encodes an ubiquitous phosphoprotein of molecular mass 51–53,000, essentially expressed in the nucleus. This gene is frequently inactivated by somatic mutation or by loss of alleles in many common human cancers. More than 25,000 such mutations have been described so far. Inherited, heterozygous mutations have been identified in about 400 families with ▶Li-Fraumeni Syndrome and Li-Fraumeni-like syndromes (LFS and LFL), characterized by the early occurrence of cancers at multiple organ sites. TP53 belongs to a ▶p53 family that also includes TP73 (1p36) and P63 (3p28). In contrast with TP53, these two genes have a restricted, tissue specific and developmental expression pattern and are not frequently mutated in cancer. The p53 protein is a latent ▶transcription factor that is activated in response to multiple forms of physical and chemical stress to exert diverse, complementary effects in the regulation of cell proliferation, genetic integrity and survival. These effects include: . Induction of ▶apoptosis, . Control of cell ▶division through regulation of ▶cell-cycle progression in G1 and G2, ▶centrosome duplication and mitosis, and . Modulation of DNA replication and ▶repair of DNA.

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TP53. Figure 1 The p53 pathway. The p53 protein is induced in response to various forms of stress and mediates a set of coordianted, anti-proliferative responses including cell-cycle arrest, control of replication, transcription, repair and apoptosis. Blue: factors that bind to p53 and that are regulated by protein interactions. Red: factors that are regulated by p53 at the transcriptional level.

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Drosophila. Several ▶gene polymorphisms are identified in the human population, with ▶allele frequencies that vary with ethnic origin. However, there is only limited evidence to prove that these polymorphisms play a role in tumor susceptibility. The TP53 gene does not contain a conventional TATA box, but is under the control of several ubiquitous transcription factors, including ▶NFkB, Sp1 and Jun. It is expressed in the form of one major transcript of 2.8 kb and several isoforms generated by alternative splicing or use of an alternative promoter, intron 4. The protein contains 393 residues and is organized in a hydrophobic, central core (residues 110–296, encoded by exons 5 to 8), flanked by an acidic N-terminus and a basic C-terminus (Fig. 2 top). The N-terminus contains

two complementary transcriptional activation domains, with a major one at residues 1–42 and a minor one at residues 55–75, specifically involved in the regulation of several pro-apoptotic genes. The central core is made of a scaffold of two β-sheets supporting a set of flexible loops and helices stabilized by the binding of an atom of zinc. These loops and helices make direct contact with DNA sequences containing inverted repeats of the motif RRRC(A/T). The C-terminus contains the main nuclear localization signals and oligomerization domains (residues 325–366). The active form of the protein is a tetramer (in fact, a pair of dimers). The extreme C-terminus has multiple regulatory functions and exerts a negative control on sequence-specific DNA binding activities. Both N- and C-terminal regions contain

TP53. Figure 2 Diagram of the p53 protein structure. c: linear structure, showing the three main structural domains. Codon numbers of the main mutation hotspots are shown as coloured boxes. Sites of post-translational modifications are shown as “P” (phosphorylations), “A” (acetylations) and “Z” (zinc binding sites). bottom: 3-D structure of the central core of p53 in complex with target DNA. Hotspot residues are shown in the same color code as above.

TP53

multiple post-translational modification sites, while only a few have been identified so far in the central core (see Table 1). Upstream of p53: Signaling of DNA Damage The p53 protein is constitutively expressed in most cells and tissues as a latent factor. Due to its rapid turnover (5–20 min), the protein does not accumulate unless it is stabilized in response to a variety of intracellular and extra cellular stimuli. Signals that activate p53 include diverse types of ▶DNA damage (strand breaks, bulky adducts, oxidation of bases), blockage of RNA elongation, ▶hypoxia, depletion of ▶microtubules, ribonucleotides or growth factors, modulation of cell ▶adhesion and alteration of polyamine metabolism. Most of the current knowledge of p53 protein activation is derived from studies using DNA strand breaks as inducing signals.

TP53. Table 1

The main regulator of p53 protein activity is ▶mdm-2, a protein which binds p53 in the N-terminus (residues 17–29); it conceals its transcription activation domain, redirects p53 from the nucleus to the cyclasm and acts as a ▶ubiquitin ligase to target p53 for degradation by the ▶proteasome. The MDM-2 gene is a transcriptional target of p53, thus defining a regulatory feedback loop in which p53 controls its own stability. The p53/mdm-2 complex is regulated by Arf (Alternative Reading Frame), a 14 kD protein encoded by the p16/CDKN2A gene. The kinetics, extent and consequences of p53 activation vary according to the nature and intensity of the inducing signals. In response to ionizing radiation, activation of the p53 protein is thought to proceed through several consecutive steps, with first phosphorylation of p53 in the N-terminus by kinases involved in the sensing of DNA damage such as Atm

Factors involved in the activation and post-translational modification of p53

Factor

Biochemical function/activated by p53

PARP

ADP-ribose polymerase/DNA strand breaks, nucleotide depletion High mobility group 1/? E6 accessory protein/ubiquitin-mediated degradation Hypoxia-inducible factor/Hypoxia Cell-cycle regulator/ionizing radiations Histone acetyl-transferases/co-activators of transcription Tyrosine kinase/irradiation, DNA-strand breaks oncogene/negative control of p53 Nitric oxide/oxidative stress, inflammation, irradiation Cell-cycle dependent kinases

HMG-1 E6AP Hif-1 14-3-3 s p300/CBP c-abl mdm-2 NO Cdc2/Cdk2Cyclin A/B cdk7-cyclin H CKII

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Component of TFIIH Kinase/UV

Interaction with p53 ADP-ribose polymers bind to p53 Binding to N-terminus or to DNA-binding domain Binding to p53 Binding to p53 Binding, C-terminus (Ser-376) Binding, N-terminus acetylation, C-terminus Binding, proline-rich region Binding, residues 13–29 Oxidation of cysteines in DNA-binding domain Phosphorylation of Ser-315; forms complexes with p53 Phosphorylation of Ser-33 Phosphorylation of Ser-389; forms complexes with p53 Phosphorylation, Thr-73 and 83 (mouse p53) Phosphorylation, Ser-15 Phosphorylation, Ser-15 and Ser-37 Phosphorylation, Ser-20 Phosphorylation, Ser-34, mouse p53 Phosphorylation, Ser-378 Phosphorylation, several N-terminal serines (including Ser-6 and Ser-9) Prevents p53-mdm2 interactions

MAPK ATM DNA-PK Chk-2 JNK/p38 PKC CKI

Mitogen-activated protein kinase/UV? Kinase/ionizing radiations Kinase/UV Cell-cycle-dependent kinase Stress-activated kinases/UV Protein kinase C Kinase/?

p19arf

Cell-cycle inhibitor, alternative product of CDKN2A Redox-repair enzyme/oxidative stress, hypoxia Reduction of cysteine in DNA-binding region binding to C-terminus

Ref-1

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TP53

(the product of the ▶ataxia telangiectasia mutated gene) and Chk-2 (a cell-cycle regulatory kinase). These phosphorylations contribute to the dissociation of the p53/mdm-2 complex and stabilize the protein. Second, p53 binds co-activators with acetyl-transferase activity such as ▶p300/CBP co-activators and pCAF. These factors acetylate p53 in the C-terminus. These processes, as well as other coordinated post-translational modifications of the C-terminus, induce conformational changes that turn the protein into an active form with a high affinity for specific DNA binding sites. The third step involves redox regulation of sensitive cysteines within the DNA-binding domain of the protein. This three-step mechanism may account for p53 induction in response to most forms of DNA damage. Downstream of p53: Cell-Cycle Control, Apoptosis and DNA Repair Once activated, p53 exerts its effects through two major mechanisms: transcriptional control (activation

TP53. Table 2

or repression of specific genes) and complex formation with other proteins. Important downstream effectors of p53 (Table 2) include regulators of ▶cell-cycle ▶checkpoints (in G1/S, G2 and during mitosis), factors involved in the signaling of apoptosis, and components of the transcription, replication and repair machineries. At the cellular level, activation of p53 most frequently results in either cell cycle arrest (mostly in G1 and/or G2/M) or apoptosis. How a given cell “chooses” between cell cycle arrest and apoptosis in response to specific stimuli may depend upon many factors, such as the nature and intensity of the stress, as well as the cell type. In many tissues, p53 plays a role in drug-induced apoptosis and is thus an important effector in the response of cancer cells to chemo- or radio-therapy. In addition, loss of p53 function results in deficient cell cycle arrest, inefficient ▶mitotic spindle checkpoint, aberrant centrosome duplication, premature re-entry into S phase, ▶genomic instability and ▶aneuploidy.

Some important downstream effectors of p53 functions

Factor Apo-1/Fas/CD95 Bax-1 Bcl-2 IGF-BP3 Killer/DR5 P85 Pig-12 Pig-3 Pig-6 IGF-I IL-6 thrombospondin-1 Gadd45 BTG2 p21waf-1 cyclin A cyclin G GPx NOS2/iNOS COX2 Pig-1 PCNA RPA ERCC2/ERCC3 P53RR2 TBP Mdm-2 MDR-1

Activity

Mode of regulation

Function

Death signaling receptor Dominant-negative inhibitor of bcl2 Repressor of apoptosis Inhibitor of IGF-I Death signaling receptor Regulatory subunit of PI3 kinase Glutathione transferase homologue Quinone oxidase homologue Proline oxidase homologue Growth factor Survival factor Inhibitor of angiogenesis Binding to PCNA Inhibitor of proliferation Inhibitor of CDK2–4 and 6 Cell-cycle regulation, S phase Cell-cycle regulation Glutathione peroxidase Inducible Nitric Oxide synthase Inducible cyclooxygenase Galectin-7 Auxiliary subunit of polymerase δ Replication protein A Helicases, TFIIH complex Ribonucleotide reductase homolog TATA box-binding protein Oncogene Multi-drug resistance

Transcriptional activation? Transcriptional activation Transcriptional repression Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional repression Transcriptional repression Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional activation Transcriptional repression Transcriptional activation Transcriptional repression Transcriptional repression Transcriptional repression Transcriptional activation Transcriptional activation Inhibition by protein binding Activation by protein binding Transcriptional activation Inhibition by protein binding Transcriptional activation Transcriptional repression

Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis Apoptosis? Apoptosis? Apoptosis? Cell-cycle arrest ? Cell-cycle arrest, G1 cell-cycle arrest, G1 and G2/M Cell-cycle arrest, G1/S cell-cycle arrest? Control of oxidative stress Control of oxidative stress Control of oxidative stress? Differentiation? DNA repair/replication DNA repair/replication DNA repair/transcription DNA repair? Inhibition of transcription Repression of p53 Resistance to chemotherapy

TP53

Clinical Relevance The TP53 gene is often inactivated by ▶missense mutations, in contrast with many other tumor suppressors such as ▶APC, ▶RB1, ▶BRCA1 or p16/ ▶CDKN2A that are inactivated by gene deletion or truncation. The mutations described to date mostly occur in the region of the gene encoding the DNA binding domain. Most of these mutations impair DNA binding by disrupting the structure of the domain or crucial contact points between the protein and target DNA. About 30% of missense mutations affect six “hotspot” codons (175, 245, 248, 249, 273 and 282) (Fig. 2 bottom). The other mutations are scattered over 300 different codons. Mutations are very common in the invasive stages of many epithelial tumors. A database of all published mutations is available at the International Agency for Research on Cancer (▶IARC TP53 database, http://www-p53.iarc.fr/).

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In many cancers, the patterns of mutations show variations, revealing clues about the mechanisms responsible for the formation of the mutations. Specific carcinogen-induced mutations have been identified in ▶hepatocellular carcinoma (mutations induced by ▶aflatoxins in sub-Saharan Africa and in south-east Asia), in skin tumors (double transitions at adjacent cytosines, a typical signature of mutagenesis by UV in squamous and in basal cell carcinomas) and in lung cancers (G to T transversions associated with exposure to tobacco smoke; ▶tobacco carcinogenesis; (Fig. 3). The usefulness of TP53 mutation detection in molecular pathology is still a matter of debate. As mutation often results in the accumulation of the protein, ▶immunohistochemistry (IHC) has often been used as a criterion to detect TP53 abnormalities. However, positive IHC does not always correlate with mutation as several common missense mutants, as well as most

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TP53. Figure 3 Prevalence of two common mutation types: G to T transversions and C to T transitions at dipyrimidine (CpG) repeats in tumors of various organs. Tumors with high prevalence of G to T transversions often have a low prevalence of transitions and vice-versa. G to T transitions are a common molecular signature of many environmental carcinogens, such as tobacco smoke components (lung and esophageal cancers) or dietary mycotoxins (liver cancer).

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TP73L

frameshift and nonsense mutants, do not result in protein accumulation. Several well-established methods have been described for the detection of mutations in the TP53 gene, including SSCP (Single Stranded Conformation Polymorphism) analysis, TTGE (Temporal Temperature Gradient Electrophoresis), yeast-based functional assays and, recently, micro-array hybridization assays. TP53 gene mutations are good markers for the clonality of tumor lesions. In many tissues, mutation correlates with bad prognosis and poor response to therapy, but TP53 mutation has been shown to behave as an independent marker of prognosis only in rare cases such as breast and head and neck cancers. Recent evidence suggests that the nature and position of the mutation may help to predict poor response to treatment. Detection of circulating anti-p53 antibodies as well as of free plasmatic DNA containing mutant TP53 may be of interest in the early detection of cancer lesions. TP53 is the target of several experimental therapeutic approaches. Gene transfer of wild type TP53 into cancer cells has been tested in several human tumors. However, the effects reported to date are limited and, at best, transient. Another approach is based on the use of cytolytic viruses selectively replicating in TP53deficient cells (▶ONYX vectors). Several pre-clinical studies have investigated the use of small lipophilic coumpounds or peptides to activate TP53 function or to restore the activity of mutant proteins. ▶p53 ▶p53 Family

tPA Definition

▶Tissue-Type Plasminogen Activator ▶Proteinase-Activated Receptors

TPA Definition 12-O-tetradecanoyl-phorbol-13-acetate. A double ester of phorbol found in croton oil, and commonly known as ▶phorbol 12-myristate 13-acetate (PMA). It is a potent tumor promoter often employed in biomedical research to activate the signal transduction enzyme ▶protein kinase C. Also can activate JNK and AP-1. ▶JNK Subfamily

TR-FRET ▶Time-Resolved Fluorescence Resonance Energy Transfer Technology in Drug Discovery

References 1. Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310 2. Levine AJ, Hu W, Feng Z (2006) P53 pathway: what questions remain to be explored. Cell Death Differ 13:1027–1036 3. Olivier M, Hussain SP, Caron de Fromentel C et al. (2004) TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci Publ 157:247–270 4. Lowe SW, Bodis S, McClatchey A et al. (1994) p53 status and the efficacy of cancer therapy in vivo. Science 266:807–810 5. Foster BA, Coffey HA, Morin MJ et al. (1999) Pharmacological rescue of mutant p53 conformation and function. Science 286:2507–2510

TP73L ▶p53 Family

TRA-8 ▶TRAIL Receptor Antibodies

Trabectedin F EDERICO G AGO 1 , S ERGIO M ORENO 2 1

Departamento de Farmacología, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain 2 Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain

Synonyms Yondelis; Ecteinascidin 743; ET-743; NSC 684766

Trabectedin

Definition A potent antitumor tetrahydroisoquinoline alkaloid in clinical development originally derived from a marine tunicate and now obtained by a synthetic process developed by PharmaMar starting from microbially produced cyanosafracin B.

Characteristics Crude aqueous ethanol extracts of the ascidian, or sea squirt, Ecteinascidia turbinata were shown to have powerful immunomodulating and antiproliferative properties as early as 1969 but the active principles were not identified until the early nineties. The first six alkaloids that were characterized received the names ecteinascidins 729, 743 trabectedin, (Fig. 1), 745, 759A, 759B, and 770, in accordance with the molecular masses ascribed to these compounds. They revealed a unique chemical structure consisting of a novel pentacyclic skeleton composed of two fused tetrahydroisoquinoline rings (subunits A and B) linked to a 10-member lactone bridge through a benzylic sulfide linkage and attached through a spiro ring to an additional ring system (subunit C) made up of either tetrahydroisoquinoline (as in trabectedin) or tetrahydro β-carboline (as in ET-736). The first two subunits bear a clear structural resemblance to microbially derived safracins and saframycins, and also to sponge-derived renieramycins, all of them less potent anticancer agents than ecteinascidins. On the other hand, a reactive α-carbinolamine or hemiaminal (N-C-OH) group is also present in naphthyridinomycins, quinocarcins, and pyrrolo[1,4]benzodiazepine antibiotics such as ▶anthramycin, sibiromycin and tomaymycin. By analogy to these related antibiotics, the potent biological activity of trabectedin and other ecteinascidins was rapidly associated with their ability to form covalent adducts to DNA following in situ dehydration of the carbinolamine group to an iminium intermediate that is covalently attached to the amino group of guanine in the minor groove.

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Ecteinascidia turbinata was first harvested from the wild and then successfully grown by the Spanish pharmaceutical company, PharmaMar, in aquaculture facilities in Spain (near Formentera island, in the Mediterranean sea). Subsequently, several synthetic schemes were developed to produce the multigram quantities required for clinical studies worldwide and overcome the limitation of the very low yield (0.0001%) of trabectedin in its natural source. The first enantioselective total synthesis of trabectedin was achieved in 1996 but industrial manufacturing was made possible through a synthetic route involving the conversion of cyanosafracin B, readily available by fermentation of the bacterium Pseudomonas fluorescens, to trabectedin in a very short and straightforward way developed by PharmaMar. Structural and Biophysical Characterization of Trabectedin-DNA Adducts Direct evidence that trabectedin alkylates duplex DNA at the exocyclic amino group of guanines was provided by a variety of experiments including gel electrophoresis, DNA footprinting, nuclear magnetic resonance (NMR) spectroscopy, and band shift assays, as well as molecular modeling studies. As a result of this work, it was found that trabectedin was protonated on N12 at physiological pH and a role for hydrogen bonding in sequence recognition and orientation in the DNA minor groove was demonstrated, with TGG, CGG, AGC, GGC, and AGA being established as the preferred DNA triplets for stable adduct formation, and much higher rates of reversibility being measured for site-directed AGT- versus AGC-containing adducts. The proposed mechanism for activation takes advantage of the increased strength of the hydrogen bond between the proton on N12 and the hydroxyl group on C21 as the trabectedin molecule approaches the minor groove and is desolvated. This proton, which is essential for both sequence recognition and adduct

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Trabectedin. Figure 1 Chemical formula and three-dimensional stick representation of the X-ray crystal structure of trabectedin.

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Trabectedin

stabilization, would then catalyze the dehydration of the carbinolamine yielding the reactive iminium intermediate that undergoes nucleophilic attack at C21 by the exocyclic amino group of the guanine. Since a similar mechanism operates in the activation of pyrrolo[1,4] benzodiazepine antibiotics, it appears that Nature ensures the reactivity of these carbinolamine-containing molecules by the inclusion of an internal catalytic proton adjacent to the leaving hydroxyl group. As a consequence of trabectedin bonding, the double helical structure is only minimally perturbed except for widening of the minor groove and a net smooth bending towards the major groove due to the introduction of positive roll. This latter feature was novel among minor groove DNA monoalkylating agents as covalent modification of N3 of adenine in AT-rich regions by (+)-CC-1065 and related compounds is accompanied by bending of the DNA into the minor rather than the major groove. Furthermore, if multiple binding sites for trabectedin are properly phased in a relatively short stretch of DNA, the imposed cumulative curvature could bring closer together specified fragments not contiguous in primary sequence, and the drug could be serving a surrogate protein function. On the contrary, binding of three trabectedin molecules in a head-to-tail fashion to three adjacent optimal binding sites would result in no net DNA curvature because the localized bends, brought about by the increase in roll at the sites of covalent attachment, would cancel out over virtually one turn of the helix. In fact, the DNA structure in one such complex (containing the sequence TGGCGGCGG) was shown to be intermediate between the canonical A and B forms of DNA, thereby strongly resembling the conformation that DNA adopts when bound to the consecutive C2H2 zinc fingers that are present in transcription factors (▶Transcription factor) such as EGR-1 and Sp-1 (which bind to the major groove of GC-rich regulatory sequences in many gene promoters) or that observed in the hybrid double helix of template DNA paired to nascent RNA in the active site of ▶RNA polymerase II (RNAPII) elongation complex. The close contacts and the hydrogen-bonding interaction network that are established between trabectedin and DNA on both sides of the covalent adduct involve both DNA strands and therefore give rise to a significant increment in the stability of the resulting drug-DNA complexes. As a consequence, notable increases in the temperature of thermal denaturation of duplex DNA and substantial blockade of the helicase activities of both simian virus (SV40) large tumor antigen (T-antigen) and bacterial UvrABC and RecBCD enzymes have been reported for DNA oligonucleotides containing trabectedin adducts. This hampering or prevention of strand separation is also expected to result in stalled replication and transcription forks, as observed for a variety of conventional interstrand crosslinkers

(e.g. nitrogen mustards, mitomycin or cisplatin). An added advantage in the case of trabectedin would be the minimal distortions inflicted on the normal DNA structure that could help evade some of the recognition and repair mechanisms used for the processing of crosslinks produced in the major groove by these other agents. Biological Activity In vitro cytotoxicity studies with trabectedin and other ecteinascidins established subnanomolar potencies against L1210 and P388 mouse leukemia cells, as well as human A549 lung cancer, HT29 colon cancer, MEL28 melanoma cells and human tumors explanted from patients. Tumor-specific responses and concentrationdependent relationships were observed when a soft agar cloning assay was used to determine the effects of a continuous exposure of trabectedin at different concentrations. These experiments clearly indicated that the duration of exposure to trabectedin was an important factor in human tumors thereby pointing to preferential administration schedules in clinical trials. In vivo activity was then evaluated in several mouse tumor models and a variety of human tumors xenografted into nude mice, including melanoma, non-smallcell lung carcinoma and ovarian cancer. Long-lasting, complete or partial regressions were observed in both chemo-sensitive and marginally cisplatin-resistant xenografts at the maximum tolerated dose (MTD) but no activity was seen in highly chemo-resistant tumors such as MNB-PTX-1, MEXF 514 and LXFA 629. Importantly, the absence or incomplete cross-resistance with cisplatin and the comparable efficacy against the ovarian carcinoma xenografts justified the clinical assessment of trabectedin in ovarian cancer. The activity parameters for trabectedin in the panel of 60 human tumor cell lines of the National Cancer Institute (NCI) Anticancer Drug Screen revealed a rather unique profile that encouraged further development as an anticancer agent. The COMPARE algorithm (▶COMPARE analysis) established a very high correlation coefficient (0.96) with ▶chromomycin A3, an aureolic acid derivative shown to give rise to a pattern of distinct bands in human metaphase chromosomes, thus suggesting similarities in their apoptotic mechanisms. Despite the fact that these two compounds display very different modes of binding to DNA (i.e. covalent versus noncovalent, carbinolamine activation versus ionmediated dimerization, etc.), both share a strong binding affinity for some common DNA sites, such as the selfcomplementary hexanucleotide TGGCCA, to which two trabectedin molecules can bind in a tail-to-tail fashion, each covalently bonded to a different strand. Furthermore, these two natural products are known to exert at least part of their cytotoxicity by interfering with DNA replication and transcription. Thus, at physiologically relevant concentrations (1–100 nM), trabectedin has been

Trabectedin

shown to effectively inhibit intracellular DNA synthesis by decreasing replication origin activity and by inducing unusual replication intermediates that may be blocked in fork progression. In addition, trabectedin is able to abrogate the transcriptional activation of a number of genes, including those encoding the multidrug resistance P-glycoprotein (MDR1), heat shock protein 70 (hsp70), the cell cycle inhibitor p21 Cip1 (p21), and collagen α1(I) (COL1A1). Nevertheless, global gene expression profiling of trabectedin-treated cancer cells has revealed rather complex patterns of both up- and down-regulation. The reported effects on MDR1 and additional in vitro data showing enhancement by trabectedin of the cytotoxicity exerted by other chemotherapeutic agents that are substrates for P-gp/MDR1 suggest that combination treatment may be valuable in the clinic. The extremely low concentrations of trabectedin that are necessary to cause cell cycle arrest and cell death are suggestive of a trans-acting mechanism that probably operates through one or more cellular DNA damage response pathways or checkpoints. In this respect, it is notable that cell sensitivity to trabectedin appears to be somehow dependent on a proficient transcriptioncoupled ▶nucleotide excision repair (TC-NER) machinery, and more specifically on the presence of selected components that are implicated in ▶xeroderma pigmentosum and Cockayne syndromes. Thus, the initial observation that hamster cells deficient in XPB, ERCC1 or CSB, as well as human XPA and XPC cells, had reduced sensitivity to trabectedin was followed by the report that a human colon carcinoma cell line selected for increased (20-fold) resistance to trabectedin (following continuous exposure to increasing concentrations of this drug for 1 year) had a truncated and inactive form of the XPG structure-specific endonuclease. Furthermore, drug sensitivity was restored in all cases upon complementation with the respective wild-type protein. These intriguing effects were recapitulated and expanded using yeast as a simpler eukaryotic model system. It was seen that trabectedin activates the G2-M and S phase DNA damage checkpoints, in good agreement with the G2/M block and S phase delay reported in human cells. Likewise, cells deficient in the XPG orthologue (rad13 in Schizosaccharomyces pombe) were shown to be much more resistant to trabectedin and underwent much less DNA damage than the corresponding isogenic wild-type strains. However, it became clear that it was not the missing endonuclease activity of this protein that conferred resistance to trabectedin but the lack of part of its DNAbinding domain in the COOH-terminal region. Furthermore, on the basis of a homology model suggesting that the rad13:DNA:trabectedin ternary complex could be stabilized through the direct interaction of subunit C of the drug with a highly conserved arginine residue, an

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S. pombe strain carrying an Arg961→Ala point mutation in rad13 was generated. This mutant displayed normal endonuclease and NER activity but was found to be strongly resistant to the drug. Haploid yeast mutants with deletions in the RAD52 epistasis group of genes encoding proteins responsible for ▶homologous recombination (HR) and hence most ▶double-strand break (DSB) repair in eukaryotic cells (e.g. rad51, rad22 (the fission yeast counterpart of mammalian rad52), and rad54) were found to be extraordinarily sensitive to trabectedin. This result reinforced other indications that the drug is giving rise (directly or indirectly) to DSBs that need to be repaired by homologous recombination, and the fact that the absence of rad13 partially rescued rad51Δ cells supports the view that a rad13-containing complex is somehow involved in the induction or irreparability of lethal DSBs. These results may have important implications for the optimal use of trabectedin in cancer therapy because patients harboring tumor cells with proficient NER and deficient HR systems would be expected to respond best to the treatment. Clinical Studies Trabectedin was selected for clinical development in preference to other related ecteinascidins because of its outstanding potency and greater relative abundance in the tunicate. Among the criteria that were taken into account for bringing it into clinical trials in early 1996 both in Europe and in the United States, we can summarize the following: (i) a novel chemical entity harboring a potential new mode of action, (ii) evidence for a positive therapeutic index, (iii) lack of complete cross-resistance with conventional chemotherapeutic agents, and (iv) feasibility of supply for clinical development. Toxicity to trabectedin so far has been shown to follow a transient-reversible pattern and to be predictable, dose-related, and mostly limited to bone marrow and liver. Following a favorable opinion adopted by the Committee for Orphan Medicinal Products (COMP) of the European Agency for the Evaluation of Medicinal Products (EMEA), trabectedin was granted by the European Commission orphan medicinal product designation for the treatment of ▶soft tissue sarcoma (STS) in April 2001 and for ▶ovarian carcinoma (OC) in October 2003. The United States Food and Drug Administration (FDA) awarded Orphan Drug Designation to trabectedin in the indication of STS in October 2004, and in OC in April 2005. Trabectedin is currently in phase III clinical trials worldwide for ovarian cancer. Extended phase II trials and comparative studies have produced evidence of long lasting responses and tumor control in advanced pretreated sarcomas, breast carcinoma, ovarian carcinoma

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Trabecular Bone

and prostate cancer. In July 2007, Yondelis received a positive opinion from the EMEA for the treatment of metastatic or advanced soft tissue sarcoma after failure to anthracyclines and ifosfamide.

TRAF-1 Definition

References 1. Rinehart KL (2000) Antitumor compounds from tunicates. Med Res Rev 20:1–27 2. Manzanares I, Cuevas C, García-Nieto R et al. (2001) Advances in the chemistry and pharmacology of ecteinascidins, a promising new class of anticancer agents. Curr Med Chem Anticancer Agents 1:257–276 3. Gago F, Hurley LH (2002) Devising a structural basis for the potent cytotoxic effects of ecteinascidin 743 In: Demeunynck M, Bailly C, Wilson WD (eds) Small molecule DNA and RNA binders: from synthesis to nucleic acid complexes. Wiley-VCH, Weinheim, Germany, pp 643–675 4. Sainz-Diaz CI, Manzanares I, Francesch A et al. (2003) The potent anticancer compound ecteinascidin-743 (ET743) as its 2-propanol disolvate. Acta Crystallogr C 59: o197–o198 5. Herrero AB, Martín-Castellanos C, Marco E et al. (2006) Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin. Cancer Res 66:8155–8162

Trabecular Bone

▶Tumor necrosis factor-associated factor 1; One of a number of adapter molecules that are involved with tumor necrosis factor receptor superfamily signaling. ▶Hodgkin Disease, Clinical Oncology

TRAF2 Definition Tumor necrosis factor-associated factor 2.

Traffic ATPases ▶ABC-Transporters

Definition Type of bone tissue with a low density and strength but very high surface area, that fills the inner cavity of long bones (also known as cancellous, or spongy bone). ▶Lead Exposure

TRAIL Definition

▶Receptor for TNF-Related Apoptosis-Inducing Ligand ▶TNF-Related Apoptosis Inducing Ligand.

Trace Elements Definition Refers to microminerals, are mineral nutrients which are typically required to be ingested by humans in amounts of a few milligrams or less per day. This category includes iron, zinc, copper, manganese, selenium, iodine, chromium, molybdenum, and vanadium. Sometimes those mineral nutrients required in amounts of a few micrograms per day are referred to as ultratrace minerals. ▶Mineral Nutrients

TRAIL Receptor Antibodies C LAUS B ELKA Department of Radiation Oncology, University Tübingen, Tübingen, Germany

Synonyms DR4 antibodies; DR5 antibodies; Lexatumumab; Mapatumumab; TRA-8

TRAIL Receptor Antibodies

Definition

▶TRAIL induces ▶apoptosis preferentially in malignant tissues. Therefore TRAIL is considered to be a potential antineoplastic drug. Agonistic TRAIL receptor antibodies have been developed as alternative pharmacological tool for apoptosis induction via the TRAIL receptors.

Characteristics After having identified TRAIL as a member of the family of cell death inducing ligands, it became obvious that TRAIL has a strong propensity for transformed or malignant tissues. Therefore TRAIL is considered to be a candidate anticancer drug. TRAIL exerts its apoptosis inducing activity via the two respective agonistic TRAIL receptors DR4 and DR5. TRAIL itself is able to induce cell death in a wide array of cancer cells in vitro or when grown in xenograft settings. The efficacy of TRAIL is increased whenever the ligand is combined with conventional cytostatic agents or ionizing radiation. Depending on the production process, there was concern that TRAIL, like ▶CD95-L, could exert considerable hepatotoxicity. Recently released data from phase I trials suggest that TRAIL can be safely administered in patients up to serum concentrations consistent with those demonstrating efficacy in tumor xenograft models. In parallel to the development of TRAIL as anticancer drug, agonistic antibodies directed against both death inducing TRAIL receptors were developed. Up to now relevant data on three agonistic TRAIL antibodies are available. The signaling pathways triggered by agonistic antibodies have not been reported to differ from the cascades triggered by TRAIL. Treatment of susceptible cells with agonistic antibodies results in the activation of ▶caspase-8, caspase-9, and cleavage of ▶PARP. As shown for TRAIL, agonistic TRAIL antibodies also induce cleavage of the antiapoptotic MCL-1 protein. How far key regulatory molecules including ▶FADD, ▶c-FLIP, and caspase10 are involved in the regulation of cell death induction via agonistic TRAIL antibodies has not been tested in detail. However, the fact that there is a cross resistance between TRAIL and the agonistic antibodies in such a way that cells being resistant towards TRAIL cannot be killed by either antibody indicates that the signaling pathways are identical or at least highly similar. The agonistic TRA-8 antibody directed against DR5 has been developed by Sankyo together with researchers from the University of Alabama (Birmingham, USA) and was the first agonistic antibody being described. TRA-8 was generated by immunizing BALB/c mice with a fusion protein containing the extracellular domain of DR5 and the Fc proportion of human IgG1. The antibody does not cross react with murine DR5. The Kd values for TRAIL or TRA-8

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binding to DR5 were estimated at 59 and 3 nM, respectively. The high specificity of TRA-8 for DR5 was documented by competition assays showing that TRA-8 efficiently competed with TRAIL for binding to DR5 but not for binding to DR4. In addition, these results indicate that TRA-8 potentially recognizes an epitope within the TRAIL-binding site on DR5. In general, TRA-8 induces apoptosis in tumor cell systems in vitro as well as in murine xenograft model systems (Jurkat and 1321N1 astrocytoma cells). In contrast to the TRAIL preparation used for comparison, the TRA-8 antibody did not induce any signs of hepatopathy in mice. In subsequent studies, the increased efficacy of multimodal approaches combining either TRA-8 with radiation, chemotherapy or other response modifiers was documented. In this regard it is important to notice that the efficacy of the tested combinations in term of growth delay was shown in ▶xenograft models for ▶cervical cancer, ▶breast cancer and ▶pancreal cancer. The overexpression of Bax using an adenoviral vector system increased the efficacy of TRA-8 in a wide array of glioma cells suggesting that bax is involved in the efficacy of the combined treatment. The increased cell death induction translated into an increased growth delay. Besides the TRA-8 antibody, Sankyo also develops an agonistic DR4 antibody 2E12. However, considerably less data are available regarding the pharmacology and efficacy of this antibody. The second group of antibodies was developed by Cambridge Antibody Technology in conjunction with Human Genome Sciences (Rockville, USA). HGS-ETR1 (Mapatumumab) is directed against DR4 and HGS-ETR2 (Lexatumumab) is directed against DR5. HGSI also develops a third agonistic TRAIL antibody (HGS-TR2, targeting DR5) that was initially developed by Kirin Brewery Ltd. Up to now only data on Mapatumumab and Lexatumumab are available. Mapatumumab is a fully humanized monoclonal antibody and was isolated from 102 different antiTRAIL receptor mAbs that were generated by phage display technology. The antibody has a high affinity for the DR4 receptor and exerts antitumor activity (EC50 values of 3.4 nM) in diverse preclinical tumors models including breast, gastrointestinal, lymphoma, ovarian carcinoma, and uterine cancers. Mapatumumab was shown to specifically recognize the TRAIL-R1 protein without any relevant interactions with the TRAIL decoy receptors. The efficacy of mapatumumab is increased by combinations with various cytostatic drugs including carboplatin, cisplatin, camptothecin, topotecan, paclitaxel as well as radiation. Xenograft models for breast, colorectal, nonsmall cell lung cancer as well as uterine cancer revealed a high activity of either the drug alone or in combination with other cytotoxic treatment approaches including radiation.

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TRAIL Receptor Antibodies

Like mapatumumab, lexatumumab is a fully humanized IgG1g antibody. The antibody was also generated using a phage display and screening with 383 short chain fragments for DR5 binding properties. In contrast to mapatumumab, no data on specificity, selectivity, and affinity of the antibody are publicly available. Similar to TRA-8, lexatumumab exert pronounced apoptotic reactions in a wide array of malignant cell systems when used alone. Importantly the drug has proven efficacy on tumor growth in xenograft systems from renal cell carcinoma, nonsmall cell lung cancer, breast cancer, and glioma. As already shown for TRA-8, the combination of lexatumumab with various chemotherapeutic agents (camptothecin, cisplatin, carboplatin, paclitaxel, doxorubicin, bortezomib) or radiation increased the efficacy in cell lines and xenografts. The underlying mechanisms of sensitization are still not completely understood. However, it seems likely that the presence of the proapoptotic Bax molecule as well as the upregulation of the respective receptor participate in the increased efficacy of the combined approach. Clinical Aspects Mapatumumab (anti-DR5) Data from several early clinical trials are available and allow a cautious judgment regarding pharmacological and toxicological aspects of mapatumumab. An open label phase Ia/b trial was conducted in 39 patients with various advanced solid tumors. During the first phase, dose escalation of mapatumumab was performed (0.01, 0.03, 0.1, 0.3, 1.0, or 3.0 mg/kg). The second phase of the trial involved administration of mapatumumab (10 mg/kg) once every 28 days or once every 14 days. The i.v. administration of mapatumumab produced dose-proportional pharmacokinetics up to a dose of 1.0 mg/kg, with a half-life of 15 days for 1.0 mg/kg. The pharmacokinetic data indicate that distribution and clearance follow a twocompartment model, with first order elimination from the central compartment. The best clinical responses reported so far are stable diseases in a proportion of the heavily pretreated patients. No data from ongoing trials combining chemotherapy with TRAIL are available at present. In addition to various phase I trials, mapatumumab was tested in a multicenter phase II trial in patients with relapsed or refractory non-Hodgkin lymphoma. Patients (n = 40) received either (3 or 10 mg/kg mapatumumab once every 21 days). Partial responses were observed in three patients, and on one patient with relapsed follicular mixed-cell lymphoma demonstrated a more pronounced regression. Two other phase II trials with mapatumumab are ongoing. One trial was conducted in patients with relapsed or refractory colorectal cancer. No data on safety, tolerability,

pharmacokinetics, tumor response, time to response, duration of response, and progression-free survival from this trial are available. The second phase II trial including patients with solids tumor was performed in 32 patients with nonsmall cell lung cancer (median of three previous treatment cycles). These patients received 10 mg/kg mapatumumab every 21 days until disease progression. Mapatumumab was well tolerated with not treatment discontinuations due to drug-related toxicity. In 29% of these patients a stable disease (median duration of 2.3 months) was observed. The most common mapatumumab related adverse events were nausea, fatigue, hypotension, myalgia, pyrexia, peripheral sensory neuropathy, diarrhea, constipation or abdominal pain, rash, hypertension, and thrombocytopenia. A clear maximum-tolerated dose had not been achieved. No antibodies to mapatumumab had been observed. The phase II trial in NSCLC patients revealed that mapatumumab administration was generally safe and well tolerated. In 97% of these patients at least one adverse event reported, however, only 44% of the patients experienced an adverse effect that was considered to be drug related. Again, no immunogenic responses were observed.

Lexatumumab (anti-DR5) Data from several phase I trials using lexatumumab are available. Results from a US dose escalation trial (0.1, 0.3, 1.0, 3.0, and 10 mg/kg i.v. lexatumumab every 2 weeks) revealed that dose responses were linear up to the 10mg/kg level with a mean half-life of 11 day at the 10-mg/kg dose level. The analysis of a similar trial performed in the UK (37 patients with advanced cancer of different organs sites treated with doses of 0.1, 0.3, 1.0, 3.0, 10, or 20 mg/kg every 3 weeks) revealed linearity over the whole dose range and a distribution model consistent with a two compartment model with first-order elimination from the central compartment (reported pharmacological values: mean parameters for the 1- and 10-mg/kg groups were: Cmax = 24.0 and 195.9 μg/ml; AUC = 190.8 and 2379 μg.days/ml; halflife = 12.0 and 15.3 days; plasma clearance = 5.7 and 4.8 ml/kg/day; V1 = 44.1 and 50.8 ml/kg and VdSS = 83.7 and 87.1 ml/kg). Clinical results from both trials have been reported with no major toxicity. Of 31 patients entered in the US trial, 10 experienced disease stabilization and 20 had disease progression. Of 37 patients entered in the UK trail, 11 experienced disease stabilization and 26 had disease progression. Data from the studies indicate that lexatumumab was well tolerated at doses up to 10 mg/kg. The most frequently reported toxicities were: fatigue, nausea, anorexia, constipation, diarrhea, tachycardia, and vomiting. The DLT for Lexatumumab was

Transarterial Chemoembolization

defined as 10 mg/kg. No data from phase II trials are available at present. TRA-8 (CS1008) Up to now no results from clinical trials have been reported with the humanized version of TRA-8 (CS1008). Perspectives TRAIL receptor based treatment strategies are currently entering clinical trials. The feared liver toxicity of TRAIL and agonistic compounds has not been documented in any of clinical trials currently available. No judgment on the definitive clinical anticancer activity of agonistic TRAIL receptor antibodies can be made although the phase I and phase II data revealed some clinical activity.

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Trans Activating Protein Definition Is a protein that alters gene expression at sites other than those adjacent to where it is being encoded. ▶Transcription Factor

Transactivation Definition Is the stimulation of gene expression by diffusible mediators, for example proteins that are usually encoded on distant regions of the genome. ▶Transcription Factor

TRAM-1 ▶Amplified in Breast Cancer 1

Transactivation Domain Definition

Trans-Golgi Network

TADs are most frequently found in ▶transcription factors. These protein modules interact with the basic ▶transcription machinery and functions to actively mediate transcription of downstream target genes.

Definition

Is part of the Golgi apparatus, which is an ▶organelle found in ▶eukaryotic cells. The primary function of the Golgi apparatus is to process and package ▶macromolecules synthesized by the cell, primarily ▶proteins and ▶lipids. Trans-Golgi network is the last station of the Golgi apparatus and it directs proteins in the secretory pathways to the appropriate cellular destination.

Transarterial Definition Through the artery.

Transarterial Chemoembolization Transabdominal Metastasis/ Dissemination ▶Transcoelomic Metastasis

Definition An interventional procedure mostly used to treat cancer. Physicians first use a needle to puncture into the artery. Then a thin catheter is threaded into the tumor through

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Transcoelomic Metastasis

the blood vessel under continuous radiological imaging guidance. Anti-tumoral chemotherapeutic agents and small ▶embolization particles are injected into the tumor to induce tumor cell death.

Transcoelomic Metastasis DAVID S. P. TAN , S TANLEY B. K AYE Section of Medicine, Institute of Cancer Research, The Royal Marsden Hospital, London, United Kingdom

Synonyms Peritoneal metastasis/dissemination; Transabdominal metastasis/dissemination

Definition Dissemination or spread of malignant tumor throughout the peritoneal (abdominal and pelvic) cavity.

Characteristics

Transcoelomic (meaning “across the peritoneal cavity”) ▶metastasis refers to the dissemination of malignant tumors throughout the surfaces and organs of the abdominal and pelvic cavity covered by ▶peritoneum. Transcoelomic metastasis can occur as a result of ▶invasion into the peritoneal cavity by: (i) a primary cancer arising from within the abdominal/pelvic cavity e.g. ▶ovarian cancer; (ii) as a manifestation of systemic metastasis following haematogenous or ▶lymphatic invasion by a primary cancer e.g. advanced ▶breast cancer; or (iii) following intraperitoneal seeding during surgical manipulation e.g. during surgical resection of a colorectal tumor. The incidence of transcoelomic metastasis is higher with tumors that arise from the peritoneal cavity e.g. ovarian (up to 70% of patients at presentation) and colorectal (up to 28% of patients at presentation). In contrast, extraperitoneal cancers e.g. breast and lung, are associated with a much lower overall incidence of transcoelomic metastasis, although certain histological subtypes e.g. infiltrating lobular breast cancers, have demonstrated a greater predilection for metastases to the gastrointestinal tract, gynecological organs and peritoneum/▶retroperitoneum. This suggests that whilst the location of the primary tumor may be a key determinant in the development of transcoelomic metastasis, the tumor phenotype is also an important factor. Hence it appears that a combination of

anatomical and tumor-specific factors is involved in the transcoelomic metastatic process. Transcoelomic metastases contribute considerably to the morbidity associated with carcinomatosis because they have the capacity to affect multiple vital organs within the abdomen. Common examples include bowel obstruction caused by lesions along the gastrointestinal tract, and renal failure caused by obstruction of the ureters. In addition, transcoelomic metastases are frequently associated with the formation of malignant ▶ascites, resulting in raised intra-abdominal pressure with consequent abdominal distension and discomfort. This results in early satiety, leading to dietary deficiency, impaired circulation of blood and ▶lymphatic vessels, and respiratory compromise secondary to diaphragmatic splinting. Hence, there are potentially significant therapeutic advantages to be gained in understanding the process of transcoelomic metastasis. Mechanisms of Transcoelomic Metastasis Models of Metastasis Two models have been hypothesized for the genetic origins of tumor metastases. The first model, often referred to as the seed-and-soil hypothesis, is that tumors are genetically heterogeneous and metastases arise from clones with a genetically acquired metastatic phenotype, which determines the final site of metastasis. The alternative hypothesis, the stochastic model, is that metastatic cells do not represent a genetically selected clone distinct from the primary tumor, but arise as a stochastic event from tumor cell clones genetically identical to the primary tumor. Recent studies exploring this question using in vivo models have suggested a combination of both models of metastasis. Regardless of metastatic model, there are certain observed characteristics that appear to be important for transcoelomic metastastic progression, in which complex cellular adaptations need to occur after cell detachment from the primary tumor mass to ensure survival within the peritoneal cavity. Cell Detachment Anchorage-independent growth and the ability to resist ▶anoikis is a vital step for the initiation of metastasis. This process appears to involve the increased expression of ▶survivin and X-linked inhibitor of ▶apoptosis (XIAP), members of the inhibitor of apoptosis protein (IAP) family, which suppress apoptosis by inhibition of ▶caspases. Other mediators of anoikis resistance include the family of ▶extracellular matrix (ECM) to ▶cell-adhesion molecules known as ▶integrins. Alterations in levels of integrin-mediated ECM-ligand binding have been found in many different tumor types and can result in decreased cell-adhesion, changes in cell morphology and increased ▶migration in vitro, and


activation of ECM degrading enzymes including ▶matrix-metalloproteinases (MMP). Peritoneal Fluid and Anatomy The peritoneal cavity is normally empty except for a thin film of fluid that keeps surfaces moist. Peritoneal fluid arises primarily from plasma transudate and ovarian exudate. Other sources of peritoneal fluid include fallopian tubal fluid, retrograde menstruation and macrophage secretions. The volume of peritoneal fluid is usually 5–20 ml, and varies widely depending on physiological or pathological conditions. Peritoneal fluid contains a variety of free-floating cells, including ▶macrophages, ▶natural killer (NK) cells, lymphocytes, eosinophils, mesothelial (peritoneal surface epithelial) cells and ▶mast cells, which are all involved in immunological surveillance. Intraperitoneal fluid flow is directed by gravity to its most dependent sites and then drawn via the paracolic gutters to the diaphragm by the generation of negative intra abdominal pressure in the upper abdomen during respiration. There is preferential flow along the right paracolic gutter, liver capsule, and diaphragm. Therefore, a natural flow of peritoneal fluid exists within the abdominal cavity, providing a route for the transcoelomic dissemination of detached tumor cells. As the epithelial surfaces of the female genital tract (i.e. ovaries, fallopian tubes and endometrium) share a common embryological lineage with the peritoneal epithelium, it has been suggested that transcoelomic metastasis from gynecological malignancies, such as fallopian tube and ovarian tumors, are not true metastases but a result of malignant transformation at multiple foci throughout the peritoneum, i.e. peritoneal ▶metaplasia. If the metaplasia hypothesis is correct, then one might expect metastatic lesions to be randomly distributed throughout the peritoneum. Alternatively, if the theory of dissemination via peritoneal/ascitic fluid is true, then one might expect that detached tumor cells would, by virtue of gravity, be more frequently implanted in the floor of the pelvis, e.g. the pouch of Douglas (the space between the rectum and back wall of the uterus), followed by the organs in the paracolic gutters, and finally on the diaphragm, i.e. along the normal route of peritoneal fluid circulation. Studies have shown that a high incidence of metastatic implants for all cancers, including ovarian malignancies, within the peritoneal cavity is found on organs where peritoneal fluid resorption occurs (▶omentum and omental appendages). In addition, the colon, greater omentum and pouch of Douglas are most often affected, with a reduced incidence of implants seen on the small bowel and its mesentery, which is free to move by peristalsis, compared to the ileocaecal area (the junction between the ileum and cecum), which is fixed to the retroperitoneum. Hence, location and topography

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with regard to the flow of peritoneal/ ascitic fluid appear to be key determinants in the process of transcoelomic dissemination for all cancers. As such, in the case of gynecological cancers, peritoneal metaplasia alone appears unable to fully account for the peritoneal distribution of carcinomatosis. Ascites: A Metastatic Milieu The development of transcoelomic metastasis is often associated with the formation of excess peritoneal fluid known as malignant ascites. It is hypothesized that, in addition to hypoalbuminaemia (low plasma albumin levels) secondary to dietary deficiency, at least three other pathological events can cause ascites: (i) reduced lymphatic drainage from the peritoneal cavity caused by the obstruction of lymphatic vessels by tumor cells; (ii) increased vascular permeability of the peritoneal cavity; and (iii) tumor neo-▶angiogenesis. While lymphatic obstruction is a well-recognized cause of ascites, the fact that massive amounts of fluid can accumulate in patients despite relatively little tumor burden suggests the involvement of other non-obstructive factors. These include ▶vascular endothelial growth factor (VEGF), a glycoprotein which induces angiogenesis and increased vascular permeability in response to hypoxia. Other immune modulators, vascular permeability factors, and MMPs secreted by both tumor cells and mesothelial cells also contribute significantly to ascites formation and stimulate tumor growth, invasion and angiogenesis. Immune Evasion Many immune cells, such as macrophages, are present in peritoneal fluid, and accumulate in so-called “milky spots” within the omentum. These omental macrophages have been found to be cytotoxic against tumor cells ex vivo. Consequently, omental macrophages might play an important role in killing tumor cells, thereby preventing development of transcoelomic metastasis and local peritoneal recurrences. Paradoxically, however, in vivo studies have shown that cancer cells seeded intraperitoneally specifically infiltrate the milky spots in the early stage of peritoneal metastasis. These studies suggest that omental milky spots are insufficient to prevent tumor progression, and that intraperitoneal metastasis requires tumor cells to possess or acquire mechanisms for evasion of immunological surveillance. Tumor-infiltrating and malignant ascites-derived lymphocytes, in particular gamma–delta T cells, from patients with metastatic ovarian and colorectal cancer, have also been shown to possess antitumor activity. Hence, it appears that metastatic tumor cells have also developed strategies to evade T cell-mediated cytotoxicity. Fas ligand (FasL) is a transmembrane protein belonging to the tumor necrosis factor superfamily

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that can trigger apoptotic cell death following binding to its receptor, Fas. Expression of FasL has been observed in renal, ovarian, colorectal, and head and neck tumors and may be responsible for the immune privilege of tumor cells by inducing apoptosis of antitumor immune effector cells within the tumor microenvironment – the “Fas counterattack”. Studies have also shown that tumor progression and metastasis is associated with increased expression of FasL. Other examples of immune evasion include the recruitment of regulatory T (Treg CD4+CD25+) cells to suppress tumor-specific T cell immunity; the presence of high concentrations of soluble forms of the complement pathway inhibitors C1 inhibitor, factor H and FHL-I on isolated metastatic ovarian cancer cells in ascitic fluid; and the phenomenon of spheroid formation observed in ▶breast, colorectal and ovarian cancer where tumor cells clump together by upregulating cell-adhesion molecules, thus resulting in increased complement resistance due to insufficient penetration of antibodies and complement into the spheroids. Tumor Implantation Although topography appears to be a key determinant in the final site of metastatic implantation within the peritoneum, the actual mechanisms behind tumor implantation remain unclear. However, there is evidence to suggest the involvement of a dynamic regulation of the tumor cell’s adhesiveness, and its interaction with the underlying peritoneal mesothelium. Potential mechanisms for the attachment of tumor cells to the peritoneal mesothelium include binding to ECM proteins like collagen type I and IV, laminin, and fibronectin via tumor cell surface integrins, and to hyaluronan expressed on the surface of human peritoneal mesothelial cells via the ▶CD44 tumor cell surface protein, of which there are 10 alternative exon splice variants (v1–v10). Upregulation of certain CD44 variants have been associated with distant metastasis in breast, colorectal and ovarian cancer. Recently, tumor antigen/marker CA125, a glycoprotein overexpressed on the cell surface and secreted by ovarian tumor cells in the majority of ovarian cancer patients, has been shown to bind to mesothelin, a glycosylphosphatidylinositol-linked cell surface molecule expressed by mesothelial cells. Upregulation of the cell adhesion molecule ▶E-cadherin may also mediate adhesion of circulating tumor cells to metastatic sites. Adhesion onto the peritoneal surface may be followed by haptotatic migration in which coordinated anti- and pro-migratory signals mediated by ECM proteoglycans confers directionality to tumor cell motility, effectively laying the tracks until a “stop” signal is encountered. Once attached to the peritoneal surface, metastatic cells proliferate and invade into the subjacent epithelium. The MMP family of proteinases and the urokinase-type

▶plasminogen activator (uPA) system appear to be major contributors to this process. Human peritoneal epithelial cells and their associated immune and stromal cells, have been shown to release regulatory ▶chemokines and cytokines, such as IL-1, ▶IL-6 and IL-8, in response to serosal inflammation and injury induced by tumor implantation, which in turn facilitate tumor angiogenesis and ascites formation (via increased secretion of VEGF), and enhanced tumor migration, attachment, proliferation and invasion. Finally, just as extraperitoneal tumors can metastasize to the peritoneum, intraperitoneal tumors can also metastasize extraperitoneally. Apart from the rich intraperitoneal network of blood and lymphatic vessels which can be invaded by tumors, peritoneal fluid is also continually being returned to the systemic circulation via the subdiaphragmatic lymphatic network and thoracic duct into the left subclavian vein, thus providing a direct “metastatic expressway” for peritoneal metastases to gain access into the lymphatic and circulatory system. Clinical Aspects Patients with transcoelomic metastasis often present with signs and symptoms of abdominal pain, abdominal distension secondary to an enlarging tumor or ascites, constipation or diarrhea, shortness of breath, fatigue, loss of appetite, and weight loss. A careful clinical history followed by thorough clinical examination is required to ascertain the likely source of the primary tumor. Investigations should include routine blood tests, including relevant tumor markers, followed by radiological investigations including ultrasound and computer tomographic (CT) scans of the chest, abdomen and pelvis to confirm the likely source of tumor and disease ▶stage. In all cases, particularly those in which there is no obvious source of primary tumor (i.e. carcinoma of unknown primary origin), a biopsy of an accessible lesion should be obtained for histopathological and immunohistochemical confirmation and diagnosis. In the past, clinical situations involving transcoelomic metastasis were treated mainly with ▶palliative intent. Increasingly, studies have shown that an aggressive approach to peritoneal surface malignancy involving ▶peritoneal debulking (cytoreductive) procedures, combined with optimal perioperative or postoperative systemic or intraperitoneal ▶chemotherapy in carefully selected patients can result in long term survival. Clinical assessment parameters that need to be considered include the patient’s ▶performance status, preoperative abdominal and pelvic CT scans to define the extent and operability of disease, including the presence of extraperitoneal metastases, and tumor histopathology. Key prognostic indicators following surgery include the completeness of ▶peritoneal debulking

Transcription Factor

surgery, the presence of intraperitoneal lymph node and visceral metastases, and tumor type. Of the various scoring systems used to assess the extent of peritoneal carcinomatosis, the most frequently quoted is the peritoneal cancer index (based on the intraoperatively observed distribution and size of intraperitoneal metastasis) and the completeness of cytoreduction score (based on the amount of residual disease following peritoneal debulking surgery), which have been found to correlate well with prognosis in ▶colorectal, ▶gastric and ▶ovarian cancer. A meta-analysis of studies comparing combined peritoneal debulking surgery and perioperative intraperitoneal chemotherapy with systemic chemotherapy alone for the treatment of peritoneal carcinomatosis from ▶colorectal carcinoma, has demonstrated improved survival in the combination therapy group. In patients with ovarian cancer and peritoneal metastasis, 2-year survival following radical resection of all macroscopic tumors is 80%, in contrast to less than 22% for the patients with residual lesions larger than 2 cm. Early aggressive treatment of minimal peritoneal surface dissemination appears to confer the most benefit. In patients with inoperable tumors at presentation, primary systemic or intraperitoneal chemotherapy is recommended, following which reassessment for surgical intervention may be possible if a good treatment response is observed. Palliative measures in the management of malignant ascites include repeated paracentesis (drainage of ascites), which provides relief in up to 90% of patients, and permanent percutaneous drains. The creation of a peritoneovenous shunt (which allows ascitic fluid to drain from the peritoneal cavity into the superior vena cava) prevents the need for repeated paracentesis. Promising experimental approaches in the treatment of transcoelomic metastasis include the use of intraoperative hyperthermic intraperitoneal chemotherapy, anti▶angiogenic agents such as the MMP inhibitors and the VEGF antagonists, as well as ▶immunotherapy approaches including antibody targeted T cell therapy and combinations of intraperitoneal immunotherapy and thermochemotherapy.

References 1. Fidler IJ (2002) Critical determinants of metastasis. Semin Cancer Biol 12(2):89–96 2. Tan DS, Agarwal R, Kaye SB (2006) Mechanisms of transcoelomic metastasis in ovarian cancer. Lancet Oncol 7(11):925–934 3. Becker G, Galandi D, Blum HE (2006) Malignant ascites: systematic review and guideline for treatment. Eur J Cancer 42(5):589–597 4. Koppe MJ, Boerman OC, Oyen WJ et al. (2006) Peritoneal carcinomatosis of colorectal origin: incidence and current treatment strategies. Ann Surg 243(2):212–222

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Transcript Definition Fragment of RNA synthesized from a given DNA template.

Transcription Definition Messanger RNA (mRNA); The process by which the genetic code is transferred from DNA to messenger RNA, so that it can be subsequently translated into a protein sequence. Is the first step in gene expression. It involves the faithful synthesis of RNA from the genetic information stored in DNA. It is carried out in the nucleus by DNA-dependent RNA polymerases. A transcriptional activator is a DNA-binding protein that stimulates transcription by interacting with DNA sequence motifs present in the regulatory sequences of the controlled gene. A transcriptional repressor inhibits transcription. ▶Orphan Nuclear Receptors and Cancer

Transcription-Coupled Repair Definition Is a DNA repair mechanism that operates in tandem with transcription. It directly repairs damaged base on the DNA strand by directly removing or repairing the damaged DNA base. Is the rapid repair of DNA damage in the transcribed strand of expressing genes. ▶Homologous Recombination Repair ▶DNA-Damage Tolerance

Transcription Factor Definition

A protein that regulates gene ▶transcription by binding to regulatory regions of DNA and/or to other

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Transcription Factory

transcription factors. The presence of these specific DNA sequences confers upon a specific gene the ability to respond in a specific cellular context, to particular stimuli, or at a defined developmental stage. The most fundamental property of all transcription factors is their ability to influence the transcriptional activity of specific genes by acting in either a positive or negative manner. In the context of cancer, there is often a dysregulation in the balance between transcriptional activation and transcriptional repression that alters the expression of critical cancer-promoting or cancersuppressing genes. Transcription factors are generally divided into two groups: (i) basal transcription factors (BTFs) and (ii) gene-specific transcription factors (GSTFs). The types of GSTFs are named based on the characteristic motifs involved in DNA binding and protein dimerization.

Transcriptional Memory Definition Mechanism to ensure that specific genes are expressed at specific times so that the genetic information is transmitted to daughter cells to maintain the differentiated cell type.

Transcriptional Regulation Definition

Regulation of gene expression by ▶transcription factors.

Transcription Factory Definition

Discrete nuclear compartment where ▶transcription takes place.

Transcriptional Silencing Definition

Repression of gene ▶transcription in a localized region via structural changes in chromatin.

Transcriptional Complex

▶Histone Deacetylases ▶Methylation

Definition A complex of proteins that directly or indirectly, affects the initiation of ▶transcription.

Transcriptome Definition

Transcriptional Coregulators

The full complement of activated genes, mRNAs, or transcripts in a particular tissue at a particular time.

Definition These are proteins that interact with transcription factors and regulate transcription. They can either be activators of transcription (enhance) or repressors of transcription (inhibit) and may act to modify chromatin structure.

Definition

▶Orphan Nuclear Receptors and Cancer

Transformation of a non-stem cell into a different cell type or the production of cells from a differentiated stem

Transdifferentiation

Transduction of Oncogenes

cell that are not related to its already established differentiation path. ▶Adult Stem Cells ▶Stem Cell Plasticity

Transduction Definition Transfer of genetic material into a cell by means of a virus ▶HSV-TK/Ganciclovir Mediated Toxicity ▶Transduction of Oncogenes

Transduction of Oncogenes J AQUELIN P. D UDLEY, J ENNIFER A. M ERTZ , S ANCHITA B HADRA Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

Synonyms Oncogene transduction; Retroviral transduction

Definition

▶Retroviruses are RNA-containing viruses that replicate through a DNA intermediate (▶provirus) using the enzyme ▶reverse transcriptase. During retroviral replication, which requires integration into the host chromosomal DNA for efficient transcription of viral RNA, some retroviruses have acquired specific cellular ▶oncogenes, usually with multiple modifications, and often with the loss of trans-acting viral functions. Inclusion of one or more oncogenes in the viral genome then imparts transforming activity on the recombinant virus independent of the site of integration in the cellular genome.

Characteristics Identification of Cellular Oncogenes In 1911, Peyton Rous described the isolation of a virus that caused fibrosarcomas in chickens. The Rous sarcoma virus (RSV) subsequently was shown to transform

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chicken embryos and the surrounding membranes and formed small tumors on the chorioallantoic membrane in proportion to the number of virus particles. Further quantitative assays were developed when RSV and other ▶retroviruses were shown to transform or induce morphological and growth behavior changes in cultured cells that mimicked tumor formation in the animal. The ability of RSV to transform cells in culture led to the conclusion that the virus encoded a gene responsible for such changes. Isolation of transformationdefective variants of RSV allowed comparisons with wild-type RSV and the discovery of the viral oncogene, v-src. Experiments from the laboratories of Harold Varmus and Michael Bishop revealed that the v-src gene is highly related to a specific cellular gene or protooncogene, c-src, which encodes a protein tyrosine kinase. Unlike the v-src gene in RSV, the cellular homologue contained introns, which could be alternatively spliced in various cell types to give different mRNAs. Further characterization showed that the product of the v-src gene, v-Src, had substitutions within several functional domains that prevented the normal regulation of the kinase activity during the process of ▶signal transduction. Shortly after the discovery of v-src, other transforming viruses were isolated and characterized. Some of these viruses were recovered by treatment of normal cells with halogenated pyrimidines to induce ▶endogenous retrovirus expression, by co-culture of primary cells with chemically transformed cells that express non-transforming ▶retroviruses, whereas others were isolated by in vivo passage of non-transforming ▶retroviruses. In each case, the transforming virus appears to be the result of recombination between a non-transforming ▶retrovirus and one or more cellular genes. The ability of ▶retroviruses to acquire cellular sequences in their genome and transmit these genes to other cells is known as retroviral ▶transduction or, in the case of protooncogenes, oncogene ▶transduction. Examples of transforming viruses and the acquired proto-oncogenes are listed in Table 1. Deregulation can occur at many steps of ▶signal transduction, leading to oncogenesis. Nevertheless, recombination events leading to the generation of a transforming ▶retrovirus appear to be rare in nature. Most of the resulting viruses are defective for replication because the acquisition of cellular protooncogenes is accompanied by the deletion of viral structural genes, which are necessary to produce viral particles. Such defective transforming viruses are not transmissible unless they successfully co-infect a cell with a related ▶retrovirus that provides the missing gene products in trans. Thus, most transforming viruses are more interesting as research tools for the identification and functional characterization of oncogenes and the process of ▶transduction than as a major cause of disease in animals and humans.

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Transduction of Oncogenes. Table 1

a b

Oncogenes transduced by retroviruses

Transforming virus

Acquired proto-oncogene

Abelson murine leukemia virus AKT8 Cas NS-1 virus Avian sarcoma virus CT10 Avian erythroblastosis virus ES4 Avian erythroblastosis virus ES4 Avian myeloblastosis virus E26 Avian retrovirus RPL30 Snyder-Theilen feline sarcoma virus Gardner-Rasheed feline sarcoma virus McDonough feline sarcoma virus Finkel-Biskis-Jenkins murine sarcoma virus Fujinami avian sarcoma virus Avian sarcoma virus 17 Hardy-Zuckerman-4 feline sarcoma virus Avian retrovirus AS42 (sarcoma) Mill Hill virus 2 (avian myelocytoma virus) Moloney murine sarcoma virus Mouse myeloproliferative leukemia virus Avian myeloblastosis virus E26 Myelocytomatosis virus 29 Avian retrovirus ASV31 (sarcoma) 3611 murine sarcoma virus Harvey murine sarcoma virus Kirsten murine sarcoma virus Avian reticuloendotheliosis virus T UR2 avian sarcoma virus S13 avian erythroblastosis virus Simian sarcoma virus SKV770 avian sarcoma virus Rous sarcoma virus Y73/Esh avian sarcoma virus

abl akt cbl crk erbA erbB ets eyk fesa fgr fms fos fpsa jun kit maf milb mos mpl myb myc qin raf b H-ras K-ras rel ros sea sis ski src yes

fes and fps are the same oncogene derived from feline and avian genomes, respectively. mil and raf are the same oncogene derived from avian and murine genomes, respectively.

Mechanism of Oncogene Transduction The majority of transforming ▶retroviruses are defective for viral replication. Since these viruses are isolated after acquisition of transforming activity, the exact steps required to form these ▶retroviruses is unknown, nor is it clear whether every transforming virus has been generated by the same mechanism. However, a general model has emerged for the formation of such viruses (Fig. 1). First, a non-transforming ▶retrovirus integrates upstream of a cellular gene, an event known to occur at a reasonable frequency. Many ▶retroviruses integrate preferentially within coding sequences or near sites of active transcription. Non-transforming ▶retroviruses

often cause tumors by insertion in or near protooncogenes, resulting in the activation of transcription or production of abnormal transcripts. These transcripts arise due to enhancer activation of the cellular promoter or activity of the viral promoter on the cellular gene. If the proviral integration results in cellular transformation, cells containing the integration site will be selected for growth. However, some ▶retroviruses integrate upstream or downstream and in the opposite orientation relative to the proto-oncogene, and it is believed that the generation of transforming ▶retroviruses requires proviral integration upstream and in the same orientation as the oncogene. Such events can result in cancer


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Transduction of Oncogenes. Figure 1 Mechanism of retroviral oncogene transduction. (See text for details.)

induction without ▶transduction of oncogenes. Thus, many proviral insertion events may lead to cancer, but not formation of transforming ▶retroviruses. Second, transcription initiating in the 5′ LTR generates a transcript that would read through the normal polyadenylation sequences in the 3′ LTR. Recent evidence suggests that increases in retroviral transcriptional readthrough also result in transductive recombination. Potentially, this readthrough transcript could be packaged into virions, although it is generally believed that transcripts longer than 150% of the genome would be accommodated poorly in the virus capsid. Alternatively, a rare deletion of cellular DNA or aberrant splicing could provide a truncated provirus or mutant transcripts that may encompass a much greater portion of the cellular transcripts. Third, hybrid oncogene transcripts may be packaged along with normal viral transcripts into virions. Retroviruses have a diploid genome that includes cis-acting sequences (usually near the 5′ end of the viral genome) necessary for packaging into the viral capsid (designated the psi sequence). Thus, hybrid transcripts including the psi sequence would be preferentially

packaged with normal retroviral sequences to give an RNA heterodimer instead of the normal RNA homodimer. However, normal cellular RNAs can be packaged into retroviral particles at low frequency, and co-packaging would allow copying by reverse transcriptase, which is not template-specific. Fourth, the hybrid oncogene transcript and the wildtype transcript both will be used as templates for reverse transcriptase, which also is incorporated into viral particles. In some cases, incorporation of the protooncogene and expression at high levels from the retroviral promoter appears to be transforming. Nevertheless, most transduced oncogenes have multiple genetic alterations. Reverse transcriptase has several properties that favor the types of genomic changes observed in transforming retroviruses. These properties include template switching, deletion formation, and the introduction of point mutations. If the resulting recombinants retain all of the cis-acting sequences needed for replication, the transforming virus will be capable of propagation in the presence of replication-competent retroviruses. Supporting the importance of readthrough transcripts for this process, transforming retroviral

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Transfection

genomes have been observed that carry poly(A) stretches typical of mRNAs at the junction between the host and 3′ viral sequences. Furthermore, the incorporated oncogenes lack introns. Lessons from Retroviral Transduction of Oncogenes Cancer is believed to be a multistep process, where several genetic events contribute to the generation of tumor cells. Some avian retroviruses are known to have transduced two oncogenes, including avian erythroblastosis virus-ES4 and -R (AEV-ES4 and AEV-R) (erbB and erbA), Mill-Hill-2 (MH2) avian myelocytoma virus (mil (aka raf ) and myc), and avian myeloblastosis virus (AMV-E26) (ets and myb). Some viruses may have transduced more than one cellular proto-oncogene to improve their transforming capacity, a process that may be similar to the acquisition of multiple genetic changes in cancer cells during tumor progression. For example, evidence suggests that erbA expression is necessary for the full transforming activity of the erbB oncogene in AEV. Also, the MH2 ▶retrovirus requires expression of both mil/raf and myc to transform neuroretinal cells from 7-day-old chicken embryos, an event that myc-expressing retroviruses cannot induce. However, AMV-E26 contains two oncogenes, but only one of them has been shown to be necessary for full transforming ability. Deletion of the ets oncogene does not diminish the transforming ability of AMV-E26 relative to wild type virus when injected in newborn chickens. Furthermore, many of the transduced oncogenes contain deletions or point mutations that reveal regulatory regions of the encoded gene products, leading to a greater understanding of their normal functions in cellular growth control. Retroviruses, including simple retroviruses that lack regulatory genes as well as lentiviruses, have become common vectors for therapeutic gene delivery. These viruses have been used to deliver genes for treatment of a variety of illnesses, including cancers and genetic disorders. Lentiviral vectors offer the advantage of being able to infect and replicate in non-dividing cell types. However, simple retroviruses may be advantageous for preferential infection of dividing tumor cells and delivery of the therapeutic gene relative to adjacent normal cells. In both cases, viral structural genes in the vector are replaced with a therapeutic gene of interest. Expression of the transduced genes can be controlled by non-viral promoters, internal ribosome entry sites or splicing. Although these vectors have shown promise as therapeutic agents, the safety of such vectors has been the overriding concern, i.e., preventing the formation of replication competent viruses and avoiding the ill effects of integration. Limitations to replication and the avoidance of multiple insertions within target cells should minimize insertional activation of oncogenes. A recent improvement to the retroviral vector system

was removal of the 3′ U3 region, thus creating a selfinactivating (SIN) virus after reverse transcription. Another possible safety concern associated with ▶gene therapy is the generation of new transforming viruses, especially in human patients. These events should be extremely rare because of several precautions included during the construction of these vectors, including removal of U3 sequences as well as viral structural genes, thus confining these retroviruses to a single round of replication. Furthermore, the absence of homologous endogenous lentiviruses in humans should reduce recombination events that lead to generation of transforming retroviruses. In the early part of this century, a major milestone in human gene therapy was achieved. To date, more than 20 children with X-linked severe combined immune deficiency (SCID) or adenosine deaminase (ADA) deficiency SCID have been successfully treated using a Moloney murine leukemia virus-based vector to allow normal expression of the defective gene. Unfortunately, in three of the patients, this vector inserted in close proximity to the LMO2 proto-oncogene, leading to dysregulation of LMO2 expression and development of leukemia. Many gene therapy clinical trials are being performed, and current efforts have focused on further improvements to vector safety. Such improvements include prevention of transcriptional readthrough or inclusion of insulator elements to block the formation of hybrid viral-cellular transcripts that may lead to oncogene ▶transduction.

References 1. An W, Telesnitsky, A (2004) Human immunodeficiency virus type 1 transductive recombination can occur frequently and in proportion to polyadenylation signal readthrough. J Virol 78:3419–3428 2. Nevins JR (2001) Cell transformation by viruses. In: Knipe DM, Howley, PM (eds) Fundamental Virology, 4th edn. Lippincott William & Wilkins, pp 245–283 3. Swain A, Coffin, JM (1992) Mechanism of transduction by retroviruses. Science 255:841–855 4. Swanstrom R, Parker, RC, Varmus, HE, et al. (1983) Transduction of a cellular oncogene: the genesis of Rous Sarcoma Virus. Proc Natl Acad Sci USA 80:2519–2523 5. Verma IM, Weitzman MD (2005) Gene therapy: twentyfirst century medicine. Annu Rev Biochem 74:711–738

Transfection Definition Introduction of DNA into live cells is referred to as: . Transformation when the cells are bacterial or yeast cells;

Transforming Growth Factor Beta

. Transfection when the cells are eukaryotic and DNA is offered to the cells as a complex . Transduction when the cells are eukaryotic and the vector is a non-self-replicating virus . Infection when the cells are eukaryotic and the vector is a self-replicating virus

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Transforming Growth Factor Beta J ORMA K ESKI -O JA Departments of Pathology and of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland

Definition

Transformation Definition Malignant transformation is the collection of events that leads to loss of normal cellular function and acquisition of tumorigenic properties. Transformation of eukaryotic cells describes the failure to observe the normal constraints of growth. It refers to their conversion to a state of unrestrained growth in culture, resembling or identical with the tumorigenic condition. Transformed cells become independent of growth factors usually needed for cell growth. In vitro cell transformation tests are used as a model for predicting in vivo ▶carcinogenesis. Hallmarks of transformation in experimental settings include ▶anchorage independent growth, cell proliferation without exogenous growth factors, loss of ▶contact inhibition and tumor formation in ▶xenograft assays.

Transforming Gene ▶Oncogene

Transforming Growth Factor Alpha Definition

TGFα; A potent ▶mitogen and ▶EGFR ligand. Initially identified as a 50 amino acid polypeptide secreted by transformed cells, it is often overproduced alongside the EGFR in tumors and promotes autonomous proliferation of cancer cells. ▶ADAM17

Transforming growth factors were identified on the basis of their ability to induce soft agar growth and morphological changes in nonmalignant cells. The original observation was of an activity, which was named sarcoma growth factor. Soon afterward the term transforming growth factor, ▶TGF, was adopted. Sarcoma growth factor was subsequently found to be composed of an epidermal growth factor like protein, which was named transforming growth factor alpha, ▶TGF-α, and of ▶TGF-β. TGF-α is a member of the epidermal growth factor family, and is unrelated to TGF-β.

Characteristics TGF-βs are multifunctional polypeptide growth factors involved in the regulation of cellular growth and differentiation and immune functions. The number of known members of this family is rapidly increasing and stands at more than 40 different growth modulating proteins. Besides TGFβs, these include bone morphogenetic proteins (BMPs), growth and differentiation factors, inhibins and activins. TGF-βs are in many senses unique among growth factors in their potent and widespread actions. Three different mammalian gene products, TGF-βs 1–3, have been molecularly cloned. Almost all types of cells in the body make some form of TGF-β, and nearly all cells have cell surface receptors for it. One of their major effects is inhibition of cell proliferation, a property needed in developmental processes, for instance. TGF-βs have important roles in the control of the pericellular proteolytic balance and in the regulation of the production and structure of the components of the connective tissues and extracellular matrices. TGF-β stimulates the transcription and synthesis of various components of the extracellular matrix like collagens, ▶fibronectin, vitronectin, tenascin and proteoglycans. TGF-βs are potent chemotactic factors for many cell types like fibroblasts, eosinophils and various inflammatory cells at very low concentrations. They also suppress matrix degradation by decreasing the expression of proteinases (▶Serine proteases (type II) spanning the plasma membrane), such as plasminogen activators (▶Plasminogen activating system), numerous metalloproteinases, and by inducing proteinase inhibitors, such as plasminogen activator inhibitor-1 and ▶tissue inhibitors of metalloproteinases (TIMPs). In addition, TGF-βs regulate cellular functions by modulating the expression of matrix receptors, the

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integrins. For these reasons the activities of TGF-βs must be tightly regulated. TGF-b Receptors and Signaling Mechanisms Members of the TGF-β superfamily have diverse functions in cell-cell signaling. TGF-βs play different roles in tissue homeostasis and at various stages of development. The mechanisms of regulation of TGF-β activity are multifaceted and complex. Three different TGF-β isoforms and the types, affinity, and signaling functions of its receptors also add complexity to the regulation of their effects. The effects of TGF-βs and the other family members are mediated from the cell membrane to nucleus through distinct combinations of type I and type II serine/threonine kinase receptors, ▶TβRI and ▶TβRII and downstream effectors, the ▶Smad proteins. The TGF-β receptors form a signaling receptor network with activin like kinase (ALK) receptors. The receptor-regulated Smads become phosphorylated by activated type I receptors, and they form heteromeric complexes with a common partner, Smad4, which gets translocated into the nucleus for gene transcription control. In addition to the signal transducing Smads, inhibitory Smads also play a role in the outcome of the signaling. They down-regulate the activation of receptor-regulated Smads. In contrast to the still growing TGF-β growth factor superfamily, relatively few type I and type II receptors or Smads have been identified. The signaling specificities between different TGF-β superfamily members vary, and a certain family member can elicit a broad spectrum of biological responses. Latency TGF-b TGF-βs are produced by the majority of cells in latent complexes unable to associate with TGF-β signaling receptors. Some primary cells and established cell lines secrete active TGF-β. TGF-βs are secreted from cells as latent dimeric complexes containing the mature C-terminal TGF-β and its N-terminal pro-domain, ▶LAP, the TGF-β ▶latency-associated peptide. The two polypeptide chains of pro-TGF-β associate to form a disulfide bonded dimer. TGF-β is cleaved from its propeptide by furin-like endoproteinase during secretion at RRXR sequence. The LAP propeptide dimer remains associated with the TGF-β dimer by noncovalent interactions. This complex is referred to as small latent TGF-β. TGF-βs are secreted in most cultured cell lines as large latent complexes, consisting of small latent TGF-β covalently bound to one of the three ▶latent TGF-β binding proteins (▶LTBPs -1, -3 or -4) covalently linked to LAP (Fig. 1). Interestingly, LTBP2 is unable to form complexes with any of the small latent TGF-βs, suggesting some other functions for this widely expressed molecule. The true LTBPs have a central role in the processing and secretion of TGF-βs,

but they evidently have other, for example, structural roles like fibrillins. The expression and secretion of LTBPs and TGF-βs is, in general, coordinately regulated. Matrix Association and Release of TGF-b LTBPs have a central role in the targeting of TGF-β to extracellular matrix structures. LTBPs are produced in excess to TGF-β, and since TGF-β secretion is very inefficient in the absence of LTBP, most secreted cellular TGF-β is in the large latent complexes (Fig. 1). Release of active TGF-β from matrix associated latent complexes appears to require two steps, the release of the large latent complex from ECM by proteolytic truncation, and subsequent activation, which can be achieved by different mechanisms like the integrins. Since TGF-β regulates the cellular production of ECM components as well as the proteolytic balance, the matrix association and activation of TGF-β complexes form a finely tuned control network for the maintenance of the organization of extracellular structures. Cancer cells produce frequently aberrant amounts of both the matrix components and TGF-β. Malignant cells do also frequently fail to deposit TGF-β complexes to the extracellular matrix, probably due to their perturbed deposition of fibronectin-collagen matrix, as well as altered LTBP production. Latent complexes of TGF-β in the ECM may provide tissues with a readily available storage form of this growth factor. The release and activation of stored growth factors by proteases or migrating cells can generate rapid and highly localized signals like in wound healing or during radiotherapy. Cell movement causes traction of the latent matrix-associated complexes and induces activation. Rapid activation of extracellular signaling mechanisms could be important in the healing of tissues after damage, in the control of cells of the immune system during acute infections and in the initial stages of angiogenesis. It is unclear how soluble growth factors could form gradients in highly cellular tissues. Matrix bound growth factors might generate this kind of an immobilized activity gradient. LTBPs: Expression and Functions Relatively few functions have been identified for LTBPs thus far. Structurally they resemble fibrillins, which are components of the extracellular microfibrils. LTBPs have a typical structure consisting of four eight cysteine (8-Cys) repeats and several EGF-like repeats. The association of latent TGF-β with the matrix is mediated by LTBPs (Fig. 1). Not only the N-terminal domains, but also a region of the C-terminus of LTBP are important in this association. The N-terminus contains transglutaminase substrate motifs, and transglutaminase is required for the covalent ECM association. In addition, TGF-β2 and -β3 become associated with LTBPs. It is thus likely


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Transforming Growth Factor Beta. Figure 1 Large latent TGF-β complex. The small latent complex contains the C-terminal mature TGF-β and its N-terminal pro-domain, LAP (TGF-β latency-associated peptide). This complex forms a disulphide-bonded complex with the third 8-Cys repeat of LTBP. LTBP associates with the ECM mainly via the 8-Cys domains and some adjoining regions.

that LTBPs mediate and target the binding of all three TGF-β isoforms to various extracellular matrices. The TGF-β1 binding region in LTBPs is located close to their C-terminus in the third 8-Cys repeat. The association between LTBP-1 and the propeptide part, LAP, is mediated by disulfide bonding. The respective 8-Cys repeats of LTBPs-3 and -4 also bind small latent TGF-βs. Of the numerous known 8-Cys repeats of the LTBPs and fibrillins only three have been found to have the capacity to associate in a covalent manner with the small latent TGF-βs. Activation of Soluble and Extracellular Matrix Forms of Latent TGF-b TGF-β can be activated in vitro by multiple mechanisms, including proteolysis, enzymatic deglycosylation and extremes of pH. Activation of latent TGF-β involves proteolytic disruption of the non-covalent interaction between the propeptide LAP and TGF-β, which releases biologically active TGF-β capable of binding to its signaling receptors. LAP may also undergo conformational changes in such a manner that TGF-β is released or exposed to its receptors. The existence of different TGF-β isoforms and latent

complexes, as well as the number of different LTBPs, suggests that there are variable mechanisms for the activation of TGF-βs. The electrostatic interaction between LAP and TGF-β can be dissociated in vitro by extremes of pH, chaotropic agents, and heat treatment. From the physiological point of view, the acidic environment in the bone (osteoclasts) or during wound healing could induce this kind of ▶TGF-β activation. In vivo analyses of tumor-bearing mice indicated that irradiation (02372) causes rapid activation of TGF-β in the tumors. This effect appears to result from the activation of existing, most probably of matrix-bound latent TGF-β. Irradiation produces reactive oxygen species leading to redox-mediated activation of latent TGF-β complexes. Redox-mediated TGF-β activation may be involved in chronic tissue processes, where oxidative stress is implicated, such as carcinogenesis. TGF-β can be activated by deglycosylation of LAP. Mechanisms involving proteolysis are, however, more diverse and more likely to operate in vivo. Various proteases can degrade LAP and release active TGF-β. Protease inhibitors prevent the activation of TGF-β in cell culture. Cell-cell contacts, targeting of TGF-β, as

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well as transglutaminase activity appear to be important in the generation of active TGF-β in vivo. The processing of pericellular matrix-associated LTBPs and activation of TGF-β are constant events in the ▶apoptosis or ▶anoikis of endothelial and epithelial cells, pinpointing the importance of pericellular latent complexes as a physiological source of TGF-β. Thrombospondin-1 (TSP-1), a platelet α-granule and ECM protein, plays a role in the activation of latent TGF-β complexes via a mechanism that does not involve cell surfaces or proteases. Using purified plasma TSP-1 or the recombinant protein it was found that it is able to activate both small and large latent TGF-β complexes. The activation mechanism is not fully understood, but seems to involve the N-terminal end of LAP and the type I repeats of TSP-1, possibly by inducing a change in the conformation of LAP and thus releasing the active TGF-β. TSP-1 interacts with LAP as a part of a biologically active complex, and this may prevent the re-association of the inactive complex of LAP with TGF-β. The expression of TSP is induced during wound healing. TGF-β may thus get focally activated at sites of injury by enhanced TSP synthesis. Accordingly, TSP deficient mice display many phenotypic features, similar to those detected in TGF-β1 deficient mice. The abnormalities in some tissues of the TSP null animals were even reverted by TSP-derived TGF-β activating peptides, further emphasizing the role for TSP in TGF-β activation. The LAP part of TGF-β contains an RGD-motif, which is recognized by integrins (▶Integrin signaling and cancer) αvβ1 and αvβ6. Integrin αvβ6 is also able to activate TGF-β. This activation model is particularly interesting, because αvβ6 integrin is expressed solely on epithelial cells, which are very sensitive to TGF-β mediated growth inhibition, and also because the overlap of the phenotypes of TGF-β1 and integrin β6 chain deficient mice. β6 integrin deficient mice show increased inflammation and decreased fibrosis, processes that are regulated by TGF-β. Hormonal effectors (▶Tamoxifen) can also affect TGF-β activation. Originally it was found that antiestrogens could induce the production and secretion of active TGF-β in cultured breast cancer cells. Activation of TGF-β has subsequently been observed in a number of cell culture models using estrogens and antiestrogens, retinoids and vitamin D derivatives. Steroid hormone superfamily members are efficient regulators of the expression of TGF-β isoforms, and TGF-βs are likely to act as local mediators of the diverse actions of steroids. Estrogens and antiestrogens regulate TGF-β1 formation in different cells and tissues like in mammary carcinoma cells and in fetal fibroblasts. TGF-β functions, for instance, as an autocrine negative growth regulator in breast carcinoma cells.

TGF-Beta and LTBP Knockout Mice The importance of the three different TGF-βs is elucidated in the gene knockout studies. Knockout of TGF-β1 results in multifocal inflammatory disease leading to the death of the animal. TGF-β2 knockout is embryonally lethal. The mice develop severe cardiac, lung and craniofacial defects. Inner ear and eye are also affected. TGF-β3 null mice develop cleft palate. Accordingly, null-mice unable to produce LTBPs develop serious physiological defects. LTBP-3 knockout mice develop multiple defects such as growth retardation, emphysema, bone malformations and abnormalities of thymys and spleen. Their lifespan is, however, normal and they are able to reproduce. Hypomorphic LTBP-4−/− mice develop early emphysema and colorectal tumors, indicative of missing growth inhibitor. The short and long splice forms have different functions. LTBP-1L null mice exhibit a cardiac phenotype, which reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgfbeta activity in heart organogenesis. Perspective Growth factors of the TGF-β family are important autocrine and paracrine regulators of cell proliferation and differentiation. The regulation levels of their activities include the expression of TGF-β receptors, availability of TGF-βs, their activities and modulation of the cellular response. Most cells secrete TGF-β in a large latent complex, which associates with the extracellular matrix and is unable to bind to the TGF-β signaling receptors. LTBPs have a central role in TGF-β secretion, extracellular matrix deposition and activation. In addition, LTBPs have structural and other functions not directly related to TGF-β signaling. Structural diversity in LTBP proteins is tremendous, and the possible functions of the different forms include, amongst others, the modulation of cell adhesion and the functions of integrins. Focal activation of latent TGF-β in the matrix by physicochemical means offers a rapid way to induce TGF-β signaling. In addition to plasmin-mediated TGF-β activation novel mechanisms have been found including other proteases, reactive oxygen species, thrombospondin and integrin mediated activation. The modulation of pericellular proteolytic activity by TGF-β supports a general cascade of events, where proteinases and latent matrix-bound growth factors are components of extracellular signal transduction machinery. This directs tissue construction and remodeling, and probably also regulates the activity of infiltrating immune cells. Disturbances in these control systems could participate in the pathogenesis of a variety of disease states like atherosclerosis, cancer, various fibrotic diseases and chronic inflammation.

Transgenic Mice

References 1. Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer initiation and progression. Nature 432:332–337 2. Sporn MB (2006) The early history of TGF-beta, and a brief glimpse of its future. Cytokine Growth Factor Rev 17:3–7 3. Massague J, Gomis RR (2006) The logic of TGF-beta signalling. FEBS Lett 580:2811–2820 4. Hyytiainen M, Penttinen C, Keski-Oja J (2004) Latent TGF-beta binding proteins: extracellular matrix association and roles in TGF-beta activation. Crit Rev Clin Lab Sci 41:233–264

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Transgene Definition A foreign gene introduced into a cell by viral or nonviral gene transfer techniques.

Transgenic Definition

Transforming Retroviruses Definition

A class of ▶retroviruses that is capable to transform host cells. Two classes of transforming retroviruses can be distinguished: 1. Acute transforming retroviruses are characterized by the presence of an oncogene in their genome that has been acquired during host cell passage by retroviral transduction. Elimination of this retroviral oncogene (v-onc) by deletion or mutations within the oncogene that affect its function (e.g. a temperaturesensitive mutation), results in the loss of the transforming activity of the virus. This demonstrates that the presence of v-onc in the retroviral genome is necessary and sufficient to induce host cell transformation. Examples for this sub-class are the Rous Sarcoma Virus (v-src), Abelson leukemia virus (v-abl) and Mill Hill 2 virus (v-myc; v-mil/raf-1). 2. Slow transforming retroviruses lack a v-onc gene, but often trigger tumorigenesis by inserting their proviral genome into genomic regions that control the expression of proto-oncogenes (insertion mutagenesis). For example, avian B cell lymphomas often contain proviral sequences derived from the Avian leukosis virus in the promotor region of the c-myc proto-oncogene. As the retroviral control elements represent strong promotors, the transcription of the c-myc gene is largely driven by the provirus and uncoupled from its tight control by the cellular signaling network. This results in Myc overexpression and malignant transformation. ▶Retroviral insertional Mutagenesis ▶Myc Oncogene ▶Transduction of Oncogenes

Transgenic animals or plants are created by introducing new DNA sequences into the germ line via addition to the egg. The use of transgenic mice carrying an alien gene in every cell is a widely used experimental system in biomedical science. They are generated by microinjection of DNA (transgene) into fertilized eggs, which are then implanted into pseudopregnant foster mothers. The offspring often carries the transgene in all cells, including germ cells. Thus, the transgene will be transmitted according to Mendelian genetics. Transgenic mice represent a tool to study gene effects in the context of the whole organism. ▶Mouse Models

Transgenic Animal Definition An animal to which new DNA has been inserted into its genome. ▶Mouse Models

Transgenic Mice Definition Are mice in which a foreign gene has been inserted in their genome. ▶Transgenic

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Transglutaminase-2

Transglutaminase-2 K APIL M EHTA The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Synonyms Tissue-transglutaminase; tTGase; Transglutaminase type-2; TG2; Cytosolic transglutaminase; TGc; Endothelial transglutaminase; Liver transglutaminase and Gh

Definition Tissue transglutaminase (▶TG2; EC 2.3.2.13) is a ubiquitous and most diverse member of the transglutaminase family of enzymes. TG2 catalyzes calciumdependent post-translational modification of proteins by inserting highly stable ▶isopeptide bonds between polypeptide chains or by conjugating ▶polyamines to proteins. In addition, TG2 exhibits ▶GTPase activity and can serve as a ▶signal-transduction G protein. Less studied functions of TG2 include its protein disulfide isomerase and ▶protein kinase activities.

Characteristics TG2 is a multifunctional protein whose expression in some cell types (e.g., endothelial and smooth muscle cells) is constitutively high. In other cell types, TG2s expression is up-regulated via discrete signaling pathways, such as those induced by certain stress factors, inflammatory stimuli, differentiation agents, and growth factors. Although predominantly a cytosolic protein, TG2 can translocate to the nucleus by “piggy-back riding” other proteins, such as ▶importin-alpha-3, or translocate to membranes in association with ▶integrins. TG2 can also be secreted outside the cell (by an

as-yet unknown mechanism), where it can crosslink extracellular matrix (ECM) proteins and promote ▶adhesion of several cell types. An important feature of TG2 is its high binding affinity for ▶fibronectin; in cancers, membrane-associated TG2 can promote a stable interaction between cell surface integrins and fibronectin and promote cell growth and survival (Fig. 1). TG2 and Apoptosis A role for TG2 in apoptosis was initially suggested by Dr. Laszlo Fesus and his coworkers in 1987 based on the observation that lead-induced hypertrophy of the liver in rats was associated with cellular expression of increased TG2. Since then, many reports have supported the role of TG2 in apoptosis. In general, the expression of TG2 is markedly increased in cells undergoing apoptosis. Forcibly increasing the expression of TG2 in several cell types results in apoptosis or makes them susceptible to death-inducing stimuli. Conversely, reducing TG2 levels by antisense RNA renders the cells more resistant to apoptosis. It is believed that TG2 promotes apoptosis by crosslinking intracellular proteins, preventing their leakage from cells and induction of an inflammatory response. These observations suggest that cells generally do not tolerate the increased expression of TG2 and that TG2 overexpression leads to apoptotic death. However, some recent reports have provided paradoxical evidence and suggest that TG2 expression and apoptosis do not always go hand in hand. For example, TG2-/- knockout mice (mice lacking all TG2 expression) did not show any genetic alterations that are suggestive of perturbed apoptosis. The possibility that some other proteins compensate for the loss of TG2 in these mice cannot be ruled out. Furthermore, various other studies have provided data suggesting that increased expression of TG2 can prolong cell survival by preventing apoptosis.

Transglutaminase-2. Figure 1 Schematic representation of various functional domains of the TG2 protein. In addition to catalyzing calcium-dependent protein crosslinking function, TG2 can catalyze calcium-independent GTPase, ATPase, protein kinase, and protein disulfide isomerase activities. TG2 can modulate the functions of other proteins by directly interacting or associating with them; examples include ▶phospholipase-δ1, members of the β-integrin family, focal adhesion kinase, fibronectin, osteonectin, ▶RhoA, multilineage kinases, and ▶retinoblastoma protein. Through these activities, TG2 plays a role in biological processes such as ▶apoptosis, wound healing, and cataract formation. Recent work suggests that TG2 can also serve as a signaling molecule and promote cell growth, drug resistance, and metastatic functions in tumor cells.

Transglutaminase-2

TG2 in Drug Resistance and Metastasis Evidence is accumulating that cancer cells that are resistant to chemotherapeutic drugs or that are isolated from metastatic sites express elevated levels of TG2. Also, there is evidence that drug-resistant and metastatic cancer cells share some common pathways. For example, cells from advanced-stage cancers accumulate a large number of genetic alterations that can render them resistant to apoptosis. Resistance to apoptosis can enable cancer cells not only to grow and survive in the stressful environment of distant tissues (i.e., to metastasize) but also to withstand the toxic effects of drugs. Moreover, cell lines selected in vitro for resistance against chemotherapeutic drugs are more metastatic in vivo, while cancer cells isolated from metastatic sites, in general, exhibit higher resistance to chemotherapeutic drugs. Based on these observations we hypothesized that aberrant expression of TG2 in drug-resistant and metastatic cancer cells may deregulate some intrinsic apoptotic pathways in order to protect cells from apoptosis. Indeed, down-regulation of endogenous TG2 by antisense RNA, TG2-specific ribozyme, or small interfering RNA (▶siRNA) could reverse drug resistance in ▶lung cancer and ▶breast cancer cells. Similarly, inhibition of TG2 by siRNA in breast cancer and malignant ▶melanoma cells augmented their response to chemotherapeutic drugs and reduced their invasiveness in laboratory experiments. In pancreatic cancer cells, inhibition of TG2 by siRNA resulted in massive accumulation of lysophagosomes and onset of ▶autophagy (type II apoptosis). These properties suggest that TG2 expression in cancer cells contributes to the development of drug resistance and ▶metastasis. Studies to elucidate the mechanisms involved in the development of TG2-mediated drug resistance in cancer cells revealed that TG2 expression augments cell survival signaling by promoting a stable interaction between cell surface integrins and the ECM proteins. Depending on the cell type, 20–30% of total TG2 can exist in complex with β-integrins (e.g., β1 β4 and β5). The association of TG2 with integrins occurs primarily at their extracellular domains and promotes their interaction with ECM ligands such as fibronectin, collagen, and vitronectin. Down-regulation of TG2 in glioblastoma cells resulted in decreased assembly of fibronectin in the ECM and cell death. Importantly, treatment of mice that had orthotopic glioblastomas with the TG2 inhibitor ▶KCC009 sensitized the tumors to chemotherapy, induced apoptosis of cancer cells, and prolonged survival of the animals. Further, the interaction between TG2 and integrins is independent of the crosslinking activity of TG2 and results in increased cell adhesion, ▶migration, and activation of downstream survival signaling pathways such as ▶focal adhesion kinase (FAK). Interestingly, TG2 can also interact directly with focal adhesion kinase and result in its

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autophosphorylation (pY397) and consequent activation of the downstream ▶PI3K and ▶Akt signaling. Activation of the ▶nuclear factor-κB (NF-κB), which plays an important role in regulating cell growth, apoptosis, and metastasis, has also been associated with increased TG2 expression in cancer cells. Tumor cells that overexpressed TG2 exhibited increased levels of constitutively active NF-κB. Activation of TG2 led to activation of NF-κB, and conversely, inhibition of TG2 activity inhibited the activation of NF-κB. Similarly, ectopic expression of TG2 caused activation of NF-κB, and inhibition of TG2 expression by siRNA abolished the NF-κB activation and rendered drug-resistant breast cancer cells sensitive to doxorubicin-induced cytotoxicity. Notably, immunohistochemical analysis of pancreatic ductal adenocarcinoma tumor samples further supported a strong correlation between TG2 expression and NF-κB activation. These observations suggest that TG2 induces constitutive activation of NF-κB in tumor cells via a novel pathway. Therefore, TG2 may be an attractive target for inhibiting constitutive NF-κB activation and rendering cancer cells sensitive to anticancer therapies. Clinical Relevance Drug resistance and metastasis are major impediments to the successful treatment of cancer. More than 90% of cancer-related deaths can be attributed to the failure of chemotherapy. On the basis of published results that drug-resistant and metastatic tumors and tumor cell lines express high levels of TG2, that TG2 expression promotes cell survival and invasion and that down-regulation of TG2 results in increased sensitivity of cancer cells to chemotherapeutic drugs and to undergo programmed cell death (apoptosis or autophagy), TG2 may offer an attractive target for treating drug-resistant and metastatic tumors.

References 1. Mehta K, Eckert R (eds) (2005) Transglutaminases: family of enzymes with diverse functions. Prog Exp Tumor Res 38:125–138 2. Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4:140–156 3. Mehta K, Fok JY, Mangala LS (2006) Tissue transglutaminase: from biological glue to cell survival cues. Front Biosci 11:173–185 4. Mehta K, Fok J, Miller FR et al. (2004) Prognostic significance of tissue transglutaminase in drug resistant and metastatic breast cancer. Clin Cancer Res 10:8068– 8076 5. Verma A, Wang H, Manavathi B et al. (2006) Increased expression of tissue transglutaminase in pancreatic ductal adenocarcinoma and its implications in drug resistance and metastasis. Cancer Res 66:10525–10533

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Transglutaminase Type-2

Transglutaminase Type-2 ▶Transglutaminase-2

Transition Metals Definition Are metallic elements that have an incomplete inner electron shell. They are characterized by multiple valences. ▶Particle-induced cancer

Transient Amplifying Cells Definition Progeny of stem cells that undergo replication, but are not able to self-renew and eventually give only one or more differentiated cell types.

Transin-1 ▶Stromelysin-1

Transit Amplifying Cells Definition Cells that are born from the asymmetric divisions of stem cells, having limited division potential. They become increasingly differentiated with successive divisions, finally giving rise to reproductively sterile, terminally differentiated cells. ▶Stem Cell Plasticity

Transition Definition Mutations from purine to purine or pyrimidine to pyrimidine.

Transitional Carcinoma ▶Nasopharyngeal Carcinoma

Transitional Cell Carcinoma JUN HYUK HONG1, S EONG J IN K IM 2 , I SAAC Y I K IM 1

1 The Cancer Institute of NJ, Robert Wood Johnson Medical School, Division of Urologic Oncology, New Brunswick, NJ, USA 2 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD, USA

Synonyms Urothelial tumor; Transitional cell carcinoma of bladder; Transitional cell carcinoma of renal pelvis; Transitional cell carcinoma of ureter; Urothelial Carcinoma, Clinical Oncology

Definition Transitional Cell Carcinoma (TCC) arises in the urothelium that covers the lining of the renal calyx, renal pelvis, ureter, bladder and part of the urethra. Although the WHO/ISUP consensus conference has determined that the term urothelial cancer is preferable to the term transitional cell cancer, the latter remains in widespread use. “Urothelial cancer” may also be confusing because cancers of other histologic types, such as squamous cancers and adenocarcinomas, also arise in the urothelium.

Characteristics Epidemiology It was estimated that in 2007, 67,160 new cases of bladder cancer would be diagnosed and 13,750 patients

Transitional Cell Carcinoma

would die of invasive bladder cancer in the United States. ▶Bladder cancer is nearly three times more common in men than in women and more than 90% of bladder cancers are TCCs. The median ages at diagnosis for TCC are 69 years in males and 71 years in females. Upper urinary tract urothelial tumors involving the renal pelvis or ureter are relatively uncommon, accounting for about 5–7% of all renal tumors and about 5% of all urothelial tumors.

Etiology One of the genetic changes that must occur for malignant transformation is the induction of oncogene. Oncogenes associated with TCC include those of the ▶RAS gene family, including P21 RAS oncogene, and up to 50% of TCCs have been claimed to have RAS mutations. Another important molecular mechanism in the process of carcinogenesis is the inactivation of tumor suppressor genes. These include that of ▶P53, the most frequently altered gene in human cancers, the retinoblastoma (RB) gene (▶Retinoblastoma Protein, Biological and Clinical Functions), and genes on chromosome 9. Overexpression of normal genes including those for EGF receptor (ERBB1) and ERBB2 (▶Epidermal Growth Factor Receptor Ligands) occur in most TCCs. Cigarette smokers have a fourfold higher incidence of TCC than do people who have never smoked (▶tobacco-related cancers). Ex-smokers have a reduced incidence of TCC, but the reduction of this risk down to baseline takes nearly 20 years. ▶Nitrosamines, 2naphthylamine and 4-aminobiphenyl are suggested as being responsible for TCC in cigarette smoke. Women treated with radiation for carcinoma of the uterine cervix or ovary, have a two- to fourfold increased risk of developing bladder cancer. Patients treated with ▶cyclophosphamide have up to a ninefold increased risk of developing bladder cancer.

Signs and Symptoms Microscopic or gross ▶hematuria is the most common presenting symptom. Patients with gross hematuria have reported rates of bladder cancer of 13–35%. With microscopic hematuria, the rates decreased to 0.5–10.5%. So, if a patient has unexplained hematuria, either microscopic or gross, cystoscopic examination is usually warranted, especially in individuals older than age 60 or younger people with a smoking history. The second most common presentation is the constellation of lower urinary tract irritative symptoms such as urinary frequency, urgency, and dysuria. These irritative symptoms usually occur with hematuria. In fact, the risk of TCC may be doubled in patients with irritative voiding symptoms that coexist with hematuria.

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Evaluations In all patients with signs and symptoms suggestive of bladder cancer, ▶excretory urography (IVU) is indicated. It is useful in examining the upper urinary tracts for associated urothelial tumors. Large bladder tumors may appear as filling defects in the bladder, but small ones may not be detected. More recently, computed tomography (CT) has replaced IVU in the evaluation of hematuria. After imaging studies, all patients suspected of having bladder cancer should have cystoscopy. Retrograde ▶pyelography should be done if the upper tracts are not visualized on IVU or CT. CT can help to assess the extent of the primary tumor and provides information about the presence of pelvic and para-aortic lymphadenopathy and visceral metastases. But CT fails to detect nodal metastases in up to 40–70% of patients who have them. MRI is not much more helpful than CT. Pelvic lymphadenectomy, which can be done with cystectomy, is the most accurate means of determining regional node involvement. The primary regions of lymphatic drainage of the bladder are the perivesical, hypogastric, obturator, external iliac and presacral nodes. As some patients with limited nodal metastases can be benefited by lymphadenectomy, bilateral node dissection should be done. The usually recommended metastatic evaluation for invasive bladder cancer includes a chest radiograph, abdominalpelvic CT, bone scan and liver function tests. A flexible cystoscope is often used for the initial diagnosis and follow-up of patients with bladder tumors. It has much less discomfort than a rigid cystoscope. Bugbee electrode devices can be inserted through a flexible cystoscope to allow destruction of small, noninvasive papillary tumors.

Ta Tis T1 T2a T2b T3a T3b T4a T4b N0 N1 N2 N3 M0

Papillary, epithelium confined Flat carcinoma in situ Lamina propria invasion Superficial muscularis propria invasion Deep muscularis propria invasion Microscopic extension into perivesical fat Macroscopic extension into perivesical fat Cancer invading pelvic viscera Extension to pelvic sidewalls, abdominal walls, or bony pelvis No histologic pelvic node metastasis Single positive node ≤2 cm in diameter, below common iliacs Single positive node 2–5 cm in greatest diameter or multiple positive nodes Positive nodes >5 cm in diameter No distant metastases

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Transitional Cell Carcinoma

Malignant urothelial cells have large nuclei with irregular, coarsely textured chromatin and can be observed on microscopic examination of the urinary sediment. This microscopic cytology is more sensitive in patients with high grade tumors or ▶carcinoma in situ (CIS). The specificity and positive predictive value of cytology are quite high. Staging (1997 AJCC-UICC, TNM Staging) As tumor stage forms the foundation for determining therapy, accurate staging is critical. The first treatment decision based on tumor stage is the presence or absence of muscle invasion. Because metastases are very rare with a superficial (non-muscle-invasive) tumor, treatment strategy can be grouped into superficial (Ta, T1 and Tis), muscle-invasive, and metastatic tumors. About 70% of bladder tumors are superficial at presentation. Of these, 70% present as stage Ta, 20% as T1, and 10% as CIS. Treatment of Superficial Bladder Cancer Local resection of a bladder tumor usually enables complete removal of the tumor and provides diagnostic information about the depth and the grade of the tumor. For this, first the bulk of the tumor and then the deep portion with some underlying bladder muscle should be resected. To detect dysplasia or CIS elsewhere in the bladder, selected site mucosal biopsies from areas adjacent to the tumor, bladder dome, trigone and prostatic urethra have been recommended. If 5-aminolevulinic acid (ALA) is administered into the bladder in conjunction with fluorescent cystoscopy, lesions invisible with normal cystoscopy can be detected. The most important issue in tumor biology of superficial tumors is recurrence and progression to higher stages. Low-grade Ta tumors recur at a rate of 50–70% and progress in about 5%. High-grade T1 lesions recur in 80% and progress in 50% of patients. The most important risk factor for progression in superficial bladder tumors is grade, not stage. Prognosis also correlates with the presence of CIS, tumor size, multiplicity, lymphovascular invasion and the configuration of the tumor (papillary vs. sessile). Of patients with CIS, 40–83% will develop muscle invasion if untreated. For T1 tumors, the depth of lamina propria invasion determined by the muscularis mucosa invasion or the extent of invasion below the urothelial surface has been known to be correlated with prognosis. CT and MRI appear to be inaccurate in determining the microscopic muscle infiltration and the minimal extravesical spread, which can also be aggravated by post-tumor recurrence (TUR) changes. To prevent recurrence and progression of bladder tumors, intravesical immunotherapy using BCG (▶Bacillus Calmette-Guerin) has been used. Treatments are generally begun 2–4 weeks after TUR and a

6-week course is usually administered. With BCG, tumor recurrence was reduced by 20–65% and progression was reduced by 23–27%. Intravesical chemotherapy using ▶Mitomycin C, ▶Doxorubicin, ▶Thiotepa, ▶Epirubicin and ▶Gemcitabine also has been administered. Treatment of Invasive Bladder Cancer The standard surgical approaches to muscle-invasive bladder cancer are radical cystoprostatectomy in the male patient and anterior exenteration in the female patient, with bilateral pelvic lymphadenectomy. Anterior exenteration in the female requires removal of the uterus, fallopian tubes, ovaries, bladder, urethra, and a segment of the anterior vaginal wall. A nerve-sparing modification has been proposed in the male patient and results in improved postoperative return of erectile function. The more prevalent the orthotopic reconstruction becomes, the stricter indications for urethrectomy have been applied. The most significant factor for the anterior urethral recurrence and local/distant failure in the male patient has been identified as prostatic urethral involvement. The estimated 5-year probability of urethral recurrence is 5% without any prostate involvement and 12–18% with prostate involvement. CIS of the bladder neck and trigone was also significantly associated with prostatic urethral involvement. In the female patient, overt cancer at the bladder neck and urethra, diffuse CIS, or positive margin at surgery should be treated by en bloc urethrectomy as a part of the radical cystectomy. The mortality rate for radical cystectomy is 1–2% and the overall complication rate is about 25%. After urinary tract diversion, bowel obstruction rate is 4–10%. Stricture of anastomosis between ureter and bowel are found in less than 3%. Depending on the type of neobladder, metabolic disorders, vitamin deficiency, and urinary tract infection can occur. As for the ▶neoadjuvant chemotherapy, recent results suggested improvement in overall survival of 5–6% among patients with locally advanced disease (stage T3 to T4a). Some reports suggest that for patients with locally advanced disease and lymph node involvement, ▶adjuvant chemotherapy may also provide a survival advantage. Due to the small numbers of patients, these results are as yet insufficient for the routine use of adjuvant therapy. Treatment of Metastatic Bladder Cancer These patients are routinely treated with systemic chemotherapy. The most commonly used agents are ▶methotrexate, ▶vinblastine, ▶doxorubicin, and ▶cisplatin (MVAC). MVAC chemotherapy produces a complete response in about 20% of patients, although long-term disease-free survival is rare. The combination of cisplatin and a newer agent, ▶gemcitabine (GC) has

Translesion DNA Polymerases

produced similar survival outcomes with less toxicity compared with MVAC. ▶Paclitaxel and ▶docetaxel have also been used in clinical trials and demonstrate response rates of 25–83%. ▶Urothelial Carcinoma, Clinical Oncology

References 1. Sengupta S, Blute ML (2006) The management of superficial transitional cell carcinoma of the bladder. Urology 67:48–54 2. Herr HW, Dotan Z, Donat SM et al. (2007) Defining optimal therapy for muscle invasive bladder cancer. J Urol 177:437–443 3. Sternberg CN, Donat SM, Bellmunt J et al. (2007) Chemotherapy for bladder cancer: treatment guidelines for neoadjuvant chemotherapy, bladder preservation, adjuvant chemotherapy, and metastatic cancer. Urology 69:62–79 4. Messing EM (2007) Urothelial tumors of the bladder. In: Wein AJ, Kavoussi LR, Novick AC, Partin AW, Peters CA (eds) Campbell-Walsh Urology, 9th edn. SaundersElsevier, Philadelphia, pp 2407–2446

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Transjugular Intrahepatic Portosystemic Shunt Definition TIPS; Artificial shunt (expandable metal stent) in the liver from the portal vein (carrying blood from the intestines to the liver) to a hepatic vein (carrying blood from the liver into the inferior vena cava). The procedure is performed via the internal jugular vein under local anesthesia with sedation. TIPS is primarily used in patients with liver cirrhosis with portal hypertension. ▶Acites

Translation Definition

Transitional Cell Carcinoma of Bladder

Process by which proteins are synthesized from messenger RNA templates. It has three stages: initiation, elongation and termination.

▶Transitional Cell Carcinoma

Transitional Cell Carcinoma of Renal Pelvis

Translation Initiation Complex Definition

▶Transitional Cell Carcinoma

Protein complex that promotes the proper association of ribosomes with messenger RNA and is necessary for the initiation of ▶translation.

T Transitional Cell Carcinoma of the Urinary Bladder Translesion DNA Polymerases ▶Urothelial Carcinoma

W. G LENN M C G REGOR James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA

Transitional Cell Carcinoma of Ureter ▶Transitional Cell Carcinoma

Definition Translesion DNA synthesis (TLS) is a highly conserved mechanism for the completion of replication of

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damaged genomes. Analogous pathways exist in bacteria, and homologs with remarkable similarity exist in all eukaryotic cells, including post-mitotic organisms such as D. melanogaster. Recent advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements, and can insert bases across from adducted templates, but with potentially mutagenic consequences.

Characteristics Most mutations induced by genotoxic carcinogens occur when a DNA template that contains residual (unrepaired) damage is replicated during S-phase of the cell cycle. Presumably, the replication complex is blocked by bulky adducts in the DNA such as those induced by ultraviolet light (UV) or a variety of chemical carcinogens (▶Adducts to DNA). As diagrammed in Fig. 1, recent advances indicate that error-prone translesion synthesis (TLS) is responsible for the majority of base substitutions induced in the DNA. TLS is defined as the incorporation of a nucleotide across from DNA damage followed by extension of the potentially mispaired primer-template. This process is undertaken by at least five accessory DNA polymerases, several of which have been purified and studied in vitro (▶DNA damage responses). The properties of these polymerases have been extensively reviewed. Based on structural homology, these polymerases fit into one of two families: the Y-family (REV1, pol η, ι, and κ), or the B-family (pol ζ). The cellular roles of this universe of polymerases are not known. In particular, the extent to which each of these polymerases participates in TLS most likely depends on the structure of a particular adduct and on the sequence context. As shown in Fig. 1, it has been suggested that pol η, ι, and/or κ inserts a base directly across from a lesion, and that pol ζ extends the mispair to form a template-primer that can be extended by pol δ. Although REV1 is a DNA polymerase, its role in mutagenesis is thought to be structural rather than catalytic. The unrestrained activity of error-prone polymerases would lead to widespread mutagenesis and genomic instability, so there are signaling mechanisms that tightly control polymerase switching events. Although not fully understood, the mechanisms used by cells to accomplish polymerase switching events at blocked primer temini have been studied most intensively in the budding yeast, Saccharomyces cerevisiae. In this organism, replication-blocking lesions in the template strand can be bypassed by proteins in the Rad6dependent DNA damage tolerance pathway. This process

Translesion DNA Polymerases. Figure 1 Model for translesion replication. The replicative polymerase complex stalls at sites of helical distortion induced by DNA damage, such as UV-induced photoproducts. The presumed ubiquitin ligase RAD18 targets a ubiquitin conjugating enzyme, RAD6, to the site of damage. There are two closely related homologs of RAD6 in higher eukaryotic cells, termed RAD6A and RAD6B. One of the targets of ubiqutination appears to be PCNA, which signals accessory polymerases in ways that are not fully understood, although at least one TLS polymerase, pol η, has a higher affinity for monoubiqutinated PCNA. Current thinking is that one of the Y-family polymerases (pol η, pol ι, or pol κ) may insert a base directly across from the lesion, but pol ζ is required to extend the resulting primer such that the pol d can continue processive DNA replication. REV1 is required for mutagenesis, but this role is probably separate from its dCMP transferase activity. Recent data indicate that REV1 may tether pol ζ to the other accessory polymerases.

prevents the collapse of stalled replication forks, and replication of the damaged template is completed by TLS with potentially mutagenic consequences, or by damage avoidance mechanisms mediated by recombination that are largely error-free. As diagrammed in Fig. 2, the ubiquitin conjugating enzyme encoded by Rad6 and the presumed ubiquitin ligase encoded by Rad18 are central to this process, since mutants cannot bypass replication-blocking lesions in the template and are sensitive to many DNA damaging agents. Insights into the biochemical function of this complex were gained when the Rad18/Rad6 complex was found to be responsible for the monoubiquitination of PCNA at K164. PCNA modified in this fashion is thought to signal translesion synthesis and further ubiquitination is thought to signal damage avoidance (Fig. 2). Although the molecular details of the signaling pathways downstream of monoubiquitination are unknown, at least one TLS polymerase, pol η, has


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Translesion DNA Polymerases. Figure 2 Regulation of lesion bypass in the budding yeast Saccharomyces cerevisiae is thought to be signaled by modification of PCNA at lysine 164 (K-164). The presence of a stalled DNA replication fork recruits Rad18, which is a presumed ubiquitin ligase, and Rad6, a ubiquitin conjugase, to the site of the replication-blocking lesion. Monoubiquination at K-164 leads to recruitment of TLS polymerases with potentially mutagenic consequences. Polyubiquitination at lysine 63 (K-63) of ubiquitin by MMS2-Ubc13 leads to damage avoidance. A proposed mechanism for damage avoidance is uncoupling of the replication fork, such that the undamaged strand is replicated for some distance beyond the blocked replication complex. The nascent strand, which has the same sequence as the damaged strand, then acts as a template for replication. The damage is thereby avoided in an error-free manner. A competing reaction is sumoylation at K164 by the SUMO (small ubiquitin modifier)-specific ligase Siz1 and conjugase Ubc9. This reaction is thought to suppress Rad52-dependent recombination and damage-induced genomic instability.

been shown to have enhanced affinity for monoubiquitinated PCNA. The strategies used by yeast cells to complete the replication of damaged genomes appear to have been conserved in higher eukaryotes, but with additional layers of complexity. For example, higher eukaryotic cells have at least two Y-family polymerases that are not found in yeast and one of these (pol κ) appears to be independent of RAD18. Human RAD18 was cloned and the protein was purified. It is a 56 kDa protein that shares 26% identity and 59% similarity with its yeast counterpart. The protein interacts with the two RAD6 homologs found in higher eukaryotes (RAD6A and RAD6B) with equal affinity and is ubiquitously expressed in all human tissues. Among the conserved regions are a RING finger motif found in the N-terminus that is required for the interaction with RAD6A/B, and a zinc finger that is presumably required for interaction with DNA. In principle, the accessory DNA polymerases and associated proteins described herein represent potential targets for antimutagenesis strategies. However, deficiency of individual polymerases may result in enhanced carcinogenesis. The most well-studied example of this is the human syndrome xeroderma pigmentosum variant, which is a skin cancer-prone condition that

results from an inherited deficiency of DNA polymerase η. This enzyme is posited to be specialized for the error-free bypass of cyclobutane dimers between adjacent thymidine bases. In its absence, recent data indicate that polymerase iota assumes its function and is error-prone when doing so. Unexpectedly, however, when both polymerases are deficient in mouse models, UV-induced skin cancer is accelerated despite reduced UV-induced mutant frequencies in the double knockout. These data support a role for polymerase iota as a tumor suppressor separate from its role in TLS.

T References 1. Friedberg EC, Lehmann AR, Fuchs RP (2005) Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol Cell 18:499–505 2. Dumstorf CA, Clark AB, Lin Q et al. (2006) Participation of mouse DNA polymerase iota in strandbiased mutagenic bypass of UV photoproducts and suppression of skin cancer. Proc Natl Acad Sci USA 103:18083–18088 3. Wang Y, Woodgate R, McManus TP et al. (2007) Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations. Cancer Res 67:3018–3026

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Translesion Synthesis

Translesion Synthesis Definition Error-prone DNA-repair process that involves a switch to low fidelity DNA-dependent polymerases to bypass an unhooked interstrand crosslink. Since both DNA strands are damaged at the site of such lesion, the antisense strand cannot serve as a template for repair. ▶Translesion DNA Polymerases

unbalanced translocations chromosomal material, often the translocated material from one of the nonhomologous chromosomes is deleted. . Translocation of proteins is the regulated movement of proteins or other molecules from one cellular compartment or organelle to another.

Translocation ETS Leukemia Gene ▶ETV6

Translin Definition TSN; A Protein encoded by the TSN gene is reported to recognize single-stranded DNA ends of staggered breaks that may occur at recombination hotspots. A role in recombination repair has been suggested, but has still to be confirmed. ▶ALU Elements

Translocation Non-homologous Definition Non-homologous translocation; rearrangement of chromosomes that results in the fusion of two chromosomal segments that are not normally attached to one another, often resulting in a microscopically visible alteration of karyotype; unbalanced translocations often involve deletion of genetic material.

Translocase Definition Is an enzyme that translocates lipids between the two leaflets of the lipid bilayer. ▶P-glycoprotein

Translocation

Translocation Reciprocal Definition Reciprocal translocation; exchange of chromosomal segments between two chromosomes from different chromosome pairs, resulting in the conservation of all participating chromosomal segments; no phenotypic change will occur when the translocation is balanced, which means at the same position of the two homologous chromosomes.

Definition

. Translocation of chromosomes is the illegitimate recombination between nonhomologous chromosomes, a rearrangement in which part of a chromosome is detached by breakage and then becomes attached to another chromosome. The translocation may or may not be reciprocal; in reciprocal balanced translocations, genetic material is exchanged without loss between nonhomologous chromosomes; in

Translocation t(14;18) Definition Chromosomal translocation commonly associated with non-Hodgkin B-cell lymphomas. This chromosomal

Transphosphorylation

translocation event places the ▶Bcl-2 gene into juxtaposition with powerful enhancer elements associated with the IgH locus, causing a transcriptional deregulation of the Bcl-2 gene and resulting in elevated levels of Bcl-2 mRNA and Bcl-2 protein production.

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Transmembrane Protein Type II Definition A transmembrane protein that exposes its amino terminus into the cytosol. ▶Endoplasmic Reticulum Stress

Transmembrane Definition Referring to the domain of a protein that is threaded through a membrane and therefore exists in the hydrophobic environment of a lipid bilayer.

Transmembrane 4 Superfamily Protein

Transmembrane Signaling Definition The plasma membrane of mammalian cells provides a protective barrier and many extracellular agonists are unable to enter the cell. Instead, they recognize and activate specific receptors at the cell surface that in turn stimulate activity of proteins (e.g., lipid-hydrolyzing enzymes) present inside the cell and in this way transmit the signal across the membrane, i.e., from extracellular to intracellular environment.

▶Metastasis Suppressor KAI1/CD82

Transmigration Assay Transmembrane Domain Definition The portion of a protein that spans the lipid bilayer of the cell membrane. ▶Prostate-Specific Membrane Antigen (PSMA)

Transmembrane Protein Type I

Definition Is an in vitro assay where cells are placed on one side of a chamber containing a porous membrane (usually coated with an artificial ▶extracellular matrix), a hormonal stimulus is applied, and the rate at which cells migrate across the membrane is determined. It measures cell invasiveness, or the ability of metastatic cells to escape from a tumor.

Transphosphorylation

Definition

Definition

A transmembrane protein that exposes its carboxyl terminus into the cytosol.

When two receptor tyrosine kinases are brought into close proximity to each other, the tyrosine kinase domain of one of the receptors phosphorylates the kinase domain on the other receptor. Likewise, the

▶Endoplasmic Reticulum Stress

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Transplacental Carcinogen

freshly phosphorylated kinase domain is now more active and can phosphorylate and activate the kinase domain on the remaining receptor.

Transpupillary Thermal Therapy Definition

Transplacental Carcinogen Definition

Near-infrared frequency laser used to concentrate thermal injury on the site of interest (e.g., tumor) leaving the surrounding tissue generally undisturbed. ▶Uveal Melanoma

A substance that is able to cross the placenta, inducing cancer in the offspring. ▶Diethylstilbestrol

Transporter Nomenclature Definition

Transurethral Resection Definition TUR; Endoscopic operation to remove tumor tissue from the bladder or the prostate via the urethra. ▶Urothelial Carcinoma, Clinical Oncology

Refers to the names used to describe the various biological transporters and pumps used to move a drug through the body. ▶ADMET Screen

Transporters Definition Are biological protein pumps which move a chemical from one place in the body to another.

Transversion Definition A point mutation in which a purine base is mutated to a pyrimidine base or vice-versa. Mutations from purine to purine or pyrimidine to pyrimidine are transitions. ▶UV Radiation

▶ADMET Screen ▶ABC Drug-Transporter

Trastuzumab Transposon Definition A mobile DNA element that is capable of moving within the genome, either by copying itself or by cutting itself out of its original site and inserting in a new location. ▶Retroviral Insertional Mutagenesis

Definition

Synonym ▶Herceptin; is a humanized IgG1κ monoclonal antibody specifically binding to HER-2. Human epidermal growth factor 2 (▶HER2/neu), also known as ErbB-2, is a member of the epidermal growth factor receptor (ErbB) family and is notable for its role in the pathogenesis of ▶breast cancer and as a target of treatment. It is a cell membrane receptor tyrosine kinase normally involved in the signal transduction pathways leading to cell growth and differentiation.

Trefoil Factors

HER2 is named because it has similar structure to human epidermal growth factor receptor, or HER1. ErbB2 was named for its similarity to ErbB (avian erythroblastosis oncogene B), the oncogene later found to code for EGFR. Gene cloning showed that neu, HER2, and ErbB2 were the same. Trastuzumab is approved for treatment of HER2 overexpressing metastatic breast cancer in combination with chemotherapy and as single-agent therapy in those patients with metastasic breast cancer who do not respond to chemotherapy. ▶Monoclonal Antibody Therapy ▶Drug Design ▶Herceptin

bTrCP Definition Beta-transducin repeats-containing proteins (βTrCP) serve as the substrate recognition subunits for the SCF complexes. βTrCP interact with substrates phosphorylated within the DSGXX(X)S destruction motifs. SCFβ-TrCP mediate ubiquitination and proteasomal degradation of phosphorylated substrates. ▶Anoxia

Treatment-refractory Germ Cell Tumors ▶Platinum-Refractory Testicular Germ Cell Tumors

Trefoil Factors C HRISTIAN G ESPACH INSERM U. 673, Paris, France; Laboratory of Molecular and Clinical Oncology of Solid tumors, Faculté de Médecine, Université Pierre et Marie Curie-Paris 6, Paris, France

Synonyms Trefoil peptides; SMAD-4/DPC4; TFF

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Definition Trefoil factors (TFF) belong to a family of heat, acid, and protease-resistant regulatory peptides ubiquitously expressed in brain, blood, and peripheral organs. In inflammatory conditions and generation of cancer lesions, they are induced, lost, or modified by gene silencing and somatic mutations. Thus, TFF overexpression or invalidation is either the consequence or the causal origin of human solid tumors and their ▶progression to metastatic situations. While the TFF receptors or recognition systems are not still clearly identified, TFF are involved in mucosal and epithelial cell cytoprotection, wound healing, cancer cell survival and ▶invasion, ▶angiogenesis, through several oncogenic pathways involved in neoplasia. Finally, TFF are now considered as multifaceted factors with beneficial and pejorative functions on inflammatory and cancer diseases, according to their dual and divergent impacts at early and late stages of these pathological states.

Characteristics TFF Discovery and Expression Since the discovery and molecular annotation of the trefoil factor pS2 (TFF1) in human breast cancer, much attention has been devoted on TFF1 and its structurally related protease and acid-resistant factors spasmolytic polypeptide (SP-TFF2) and intestinal trefoil factor (ITF-TFF3). These TFF contain either one (TFF1 and -3) or two (TFF2) trefoil domains delimited by three disulphide bridges. TFF are involved in the stabilization of the mucus layers secreted by mucosal epithelial cells. The three human trefoil genes are located in a cluster region of 55 kb on chromosome 21q22.3. A novel two trefoil domains Bm-TFF2 protein activating platelet aggregation has been recently purified from the frog Bombina maxima skin secretions. TFF are widely expressed in brain, the urogenital system (breast, kidney, ▶prostate), the lymphoid tissue, the respiratory and the digestive tract (esophagus, ▶stomach, intestine, exocrine and endocrine ▶pancreas, and liver), in conjunctival goblet cells and pterygium. TFF1 and -2 are predominantly detected in the normal stomach whereas TFF3 is found mainly in the small and large intestine. TFF are regulated via genetic, ▶epigenetic, and tissue-specific mechanism including amplification of the chromosomal region 21q22 harboring TFF family genes in ▶cholangiocarcinoma, promoter methylation, chromatin modification, histone H3 acetylation, and transcription factors downstream signaling pathways involved in cellular ▶stress responses, ▶inflammation, and cancer. These pathways include gastrin and bFGF growth factors, the ▶interleukin 6 family cytokine receptor gp130/▶STAT1–3 and ▶SHP-2/▶ERK cascades, ▶ras, the ▶hypoxiainduced HIF-1α transcription factor, allergens in lungs, nuclear ▶estrogen receptors and ▶GATA-6, ▶NF-κB,

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peroxisome proliferator-activated receptor gamma (PPAR-γ), hepatocyte nuclear factor 3 (HNF3), the homeodomain transcription factor CDX2, and the activator protein ▶AP-1 via the negative control of COBRA1, the cofactor of ▶BRCA1 (breast cancerassociated protein 1) involved in ▶DNA damage repair. TFF are secreted by the gastrointestinal mucosa in a mucus, inflammation, and ulcer-associated cell lineage (UACL)-dependent manner. Chronic inflammation and ulceration in the gastrointestinal tract is associated with the development of the reparative UACL from mucosal ▶stem cells. UACL was originally described as pyloric metaplasia in the ileum, reflux esophagitis associated with Barrett’s esophagus, peptic ulcer in the stomach, and chronic cholecystitis. TFF display multifaceted roles in mucosal repair and during cancer progression. TFF in Mucosal Repair and Protection It is now well accepted that TFF are involved in maintenance and repair of the mucosal barrier, wound healing, and cytoprotection during hypoxia and transient inflammatory situations in experimental ulcerative colitis. Local administration of recombinant TFF and ectopic expression of TFF3 in cellular models and transgenic animals supported this general idea of a cytoprotective role for TFF in mucosal repair. Both epithelial and stromal cells contribute to wound healing and mucosal repair. Consistent with a signaling role of TFF in mucosal protection, the tetraspanin family member Vangl1 is involved in the migratory response to TFF3 through Ser/Thr phosphorylation in intestinal epithelial cells. In addition TFF3 improved intestinal crypt stem cells survival following combined radiation and ▶chemotherapy in both wild-type and TFF3−/− knockout transgenic mice. TFF in Chronic Inflammation and Cancer Progression Although some studies argue for a therapeutic potential of TFF in mucosal injury and wound healing, recent advances in the field support their adverse effects during chronic inflammation and cancer progression. Persistent inflammatory situations initiate several genetic, molecular, and cellular dysfunctions associated with tumor promotion and cancer progression. Notably, selfinduction and cross-talk between TFF at their regulatory sequences have been described as a molecular signature of chronic inflammatory situations and neoplasia. For example, TFF1 exerts divergent functions in the digestive mucosa. In the stomach, gastric TFF1-deficient mice develop antropyloric adenomas and carcinomas, suggesting that TFF1 is a candidate gastric-specific ▶tumor suppressor gene to protect the mucosa against repetitive injury from digestive secretions, ulceration, and chronic inflammation induced by acid, proteases, and pathogens, such as ▶Helicobacter pylori. Somatic mutations and loss of heterozygosity (LOH) of the TFF1

gene is observed in human gastric cancers, in association with TFF3 overexpression. In coherence with these observations, ▶cyclooxygenase (COX)-2 was strongly induced in pyloric adenomas induced by genetic ablation of the TFF1 gene in mice. Similarly, COX-derived products are reported to exert beneficial roles in mucosal protection and wound healing, but deleterious functions during chronic inflammation and neoplasia. Conversely, in the normal human colon TFF1 is absent but is induced at high levels in Crohn disease, colitis, and colorectal cancers. It is therefore likely that TFF1 is a cancer progression factor in the human colon, according to its aberrant expression and transforming functions at the adenoma and carcinoma transitions (Fig. 1). Thus, TFF exert opposing functions, one counteracting transient inflammatory situations and the other linked to pejorative functions, in cooperation with other dominant genetic and molecular alterations during the neoplastic progression in the human colon. These include the cancer predisposition pathways controlled by ▶Wnt/▶APC/ β-catenin, ▶TGF-β/▶SMAD-4, ras, ▶src, and deleted in colon cancer (DCC). Validation of this model can be explored in transgenic animals harboring selectively these oncogenic alterations, in cooperation with forced expression of TFF1 in intestinal stem cells, through intestinal promoters that are functional in this cellular compartment, such as the villin promoter (pVIL) and the carcinoembryonic antigen (p▶CEA) regulatory regions. The emergence of colorectal adenocarcinomas (ADK) is a complex multistep process linked to genomic and ▶chromosomal instability (CIN) and LOH, ▶microsatellite instability (MSI), DNA ▶aneuploidy, and generalized deregulation of gene expression and signal transduction pathways. The first mechanism, which accounts for 80% of sporadic cases, is connected with CIN and LOH targeting the tumor suppressors APC (5q), ▶TP53 (17p), DCC (18q), and TGFβ pathway signaling elements SMAD-2 and SMAD-4 (▶SMAD-4/DPC4) at 18q. Sporadic MSI tumors are frequently mucinous, predominantly localized in the right colon, and generally diploid. In MSI patients, alterations in TGFβ-RII, IGF receptors IGF-RII, β-catenin, TCF-4, and E2F transcription factors, as well as loss of the ▶PTEN tumor suppressor, and the ▶apoptosis regulator BAX are frequently reported. Sporadic cancers are also driven by epigenetic mechanisms, hypo and ▶hypermethylation of promoter genes encoding cancer markers and/or effectors, such as TFF, MUC-5AC, COX-2, ▶proteaseactivated receptors PAR-1, ▶IGF, ▶p21/p16/cyclin D1. Dominant activation of protooncogenes by point mutations or constitutive activation by other oncogenic pathways are also frequently observed at early stages (▶ACF, polyps: src, ras, ▶c-myc) and late stages concomitant with cellular invasion, angiogenesis, and ▶metastasis (▶c-Kit, ▶EGF-R, ▶VEGF, the hepatocyte growth factor receptor ▶MET, src, and many others).

Trefoil Factors

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Trefoil Factors. Figure 1 Genetic and molecular alterations linked to the multistep progression of familial and sporadic human colorectal cancers.

Familial adenomatous polyposis (FAP) is induced by mutations of the APC gene, a defect that contributes to CIN and appearance of more than 100 colorectal adenomas. Molecular alterations in other elements of the Wnt pathways are also concerned in sporadic ▶colon cancers, including Wnt-2, Axin, β-catenin, and TCF4 transcription factor. Nonpolyposis form of the hereditary colon cancer (HNPCC) is more frequent than FAP, and is caused by germ cell mutations that invalidate the DNA repair systems. DNA ▶mismatch repair is deficient in 90% of the HNPCC patients. The mutations concern mostly the hMSH2 and hMLH1 DNA repair enzymes, less frequently hPMS1 and hPMS2. Such genetic and molecular changes lead to the formation of aberrant crypt foci (ACF), which precede the appearance of ▶premalignant adenomas anchored in the colon mucosal wall. The next stage is the evolution of the adenoma toward more aggressive lesions (ADK), and irreversible acquisition of dominant and anarchic functions, chronic inflammation, oxidative ▶DNA damage, autocrine and paracrine regulatory loops linked to IGF-R, VEGF-R, EGF-R ligands, induction of thrombin protease-activated receptors PAR-1, trefoil factor pS2 (TFF-1), and the immediate response gene COX-2. Aberrant Expression of TFF as Clinical Markers of the Neoplasia TFF are involved in the neoplastic progression in human epithelial tumors according to their ability to confer several transforming functions including

resistance to apoptosis, induction of cellular scattering, anchorage-independent growth in soft agar, proinvasive and proangiogenic activities in vitro and in vivo. Both TFF1 and TFF3 reduced apoptosis induced by serum privation and loss of cellular adhesion (▶Anoikis), a major response linked to cancer cell transformation, survival, and dissemination. Accordingly TFF are connected with several oncogenic and tumor suppressor elements such as ▶E-cadherins, EGF-R, the ▶RhoA-▶ROCK axis, ▶PI3-kinase (▶PI3K), phospholipase C, COX-2, nitric oxide synthase 2, NFkB, STAT3, and ▶Cdc25. The nuclear phosphatases Cdc25A and B are associated with hypergrowth activity and control of the ▶G2/M checkpoint in response to DNA damage and repair. This is supported by clinical investigations on aberrant expression of TFF in human solid tumors of the prostate (TFF3), premalignant▶ changes and neuroendocrine differentiation in human prostate cancer (TFF1), human hepatocellular carcinomas (promoter hypomethylation of the TFF3 gene), hepatolithiasis, and cholangiocarcinomas (TFF1–MUC5AC), primary mucinous carcinomas of the skin (TFF1–TFF3), and ulcerating Barrett esophagus, a precancerous lesion considered as a gastric-type metaplasia. Barrett esophagus is characterized by the specific expression of the gastric-type markers TFF1 and MUC5AC with high levels and strong colocalization in the surface epithelium. In contrast, TFF3, MUC6, and MUC5B were found in the deeper glandular structures. Similarly, gastric metaplasia of the duodenum (GMD) is characterized by replacement of the intestinal epithelium with gastric-type mucus cells, villus

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damage and atrophy, and is frequently found in association with inflammation and gastritis induced by H. p. GMD expressed TFF1, TFF2, and the colon cancer associated ▶mucin MUC5AC, a marker of ACF now considered as precancerous lesions in the large bowel. Progressive loss of TFF1 and TFF2 associated with reciprocal induction of TFF3 is likely to be involved in the early stages of gastric ▶carcinogenesis. In the normal gastric mucosa, TFF2 is expressed in surface mucus neck cells. Decreased TFF2 expression in chronic atrophic gastritis possibly attributes to the decrease in the number of surface epithelial cells expressing TFF2. Reexpression of TFF2 in gastric epithelial dysplasia implies that TFF2 possibly contributes to the progression of ▶gastric carcinoma. Recently, it has been reported that TFF3 induction in gastric tumors correlated with an aggressive phenotype with advanced stages, infiltrative growth pattern and positive lymph nodes. TFF3 is now considered as a marker of poor prognosis in human gastric carcinomas and is associated with aggressive behavior and lethality of colon cancer cells in rats. Ectopic expression of TFF3 promoted the invasive phenotype in Rat-2 fibroblast cells associated with upregulation of β-catenin, MMP-9 matrix ▶metalloproteinase, and downregulation of the tumor suppressor gene product ▶E-cadherin involved in β-cateninassociated ▶adherens junctions. In fact, several reports suggest that TFF participate to morphogenesis and differentiation programs for epithelial cells in breast, gastrointestinal tract, and lungs. Conversely, depletion of TFF3 in the human gastric cancer cell line SNU-1 that expresses TFF3 resulted in decreased ability to form colonies in soft agar and in a marked increase in apoptosis and chemosensitivity to anticancer agents. The situation is probably more complicated since both TFF2 and TFF3 are induced in advanced gastric carcinomas and linked with neoangiogenesis, thus having a negative impact on patient survival, and are independent predictor of disease recurrence. Both TFF1 and TFF2 are strongly expressed in diffuse-type gastric cancers, suggesting that the academic definition of TFF1 as a gastric-specific tumor suppressor gene should be applied in relation with the corresponding status of TFF2/TFF3, and the clinicopathologic context and oncogenic status of human gastric tumors. Several reports indicate that TFF2 is a gastric marker of tumor metastasis frequently upregulated in diffuse gastric cancers in correlation with decreased survival. TFF2-expressing cells are upregulated in the stomach of Helicobacter-infected mice and seem to give rise to invasive cancerous lesions. Consequently, both TFF2 and COX-2 are overexpressed in patients with H. ▶pylori-induced chronic fundic gastritis in association with dysplasia. In the established H. felis/C57BL/6 mouse model of gastric cancer induced by chronic infection with Helicobacter felis, bone marrow-derived mesenchymal progenitor cells, but not

hematopoietic stem cells, are recruited to site of gastric mucosa injury and inflammation. This proliferative zone gives rise to spasmolytic expressing metaplasia (SPEM) and differentiation toward an epithelial phenotype, evidenced by positive staining for TFF2 and the epithelial cell cytokeratin KRT1–19 in deep antral and fundic glands. Thus, experimental Helicobacter infection can give rise to a new mucosal microenvironment in the infected gastric mucosa following upregulation of the stem cell factor SCF-1 (the ligand of the c-kit tyrosine kinase) and the ▶chemokine ▶SDF-1 binding the ▶G-proteins coupled receptor CXCR4, two key factors involved in the mobilization of bone marrow progenitors and cancer metastasis. It remains to be elucidated whether subpopulations of human gastric cancers may originate from the neoplastic transformation of bone marrow progenitors with gastric mucosal cell gene expression pattern. In addition, SPEM was suppressed by invalidation of the ▶TNF-α gene in Tnf−/− K19C2mE transgenic animals expressing simultaneously COX-1/-2 in gastric mucosa via the cytokeratin 19 gene promoter. Finally, we cannot exclude the possibility that illegitimate and constitutive expression of TFF2 by mesenchymal bone marrow stem cells may also target the gastric progenitor niche for metaplasia and dysplasia. Notably, SPEM is associated with gastric H. ▶pylori infection, aberrant expression of the mucin 6 (MUC-6) gene, and progression of human gastric adenocarcinoma. Therefore, the combined loss of the gastric-specific tumor suppressor gene TFF1 with induction of TFF2 and TFF3 provide insights into the complex mechanisms underlying the biological significance and versatility of TFF in gastric cancer progression and neoplasia. In breast cancer, TFF1 and TFF3 but not TFF2 were identified as informative markers for the detection of ▶micrometastases in axillary lymph nodes and blood. Significance analysis of microarrays identified a positive correlation between TFF1 overexpression and breast cancer metastasis to bone in a cohort of 107 patients with primary breast tumors who were all lymph node negative at the time of diagnosis. The involvement of the FGF signaling pathway was also incriminated in preference of tumor cells that relapse to bone. The fact that TFF1 may contribute to tumor relapse to bone is underscored by its abundant presence in breast cancer micrometastases. In addition, morphogenetic effects have been attributed to TFF2 in human breast cancer cells following the induction of highly complex branched ductular structures typical of migratory, invasive, and survival functions, in a TFF1-dependent manner. TFF3 was also expressed in breast ductal and lobular breast carcinomas in situ, and invasive lobular carcinomas. While TFF1 is a surrogate indicator for the response to antihormonal therapy and favorable outcome in estrogen receptor-α (ERα)-positive and well-differentiated breast cancers, its deregulated expression is now considered to contribute

TRF1-interacting, ankyrin-related ADP-ribose polymerases

to the progression of both ER-α-positive and -negative human breast cancers. Of note, plasma levels of TFF were found elevated in patients with advanced prostate cancer. Conclusions TFF are now considered as valuable therapeutic tools for the treatment of injured mucosal epithelial cells and protection against mucosal and tissular damages following transient injury and other damages caused by radiation therapy and chemotherapy. Selective loss, induction, and overexpression of TFF observed during inflammatory processes and neoplasia deregulate TFF signaling cross-talks and signals, and compensatory functions linked to TFF. The molecular complexity of TFF is further illustrated by their ability to form covalent disulphide-linked dimers in vitro and in vivo. The possibility that TFF could form heterodimers adds further complexity for their relevance in receptor and signal transduction. Despite increasing interest on TFF in molecular research and clinical applications, TFF are still orphan signaling peptides facing unknown receptors in the classical definition of receptor-mediated signal transduction pathways from the plasma membrane, cytoplasmic and nuclear domains, and vice versa. Recent advances in the field pointed the discovery of new TFF-binding proteins apparently linked to mucosal protection, such as MUC-5AC and the gastrokine-1-like peptide blottin. It is conceivable that TFF are not released via normal secretory pathways in inflammatory situations and neoplasia. The epithelial cell polarity and its normal microenvironment with stromal and vascular cells, immune cells, and extracellular matrix components and their receptors, are lost during cancer progression. In this case, it is tempting to assume that TFF are in the abnormal situation interact with key signal transducers and cancer-associated stromal cell lineages during cancer progression, via illegitimate, pejorative, and persistent mechanisms. In this scenario, TFF were shown to signal through distinct transduction pathways following external addition of the peptides versus ectopic expression. Future attempts and new strategies to identify the stricto sensu TFF receptors and direct transducers are therefore expected in order to exploit the positive facets of TFF for therapeutic purpose and to fight against their deleterious functions in pathological states.

4. Thim L, May FE (2005) Structure of mammalian trefoil factors and functional insights. Cell Mol Life Sci 62 (24):2956–2573 (Review) 5. Rodrigues S, Rodrigue C, Attoub S et al. (2006) Induction of the adenoma-adenocarcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells. Oncogene 25:6628–6636

Trefoil peptides ▶Trefoil Factors

Treg Definition

▶T Regulatory cells; are a subset of CD4+ T lymphocytes responsible for the regulation of immune responses. Treg have a defined phenotypic profile (CD4+ CD25highFoxp3+) and can suppress functions, (e.g. proliferation or cytokine production) of other (responder) cells. Treg are expanded in the periphery and at tumor sites of patients with cancer. In patients with autoimmune disease, Treg are few or, if present, mediate no suppressor functions. Thus, the quantity and quality of Treg control the immune responsiveness in health and disease. ▶Autoimmunity and Prognosis in Cancer ▶Regulatory T Cells

Tremolite Definition

Is an amphibole form of ▶asbestos with a basic composition of calcium and magnesium silicates. Upon increased iron content, its color changes from C reamy white to dark green. The iron-containing samples are carcinogenic.

References 1. Lefebvre O, Chenard MP, Masson R et al. (1996) Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 274:259–262 2. Taupin D, Podolsky DK (2003) Trefoil factors: initiators of mucosal healing. Nat Rev Mol Cell Biol 4:721–732 3. Emami S, Rodrigues S, Rodrigue CM et al. (2004) Trefoil factors (TFFs) and cancer progression. Peptides 25:885–898 (Review)

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TRF1-interacting, ankyrin-related ADP-ribose polymerases ▶Tankyrases

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TbRI and TbRII

TbRI and TbRII

Trichothiodystrophy

Definition

Definition

Type I and type II serine/threonine kinase receptors of ▶TGF-β.

PIBIDS, this is an acronym for photosensitivity, ichthyosis, brittle hair and nails, intellectual impairment, decreased fertility and short stature. Disease related to ▶nucleotide excision repair. Patients with PIBIDS have mutations in the ▶xeroderma pigmentosum (XP) genes XP-B or XP-D.

▶Transforming Growth Factor Beta

Tribbles Homologue 3 Definition TRB3; Also synonym TRIB3, NIPK and SKIP3. A pseudokinase that has been shown to regulate several targets potentially involved in controlling tumor growth including Akt. ▶Cannabinoids ▶Akt Signal Transduction Pathways

Trident ▶Forkhead Box M1

Trifunctional Antibody Definition A hybrid bispecific IgG antibody containing 2 Fab segments with specificity for different antigens and Fc portions that may be derived from different species.

Trichilemmoma Definition Benign tumor of the hair follicle infundibulum. Multiple trichilemmomas are present on the face in ▶Cowden Syndrome.

Triglyceride Definition A glyceride in which the glycerol is esterified with three fatty acids. It is the main constituent of vegetable oil and animal fats.

Trichostatin A

▶Fatty Acid Synthase

Definition TSA; Is a potent reversible inhibitor of the family of ▶histone deacetylases (HDAC). Was originally reported (1976) as a fungistatic antibiotic obtained from a culture broth of Streptomyces platensis. TSA has some uses as an anti-cancer drug.

4´,5,7-Trihydroxyisoflavone ▶Genistein

TRK-A

3,4´,5-Trihydroxystilbene ▶Resveratrol

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Trivalent Chromium (Cr+3) Definition Chromium ion in the highly reduced state, with a valence of +3. This is thermodynamically the most stable state of chromium ion.

Trinucleotide Repeat Definition Any three nucleotide sequence that is repeated. CGG, CAG, and GAA expansions are associated with human genetic disorders. ▶Fragile Sites

Triple Negative Breast Cancer Definition Breast cancer that lacks expression of estrogen receptor and progestaron receptor and amplification/overexpession of HER2; a feature of many basal-like breast tumors that is often used as a synonym for basal-like breast cancer. ▶Basal-like Breast Cancer

Tripterine ▶Celastrol

Trisomy

▶Chromium Carcinogenesis

Trk Family Tyrosine Kinase Receptors Definition Includes Trk (synonym TrkA), TrkB, and TrkC; they are responsible for mediating the tropic effects of the ▶NGF family of ▶neurotrophins. Nerve growth factor (NGF) specifically recognizes Trk, a receptor identified in all major NGF targets, including sympathetic, trigeminal, and dorsal root ganglia as well as in cholinergic neurons of the basal forebrain and the striatum. ▶Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) specifically activate the TrkB tyrosine kinase receptor. TrkB transcripts encoding this receptor are found throughout multiple structures of the central and peripheral nervous system. Trk was originally cloned as an oncogene fused with the tropomyosin gene in the extracellular domain. The rearranged Trk oncogene is often observed in non-neuronal neoplasms such as ▶colon cancer and ▶papillary thyroid carcinoma, while the signals through the receptors encoded by the proto-oncogene Trks regulate growth, differentiation and apoptosis of the tumors with neuronal origin such as ▶neuroblastoma and ▶medulloblastoma. The intracellular Trk signaling pathway is also different depending on the Trk family receptors, cell types and the grade of transformation Furthermore, developmentally programmed cell death of neuron, which is largely regulated by ▶neurotrophin signaling is at least in part controlled by tumor suppressors ▶p53 and ▶p73. Thus, Trk and its downstream signaling function in both ontogenesis and oncogenesis.

TRK-A Definition

Definition

Member of the Trk Family Tyrosine Kinase Receptors.

Presence of three copies of a particular chromosome instead of the normal two.

▶TRK

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Troglitazone

Troglitazone Definition A synthetic that can suppress cancer expression as a ligand for peroxisome proliferator-activated receptor-γ. ▶Fucoxanthin

Trousseau Syndrome Definition Armand Trousseau initially described the phenomenon that malignancies are sometimes associated with spontaneous thrombotic events. Unexplained thrombotic episodes may occasionally lead to the diagnosis of cancer ▶Tumor-Endothelial Cross-talk

TROP-1 ▶EpCAM

Trophoblast

Trp63 ▶p53 Family

Definition Extra embryonic layer of epithelium that forms around the mammalian blastocyst and attaches the embryo to the uterus wall. ▶Cancer-Germline (CG) Antigens

Tropism

Trp73 ▶p53 Family

Trx

Definition Generally movement of an organism or cell; for viral vectors: Targeting of specific tissues or cell types within an organism. The predilection of a virus or another pathogen to invade, and replicate in, a particular cell type or tissue or organism.

Definition

▶Thioredoxin.

▶Oncolytic Virotherapy

Trypsin Tropomyosin Definition Structural protein involved in muscle contraction.

Definition Serine proteinase expressed not only in the pancreas for food digestion but also by uPA (urokinase-type plasminogen activator). ▶Proteinase-Activated Receptor

tTGase

Tryptase Definition A protein secreted by mast cells and related blood and tissue cell types, involved in allergic reaction, and used as a marker for mast cell activation. ▶Mastocytosis

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TSH Receptor Definition The receptor for thyroid stimulating hormone (TSH), which is also called thyrotropin. Encoded by a gene on chromosome 14q, TSHR is largest of all known glycoprotein hormone receptors. It is one of the primary antigens in autoimmune thyroid disease. Autoantibodies to TSHR act as TSH agonists in ▶Graves disease and as TSH antagonists in ▶Hashimoto thyroiditis. ▶Thyroid Carcinogenesis

TSC Definition

▶Tuberous sclerosis complex. ▶Tuberous Sclerosis Complex

TSP Definition Thrombospondin.

TSC1 ▶Hamartin

TSR Definition

▶Thrombospondin type 1 repeat. ▶Thrombospondin

TSH Definition Thyroid-stimulating hormone; Synonym thyrotropin; Is secreted from cells in the anterior pituitary called thyrotrophs, finds its receptors on epithelial cells in the thyroid gland, and stimulates that gland to synthesize and release thyroid hormones. ▶Thyroid Carcinogenesis

TTF-1 Definition Thyroid transcription factor 1 is a marker of thyroid and lower respiratory epithelium often used in immunohistochemistry to detect tumors arising from the thyroid or lung. ▶Hurthle Cell Adenoma and Carcinoma

TSH Definition Thyroid-stimulating Hormone.

tTGase ▶Transglutaminase-2

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Tuberin

Tuberin Definition

Heterodimer partner of ▶hamartin, forming the ▶Tuberous sclerosis complex (TSC). Hamartin is encoded by the TSC1 gene, tuberin by TSC2. These two proteins function within the same pathway(s) regulating ▶cell cycle, cell growth, ▶adhesion, and ▶vesicle trafficking.

Tuberous Sclerosis Complex A NDREW R. T EE , J ULIAN R. S AMPSON , J EREMY P. C HEADLE Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, UK

Synonyms TSC

Definition

▶Tuberous sclerosis complex (TSC): An autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 genes and characterized by the development of hamartomatous growths in multiple organ systems.

Characteristics Clinical Aspects Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that occurs in up to 1 in 6,000 live births, without apparent ethnic clustering. TSC is characterized by the development of unusual tumor like growths, called hamartomas, in a variety of tissues and organs. Subependymal giant cell tumors in the brain occur in about 1 in 10 individuals with TSC, with reported incidence ranging from 6.1 to 18.5%. On serial imaging, these tumors appear to arise from subependymal nodules which are believed to be present in 88–95% of individuals with TSC. Involvement of the brain is associated with some of the most problematic clinical manifestations of TSC, including intellectual handicap, epilepsy, and abnormal behavioral phenotypes, particularly autism and attention deficit disorder with hyperactivity. Other organs commonly and significantly involved in TSC include the skin, kidneys and heart, where associated hamartomatous growths include facial angiofibromas, subungual fibromas, forehead plaques, shagreen patches, renal angiomyolipomas and cysts and cardiac rhabdomyomas (Fig. 1).

Renal cell carcinoma (RCC) occurs at an earlier age and is more frequently multifocal in individuals with TSC than in the general population, although it is still uncommon (100–280 nm).

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UV Radiation

Characteristics Sunlight consists of visible light (400–700 nm), infrared radiation (>700 nm) and UV radiation. The quality (spectrum) and quantity (intensity) of sunlight are modified during its passage through the atmosphere. Solar UV radiation at ground level represents about 5% of the total solar energy; the radiation spectrum is between 290 and 400 nm, and is comprised of approximately 95% UVA and 5% UVB; UVC is completely filtered out by the Earth’s atmosphere. The spectrum of solar UV radiation to which an individual may be exposed varies with latitude, altitude, ground reflectance, season, time of day, weather, stratospheric ozone and other atmospheric components such as air pollution. For most individuals, solar radiation is the major source of exposure to UV radiation. ▶Sunbeds and sunlamps used for tanning purposes are the main source of deliberate exposure to artificial UV radiation. Solar radiation is classified as a Group 1 carcinogen by the International Agency for Research on Cancer. Cancers of the Skin Skin cancer (see ▶skin carcinogenesis) is the most frequent form of cancer worldwide. Skin cancers can be classified into three major histological types: ▶malignant cutaneous melanoma and non-melanoma skin cancers, which comprise ▶basal-cell carcinoma and ▶squamous-cell carcinoma. Basal-cell carcinoma, which represents 70% of all skin cancers, develops from the basal cells of the epidermis, grows locally and never metastasizes. Squamous-cell carcinoma, which represents 20% of all skin cancers, develops from the epithelial cells of the epidermis, has a more aggressive behavior, can metastasize, but is rarely fatal. Cutaneous melanoma develops in the melanocytes within the basal layer of the epidermis; it is the least common form of skin cancer, but is the most serious as it readily metastasizes and is only curable if detected at an early stage.

. . . .

developing a light tan) have a much higher risk for skin cancer than subjects with a skin phototype IV (never burn and always develop a deep tan). Intermediate risk categories are subjects who sometimes burn and always develop a tan (skin phototype III). Subjects with skin phototypes V and VI belong to populations with natural brown or black skin, and are resistant to sunlight. Freckles (ephelides) on the face, arms or shoulders. The risk for non-melanoma skin cancer increases with increasing sensitivity to freckling. Skin color: pale color, followed by increasing depth of pigmentation. Eye color: blue, followed by grey/green eyes, then by brown eyes. For melanoma, a large number of common naevi and the presence of a typical naevi.

Subjects with red hair, many freckles, fair skin and who never tan are at particularly high risk for skin cancer.

UV Radiation as an Environmental Risk Factor The body of evidence from epidemiological studies indicates that there is a causal relationship between exposure to solar radiation and skin cancer.

Epidemiological Evidence of the Carcinogenic Potential of UV Radiation Host Factors There is a considerable range of susceptibility of the human skin to UV radiation. Susceptibility to UV radiation is closely related to pigmentary traits, and subjects who have the following characteristics are at increased risk for developing skin cancer (melanoma, squamous-cell carcinoma and basal-cell carcinoma):

1. These cancers affect more specifically individuals with fair skin. 2. These cancers occur predominantly on the parts of the body most commonly exposed to the sun such as the face, ears, neck and forearms; however, the distribution of melanomas and basal-cell carcinomas is not as closely related to the distribution of exposure to the sun as that of squamous-cell carcinomas. 3. There is a strong inverse relationship between latitude of residence and the incidence of and mortality from skin cancer. 4. There is a positive relationship between measured or estimated ambient UV radiation and the incidence of and mortality from skin cancer. 5. Migration to a country with high solar irradiation (e.g. Australia) is associated with an increase in risk. 6. There is a dose–response relationship between total cumulative dose and risk for skin cancer, although for melanoma this is not the only determining factor (see below). 7. Actinic keratosis, a sun-induced skin damage, is a precursor lesion of squamous-cell carcinoma. 8. Treatment with PUVA therapy (UVA radiation) increases the risk for melanoma and squamous-cell carcinoma.

. Red hair, followed by blond hair, followed by light brown hair. . Skin phototype: subjects with a skin phototype I or II (always burn and never tan or always burn before

However, the type and conditions of exposition (chronic or intermittent exposure, high exposure of short duration, exposure at young age) influence the type of skin cancer induced.

UV Radiation

– Squamous-cell carcinoma develops at the sites of the body that receive chronic exposure; in contrast, basal-cell carcinoma and melanoma are associated with intermittent exposure. – The risk for squamous-cell carcinoma is cumulative; consequently, people who work outdoors tend to have a higher incidence of squamous-cell carcinoma. – For melanoma, the cumulative dose is not the only determining factor. In particular, the risk is correlated with ambient UV radiation at the place of residence during childhood. In fact, exposure to UVB during childhood is a necessary event for the development of melanoma. – The risk for melanoma is inversely correlated with intense and continuous exposure to UV. – Squamous-cell carcinoma is induced by UVB radiation. The wavelengths primarily involved in the development of melanoma and of basal cell carcinoma have not been firmly established. Intraocular Melanoma The results of studies on exposure to solar radiation and ocular melanoma would suggest that there is an association, but the data are inconsistent and their interpretation is difficult. Biological Effects of UV Radiation Relevant to Carcinogenesis DNA Damage The biological effects of UV radiation vary enormously with wavelength. In addition, there is an estimated 1,000fold variability in DNA repair capacity after exposure to UV radiation in humans (▶photocarcinogenesis). UVB is a complete carcinogen that is absorbed by DNA and can damage DNA directly. The DNA damage induced by UVB irradiation typically includes the formation of ▶cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts. If these lesions are not repaired correctly, mutations are likely to occur. These mutations are C ⇒ T and CC ⇒ TT ▶transversions, commonly referred to as “UVB fingerprint” or “UVB signature” mutations. UVB can also induce oxidative stress and the formation of singlet oxygen species (O2− ), and can thus also cause DNA damage indirectly. UVA is not readily absorbed by DNA and thus has no direct impact on DNA. Instead, UVA induces DNA damage indirectly through the absorption of UVA photons by ▶chromophores, with the formation of ▶reactive oxygen species (such as singlet oxygen and hydrogen peroxide [H2O2]) that can transfer the UVA energy to DNA via mutagenic oxidative intermediates such as 8-hydroxydeoxyguanosine (▶8-OHdG). DNA damage by UVA radiation typically consists of T ⇒ G transversions, called “UVA fingerprint” or “UVA signature” lesions. The possibility that indirect DNA

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damage induced by UVA could play a major role in the development of melanoma is underlined by reports showing that patients who are genetically highly susceptible to oxidative agents develop multiple cutaneous melanomas. Cellular Damage Both UVA and UVB lead to altered expression of ▶p53 and ▶bcl-2 proteins, which may play an important role in regulating UV-induced apoptosis. Irradiation of melanocytes with UVA or UVB leads to the alteration of different intracellular proteins, which suggests that UVA and UVB may initiate the development of melanoma via separate intracellular pathways. Differential Effects of UVA and UVB UVA radiation is of longer wavelength and is therefore weaker but more penetrant. In humans, UVA penetrates deeper into the skin than UVB. The basal layer of the epidermis receives 50% of the total UVA and 10% of the total UVB to which the skin is exposed. Since solar radiation at ground level comprises a maximum of 5% UVB, the basal cells receive approximately 100 times more UVA than UVB from exposure to the sun. In contrast, the induction of an erythemal response requires 100–1,000 times more UVA than UVB. As a result, UVA fingerprint mutations are mostly detected in the basal germinative layer of these lesions, whereas UVB fingerprint mutations are found predominantly more superficially in these lesions. In addition, UVB produces numerous immediate mutations whereas UVA produces fewer immediate mutations and more delayed mutations than UVB. Consequently, squamous-cell carcinoma is clearly associated with the carcinogenic effects of UVB. In contrast, UVA could play a significant role in the causation of melanoma and basal-cell carcinoma. Changes in the Immune Response Both UVA and UVB can affect the immune system, but the two types of radiation seem to act differently. UVB can induce immune suppression at both local and systemic levels whereas UVA does not suppress the systemic immune response. In addition, UVA radiation may affect the local immune response differently from UVB. Drug-Induced Photosensitivity A variety of commonly used drugs such as diuretics, antibiotics and non-steroid anti-inflammatory drugs (▶NSAIDs) increase cutaneous sensitivity to UV radiation, and are therefore predicted to increase the risk for skin cancer. Most drugs have a phototoxic rather than a photo-allergenic effect. Topical agents that have a

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UVC Light

phototoxic effect include plant-derived photosensitizers (psoralens) such as bergamot, which is widely used in perfumed products. Cancer Preventive Effects of UV Radiation Several ecological studies have suggested that exposure to UV radiation may reduce the incidence of or the mortality from certain cancers (breast, colon, prostate) and of lymphomas. UV radiation induces the formation of vitamin D from its precursor in the body. Daily exposure of the face, forearms and hands to moderate sunlight for 10–15 min is generally sufficient to maintain vitamin D levels. Any vitamin D deficiency should be alleviated through dietary supplements. Indoor Tanning and Cancer The UV spectrum of indoor tanning facilities has varied over the years. The first fluorescent tubes designed for tanning purposes emitted up to 5% of the UV output as UVB. As a result of the growing concern about the carcinogenic potential of UVB, the UV output was then shifted towards UVA. Lamps that produce large quantities of long-wave UVA (>335–400 nm) per unit of time were marketed; these lamps can emit up to 10 times more UVA than that present in sunlight. More recently tanning appliances have been equipped with fluorescent lamps that achieve a balance between total UV, UVB and UVA similar to tropical sun. The UV output and spectral characteristics of tanning appliances vary considerably as a result of differences in tanning appliance design (e.g. type of fluorescent tubes, materials that compose filters, distance from canopy to the skin), tanning appliance power and tube ageing. A ▶meta-analysis of epidemiological studies showed that ever-use of sunbeds is positively associated with an increased risk for melanoma (relative risk, 1.15; 95% confidence interval, 1.00–1.31), and there is a prominent and consistent increase in risk for melanoma in people who first used sunbeds in their twenties or teen years (relative risk, 1.75; 95% confidence interval, 1.35–2.26). Limited data suggest that the risk for squamous-cell carcinoma is similarly increased after first use as a teenager (relative risk, 2.25; 95% confidence interval, 1.08–4.70). Data also suggest detrimental effects from the use of sunbeds on the skin’s immune response and possibly the induction of ocular melanoma. Radiation emitted by lamps used in tanning appliances (mainly UVA) significantly increase the carcinogenic effect of broad-spectrum UV radiation, which indicates the possibility of a complex interplay between UVA and UVB radiation in human skin. The few studies that have addressed the biological changes in the skin induced by indoor tanning have shown that they are similar to those induced by sunlight.

References 1. AFSSET (2005) UV radiation. State of the knowledge on exposure and health risks. Agence française de Sécurité Sanitaire de l’Environnement et du Travail. Available at www.afsset.fr (in English) 2. Griffiths HR, Mistry P, Herbert KE et al. (1998) Molecular and cellular effects of ultraviolet light-induced genotoxicity. Crit Rev Clin Lab Sci 35:189–237 3. IARC (1992) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 55, Solar and ultraviolet radiation. International Agency for Research on Cancer, Lyon 4. IARC (2006) IARC Working Group Reports, vol. 1, Exposure to artificial UV radiation and skin cancer. International Agency for Research on Cancer, Lyon 5. Ullrich SE (2005) Mechanisms underlying UV-induced immune suppression. Mutat Res 571:185–205

UVC Light Definition Short-wave ultraviolet light, predominantly induces cis–syn cyclobutane pyrimidine dimers and (6–4) pyrimidine–pyrimidone photoproducts between adjacent bipyrimidines. ▶Fragile Histidine Triad ▶UV Radiation

Uveal Melanoma J USTIS P. E HLERS , J. W ILLIAM H ARBOUR Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA

Synonyms Choroidal melanoma; Ciliary body melanoma; Intraocular melanoma

Definition Uveal melanoma is an intraocular melanocytic neoplasm that originates from within the ▶uveal tract of the eye. The tumor is thought to arise most frequently from melanocytes within a nevus, but may also occur de novo. It is the most common primary intraocular malignancy in adults.

Uveal Melanoma

Characteristics Epidemiology Worldwide, the incidence of uveal melanoma is highest in northern Europeans. Uveal melanoma has an incidence rate of around 6/1,000,000 in the United States. Similar to cutaneous melanoma, the incidence of uveal melanoma is much higher in whites than nonwhites. There is a slight male preponderance. The average age at diagnosis is 50–60, but any individuals of any age can be affected. Risk Factors Certain patient characteristics have been associated with increased risk of uveal melanoma, including light eye color, light skin color, and ability to tan. Individuals with ocular and ▶oculodermal melanocytosis (nevus of Ota) are at increased risk of uveal melanoma. Interestingly, although these conditions can occur in any race, the increased rate of uveal melanoma appears to be primarily in whites. Clinical Diagnosis Signs and Symptoms Uveal melanoma can be asymptomatic or associated with a variety of symptoms including blurred vision, visual field defects, distorted vision, flashes and floaters. On clinical examination, uveal melanomas can range in pigmentation from light tan to dark brown, and they are typically dome- or mushroom-shaped (Fig. 1). Features associated with uveal melanoma on fundus examination include orange lipofuscin pigmentation on the tumor surface, exudative retinal detachment, and “collar button” formation where the tumor herniates through the overlying Bruch’s membrane, which can cause intraocular hemorrhage. Ciliary body melanomas are located near the crystalline lens and can induce subluxation, cataract formation, and astigmatism (from tilting the lens). Dilated episcleral feeder vessels, so-called “▶sentinel vessels,” can be associated with an underlying ciliary body melanoma. In larger tumors, neovascular

Uveal Melanoma. Figure 1 Elevated choroidal melanoma adjacent to the optic nerve.

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glaucoma and necrosis may occur. Pain may be present if these complications develop. Clinical features associated with increased risk of metastasis include advanced patient age, increased tumor size, and ciliary body involvement. Diagnostic Modalities Ophthalmoscopy remains the mainstay of diagnosis. Several adjunctive modalities can be used to further characterize the tumor and aid in diagnosis for ambiguous cases. Both ▶A-scan and B-scan ultrasonography are critical for the complete evaluation of a suspected uveal melanoma. A-scan provides a single dimensional image characterizing the echogenic interfaces within the tumor. This provides an accurate way to measure the tumor height as well as confirming the typical homogenous characteristics of uveal melanoma. B-scan provides a two-dimensional image that allows for a cross-sectional view of the tumor. Most tumors are dome- or mushroom-shaped, the latter being nearly pathognomonic for uveal melanoma (Fig. 2). ▶Fluorescein angiography is also a useful adjunct in to further characterize the tumor exclude other mimicking disorders. Common angiographic findings include early hyperfluorescence, pinpoint leakage and occasionally a double circulation pattern from vessels within the tumor (Fig. 3). Fine needle aspiration biopsy (FNAB) is required in about 5% of intraocular tumors for the purpose of confirming the diagnosis of a melanocytic tumor. In the setting of an experienced surgeon and cytopathologist, FNAB can yield a diagnosis in over 90% of cases. In addition, FNAB is increasingly being used to assess the metastatic potential of melanomas using molecular tools described below. Other imaging modalities such as CT and MRI may provide further information in selected settings, such as when orbital or optic nerve invasion of an intraocular tumor is suspected. Treatment Options Several treatment options exist for uveal melanoma. The appropriate choice is based on patient age, health and preference, and on tumor characteristics. Observation may be appropriate for small lesions where the diagnosis is in doubt, and in elderly or infirmed individuals with limited life expectancy. Choroidal melanocytic tumors less than about 3 mm in thickness and less than about 12 mm in diameter are usually considered suspicious nevi until growth is documented, at which point treatment is usually recommended. Serial fundus photography and ultrasonography are used to detect tumor growth. ▶Transpupillary thermal therapy (TTT) has been advocated as primary therapy for smaller tumors and as an adjunctive treatment following radiotherapy. In

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Uveal Melanoma

Uveal Melanoma. Figure 2 Ultrasound (B-Scan) of dome-shaped (a) and mushroom-shaped (b) melanoma. Thin arrows identify the tumor; thick arrows identify (a) the optic nerve and (b) the detached retina.

Uveal Melanoma. Figure 3 Fluorescein angiogram showing double circulation. The thin arrow shows a normal retinal arteriole; the thick arrow shows the intrinsic tumor vessel.

recent years, enthusiasm for primary TTT has waned as longer follow-up has revealed a high rate of local tumor recurrence. Most centers now reserve primary TTT for small tumors in patients who are unable to undergo surgery. The most widespread use of TTT today is as an adjunctive treatment following plaque or charged particle radiotherapy in tumors that are at high risk for recurrence, such as those located adjacent to the optic disc. In this setting, TTT appears to be effective at hastening the regression and reducing local recurrence risk compared to radiotherapy alone. Complications from TTT include retinal vascular occlusion, retinal fibrosis, retinal neovascularization, cystoid macular edema, retinal tears, retinal detachment and vitreous hemorrhage. These complications are much more common with primary TTT than with adjunctive TTT.

▶Enucleation was once the mainstay of treatment for uveal melanoma. Today, however, enucleation is usually reserved for tumors that are very large or have demonstrated extensive local invasion (e.g., transscleral or optic nerve invasion). Enucleation may also be preferred in eyes with poor visual potential. At the time of enucleation surgery, a spherical implant is placed in the orbit and attached to the extraocular muscles to imitate the movement of the eye. Subsequently, the patient is fit with a prosthetic eye, which is a shell painted to match the other eye. Plaque radiotherapy is the most common treatment for uveal melanoma. This treatment approach allows a large dose of radiation (85–100 Gy) to be delivered to the tumor over 4–5 days while minimizing radiation toxicity to the rest of the eye and surrounding structures. Various radioisotopes have been used, including 125Iodine, 106Ruthenium, 103Paladium, but 125 Iodine is the most common choice. The medium tumor trial of the Collaborative Ocular Melanoma Study compared 125Iodine plaque radiotherapy to enucleation and found no difference in survival. The plaque is surgically implanted on the scleral surface of the eye overlying the tumor. Treatment outcomes are significantly enhanced when intraoperative ultrasound is used to verify accurate placement of the plaque over the tumor. Local tumor control following plaque radiotherapy is greater than 90% in most centers. Complications include cataract, radiation retinopathy and papillopathy, neovascular glaucoma, vitreous hemorrhage, and rarely scleral necrosis. Incidence and severity of these complications are dose and location dependent. Focal delivery of high dose radiotherapy can also be achieved with ▶proton beam radiotherapy and other forms of charged particle therapy. A surgical procedure is required to place tantalum rings on the scleral surface overlying the tumor to direct the charged particle beam. Local tumor control appears to be slightly better with charged particle therapy than with plaque radiotherapy, especially in centers that do not use intraoperative

Uveal Melanoma

ultrasonography to localize their plaques, but complications are also more common with charged particle therapy. Complications include eyelash loss, dry eye, cataract, neovascular glaucoma, and radiation retinopathy and papillopathy. Newer radiosurgery techniques such as Gamma Knife and CyberKnife are being evaluated for their role in uveal melanoma. Local tumor resection was once a popular alternative treatment that avoided radiotherapy and its attendant complications. However, as more experience has been obtained, it has become clear that local resection has its own set of serious side effects, including a high rate of local tumor recurrence, retinal detachment, vitreous hemorrhage and proliferative vitreoretinopathy. Today, local resection is usually reserved for selected tumors with small basal diameter and anterior location. There are many variations on this surgical technique, but most involve the creation of a partial thickness scleral flap, through which the tumor is resected en bloc. Adjunctive radiotherapy is often used postoperatively. Pathology The Callendar classification is the most widely accepted histopathologic grading system for uveal melanoma and is based on the spindle and epithelioid cell types (Fig. 4). Spindle A cells have narrow nuclei with a longitudinal fold. Spindle B cells have larger nuclei with a defined nucleolus. Epithelioid cells are larger and polygonal in shape with large nucleoli. Uveal melanomas are usually described as predominantly spindle, epithelioid, or mixed. Spindle cells portend a better prognosis, and epithelioid cells a worse prognosis. Other pathologic features that have been associated with worse prognosis include transscleral extension, increased number of mitotic figures, increased nucleolar area, and presence of looping extracellular matrix patterns. Systemic Evaluation and Metastasis Metastatic uveal melanoma involves the liver in approximately 90% of cases. Other sites include lung, subcutaneous tissues, and bone. Systemic evaluation and monitoring should include liver function tests (especially lactate dehydrogenase, alkaline phosphatase, and gamma-glutamyl transpeptidase), and imaging

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of the abdomen with CT or MRI. Median survival after clinical diagnosis of metastasis is about 5–7 months. Metastatic disease limited to the liver can occasionally be treated with partial hepatectomy or hepatic arterial chemoembolization. Systemic chemotherapy and immunotherapy have met with limited success. Prognostic Factors As mentioned in the previous sections, there have been many clinical and pathologic features of uveal melanoma that are statistically associated with metastasis. However, none of these features has demonstrated predictive accuracy sufficient for use in making treatment decisions in individual patients. This has led many researchers to investigate genetic features of the primary tumor that may predict metastasis with greater accuracy. The chromosomal alteration that is most closely linked to metastasis is loss of one copy of chromosome 3 (monosomy 3), which is found in about half of primary uveal melanomas. Various techniques have been employed to assess chromosome 3 status in clinical settings, including karyotype analysis, comparative genomic hybridization, fluorescence in situ hybridization, and loss of heterozygosity for polymorphic markers across the chromosome. More recently, ▶microarray gene expression profiling has identified two molecular subgroups of primary uveal melanoma, referred to as class 1 and class 2 (Fig. 5). Virtually all of the metastatic deaths occur in class 2 tumors. The class 2 gene expression signature is closely linked to monosomy 3, but there are some exceptions, and the former outperforms the latter in terms of predictive accuracy. Consequently, the gene expression-based classifier has been refined to require less than ten genes and a small number of tumor cells that can be obtained by FNAB. Clinical studies are now underway to determine the best combination of molecular testing approaches for stratifying patients based on metastatic risk for inclusion in clinical trials and implementation of preemptive, adjuvant systemic therapy. Systemic Therapy and Future Directions Recent work on tumor doubling times, disease-free interval and metastasis strongly imply that those uveal

Uveal Melanoma. Figure 4 Uveal melanoma histopathology showing spindle cells (a) and epithelioid cells (b).

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Uveal Tract

Uveal Melanoma. Figure 5 Gene expression profiling is the most accurate predictor of metastasis currently available for uveal melanoma. (a) Unsupervised principal component analysis showing clustering of tumors (spheres) in to class 1 (blue) and class 2 (red), with low and righ risk for metastasis, respectively. (b) Heatmap with supervised analysis with only nine discriminating genes (rows), which classify all tumors correctly (columns). Since a small number of genes can be used, this test is being adapted for use as a clinical test that can be applied to enucleation and biopsy samples.

melanomas that have the capacity to disseminate (e.g., class 2 tumors) have already done so prior to ocular treatment in most cases. This would explain why successful local treatments have not been associated with a demonstrable improvement in survival. Consequently, it may only be possible to improve survival in uveal melanoma by identifying high risk patients and treating them preemptively with systemic therapies that delay or prevent the development of clinical metastasis by maintaining micrometastases in a dormant state. The centerpiece of such a preventative strategy will be the newly emerging molecular prognostic tests described here. This strategy will also require the identification of new therapeutic approaches targeting the micrometastastic cell population.

References 1. Ehlers JP, Harbour JW (2006) Molecular pathobiology of uveal melanoma. Int Ophthalmol Clin 46(1):167–180 2. Harbour JW (2003) Clinical overview of uveal melanoma: introduction to tumors of the eye. Albert DM, Polans A (eds) Ocular oncology. Marcel Dekker, New York, pp 1–18 3. Onken MD, Ehlers JP, Worley LA et al. (2006) Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas. Cancer Res 66:4602–4609 4. Onken MD, Worley LA, Ehlers JP et al. (2004) Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res 64(20):7205–7209 5. Onken MD, Worley LA, Davila RM et al. (2006) Prognostic testing in uveal melanoma by transcriptomic profiling of fine needle biopsy specimens. J Mol Diagn 8:567–573

Uveal Tract Definition Pigmented highly vascular layer of the eye consisting of the choroid, iris, and ciliary body. ▶Uveal Melanoma

Uvomorulin ▶E-Cadherin

UV-Signature Mutation Definition

A C → T single or tandem transition mutation that is typically formed at ▶DNA photoproducts and commonly observed in UV-induced, but not other cancers. ▶Solar Ultraviolet Light ▶UV Radiation

V

V-erb-B2 ▶HER-2/neu

V-raf Murine Sarcoma Viral Oncogene Homolog B1 ▶B-Raf Signaling

Vaccine Therapy Definition Is an active, specific immunotherapy (ASI) mediated by stimulating the host’s immune system to generate antibodies and/or immune cells (▶cytotoxic T-cells) specifically against the target antigen to destroy a tumors or infectious microorganisms. A therapeutic (treatment) vaccine is given after the onset of disease and is intended to reduce or arrest disease progression. A preventive (prophylactic) vaccine is intended to prevent the initial onset of disease. Tumor vaccines include both native and artificial (mimic) tumor-associated antigens or tumor markers. ▶Immunotherapy

Valproic Acid M ARTA KOSTROUCHOVA Laboratory of Molecular Biology and Genetics, Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University, Prague, Czech Republic

Synonyms 2-Propylpentanoic acid; Dipropyl-acetic acid

Definition Valproic acid is a short chain branched fatty acid. It was first synthesized by B.S. Burton in 1882. During the last 40 years valproic acid has been extensively used for the treatment of epilepsy, bipolar and other neurological disorders. In the human brain, valproic acid affects the function of the neurotransmitter ▶gamma aminobutyric acid (GABA) (mainly as a GABA transaminase inhibitor). Valproic acid has ▶histone deacetylase inhibitory activity and affects behavior and growth of various cancers and ▶cancer cell lines. Valproic acid induces cell differentiation and ▶apoptosis of diverse cancer cell lines, increases ▶recognition of cancer cells by the immune system, inhibits cell proliferation, decreases ▶metastatic and angiogenic potential of cancer cells. The anticancer effect of valproic acid is based on its histone deacetylase inhibiting activity, modulation of ▶MAPK signaling and ▶beta-catenin pathway. Other mechanisms of action of valproic acid were also proposed. The relative safety of administration of valproic acid and its involvement in multiple pathways are making it a valuable lead in search for more potent and more specific drugs with anti-cancer activities. Molecular Formula: (CH3CH2CH2)2CHCOOH Molecular Mass: 144.21

Characteristics The name valproic acid originates from its descriptive chemical name 2-propylvaleric acid (valeric acid is a synonym for pentanoic acid that was isolated from the flowering plant valerian (Valeriana officinalis)). Valproic acid was used as a solvent of neurologically active compounds and its therapeutic potential was discovered by chance. Valproic acid is a relatively safe drug, with only infrequent major side effects that include hepatotoxicity, thrombocytopenia, and prolonged ▶coagulation time. In about 5% of pregnant users, valproic acid can cause congenital anomalies such as ▶spina bifida. Valproic acid inhibits proliferation and induces differentiation of neuroectodermal tumor cells. Valproic acid and its analogs modulate the behavior of various tumor cell types by inducing apoptosis and differentiation, inhibiting proliferation, decreasing

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▶angiogenetic potency and increasing immunogenicity of cancer cells. Valproic acid induces apoptosis in two ways: through the effect on ▶caspases and ▶chromatin fragmentation and trough its effect on membranes linked to phosphatidylserine externalization and release of cytochrome c from mitochondria. Valproic acid induced apoptosis in various animal cell lines including rat hepatoma cell line FaO, many human leukemia cell lines of B-, T-, and myeloid lineage, murine B-lymphoid cell lines, MV4-11 and KOCL-44, prostate carcinoma cell line, human thyroid cancer cells and other cells. Treatment with valproic acid leads to inhibition of cell proliferation and induction of cell differentiation in different cell lines, mainly of neuroectodermal and leukemic origin. Valproic acid increased differentiation of human ▶neuroblastoma cells (SJ-N-KP, AF8), which was documented by neurite extension and up-regulation of ▶neuronal markers. The decreased net proliferation rate after valproic acid administration was observed in ▶prostate cancer cells – androgen receptor positive (LNCaP and C4-2) and androgen receptor negative (DU145 and PC3). In leukemic cell lines HL-60 and MOLT-4, valproic acid induced cell differentiation was marked by an increase of CD 11b and co-stimulatory/ adhesion molecule CD86. ▶Hepatocellular carcinomas (HCC) that are resistant to conventional ▶chemotherapeutics showed decreased proliferation in response to the treatment with valproic acid. A down regulation of anti- and up regulation of pro-apoptotic factors indicated that modulation of intracellular pro- and anti-apoptotic proteins is a key event in valproic acid induced tumor cell death. The influence of valproic acid in the ▶cell cycle was determined to be in the G1 phase. Valproic Acid is a Potent Inhibitor of Tumor ▶Angiogenesis. Valproic acid caused inhibition of proliferation, migration and tube formation of endothelial cells and also caused a decrease of endothelial nitricoxid synthase (eNOS) protein level. The inhibition of angiogenesis in vivo was documented on the chicken chorioallantoic membrane assay and the matrigel plug assay in mice. Valproic acid also caused disturbed vessel formation. The indirect effect of valproic acid was observed in neuroblastoma cells. A higher production of antiangiogenic-molecules thrombospondin-1 and ▶activin A were detected. Similarly the treatment of colon adenocarcinoma cell line Caco-2 caused significant reduction of ▶vascular endothelial growth factor (VEGF) secretion, down-regulation of protein expression and mRNA of VEGF, basic fibroblast growth factor (bFGF) protein level and inhibition of the ▶ubiquitin-proteasome proteolytic system activity. Valproic Acid Increases Immunogenicity of Cancer Cells. Treatment of human ▶hepatocellular carcinoma cells with valproic acid mediated recognition

of cancer cells by cytotoxic lymphocytes via the immunoreceptor NKG2D. Valproic acid induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by natural killer cells. In primary human hepatocytes, valproic acid treatment did not induce MIC protein expression indicating that valproic acid mediates specific priming of malignant cells for innate immune effector mechanisms. Valproic Acid was Shown to Modulate Behavior of Numerous Tumors. Valproic acid was repeatedly used in patients with acute myeloid leukemia (AML) and combined with all-trans retinoic acid (ATRA). In some subtypes, the AML is connected with translocations that generate fusion genes including ▶retinoic acid receptor alpha (RARα) which functions as an oncoprotein that inhibits differentiation pathways of myeloid lineage. ATRA was shown to partially release the differentiation block by binding to the RARα part of the fusion protein with subsequent increased expression of target genes (likely connected with disruption of binding of transcriptional co-repressors and induced degradation of the fusion protein). Valproic acid markedly increased the efficacy of the treatment by ATRA and resulted in transient disease control in subsets of patients with AML. The antitumor efficacy of valproic acid was observed in ▶medulloblastoma and supratentorial primitive neuroectodermal tumor (sPNET), which are the most common malignant brain tumors in children with poor prognosis. Two medulloblastoma (DAOY and D283-MED) and one sPNET (PFSK) cell lines were treated with valproic acid with resulting potent growth inhibition, cell cycle arrest, apoptosis, senescence, differentiation, suppressed colony-forming efficiency and tumorigenicity in a time- and dose-dependent manner at clinically safe concentrations (0.6 and 1 mmol/l). Valproic Acid Affects Cell Behavior by Multiple Mechanisms Valproic Acid Inhibits Histone Deacetylase Activity The effect of valproic acid on behavior of cancers and cancer cell lines initiated studies directed at its mode of action. Behavior of cells depends on their gene expression that in turn is regulated by the basic transcription machinery, cell and tissue specific ▶transcription factors and ▶co-factors and by the organization of chromatin. ▶Post-translational modification of chromatin, including histone acetylation, methylation, phosphorylation, ubiquitination and other modifications is a critical part of regulation of gene expression. Valproic acid was shown to down-regulate the HDAC activity in ▶teratocarcinoma and neuroblastoma cells. Nevertheless, this effect may have been caused by a

Valproic Acid

direct action of valproic acid on enzymes involved in adding or removing acetyl residues on lysines of nucleosomal histones (mostly histones H3 and H4) or by a modulation of other targets. It was shown that valproic acid affects the in vitro deacetylation assay and acts as an HDAC inhibitor. Inhibition of HDAC activity was analyzed by measuring the acetylation of core histones H3 and H4 in the leukemic K562 and U937 cell lines. Valproic acid and its analogs (2-methyl-2n-propylpentanoic acid (2M2PP), 4-pentenoic acid (4PA), 2-methyl-pentenoic acid (2M2P), 2-ethylhexanoic acid (2EH), and valpromide (VPM)) were shown to inhibit class I HDAC (HDACs 1–3), and class II HDAC (HDACs 4, 5, and 7). Valproic acid was the strongest inhibitor. The mechanism by which valproic acid affects behavior of cancer cells differs from its antiepileptic effect since the efficacy of antineoplastic and antiepileptic effects differ between particular analogs. Valproic acid did not inhibit the activity of class II HDAC 6 and 10, in contrast to another HDAC inhibitor, ▶trichostatin A (TSA). This implies a more selective effect of valproic acid on HDAC inhibition compared to ▶TSA. Although valproic acid affects both class I and class II HDACs, the effect on these two classes differs. Valproic acid has been shown to inhibit the catalytic activity of class I HDACs and induce the proteasomal degradation of class II HDACs in contrast to TSA. Valproic acid and its analogs induce expression of multiple exogenous reporter genes which are associated with HDAC inhibition, i.e. SV40, p21 and gelsolin. Studies directed at the genome wide expression pattern showed that valproic acid affects expression of selective groups of genes (by their induction or repression).

Valproic Acid Interferes with MAPK Signaling Valproic acid increases the DNA binding and transactivation activity of the transcription factor ▶AP-1. In vitro studies showed that valproic acid specifically triggers the phosphorylation of ▶ERK, the upstream modulator of AP-1, but does not act via the JNK (c-jun N-terminal kinase) and p38 pathways. Valproic acid and its derivatives (named above) activate MAPK. In clear contrast to their effect on inhibition of class I HDACs, the analogs of valproic acid have a stronger effect on MAPK activation than valproic acid itself. This may reflect a possibility that the effect on MAPK is not mediated by HDAC inhibition. Therefore valproic acid-induced increases in AP-1 binding and function are likely due, at least in part, to activation of ERK followed by phosphorylation and increase in expression of c-Jun. Expression and phosphorylation of c-Jun in ERK pathway is required to direct cellular differentiation in poorly differentiated cells. Nevertheless, it seems likely that the way valproic

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acid affects cell behavior may to a large extent depend on the particular cell type. Valproic Acid Affects the Beta-Catenin Pathway GSK-3beta (▶glycogen synthase kinase-3 beta) is a negative regulator of the ▶Wnt signaling pathway, which regulates numerous processes, including cellular proliferation, cell migration, cell polarity, organo- and carcinogenesis. GSK-3 beta phosphorylates beta-catenin and this leads to its rapid degradation. Inhibition of GSK-3 beta by Wnt signaling leads to stabilization and accumulation of the beta-catenin protein. Consequently, betacatenin translocates to the nucleus where it activates transcription of Wnt dependent genes by binding to factors ▶Tcf/Lef (T cell factor, Lymphoid-enhancer factor). Valproic acid has been reported to inhibit GSK-3 beta-mediated phosphorylation of a peptide derived from ▶CREB protein in human embryonic kidney 293T and murine Neuro2A ▶neuroblastoma cells and increase levels of beta-catenin in human neuroblastoma cells. Nevertheless, it was also proposed that valproic acid activates Wnt-dependent gene expression through the inhibition of histone deacetylase activity. The involvement of histone deacetylase inhibition by valproic acid in beta-catenin dependent regulation is supported by its effect on the expression of E-cadherin. Expression of numerous ▶tumor suppressor genes including E-cadherin is linked to hypermethylation of specific regions of DNA and may be partially reverted by ▶increased histone acetylation. Valproic Acid may Influence Additional Pathways Valproic acid was shown to be involved in other regulatory pathways. In many of them, it is likely that the effect is mediated primarily by the inhibition of histone deacetylase activity but some may be distinct. Valproic acid increases the levels of 5-▶lipoxygenase (5-LOX) protein in murine hippocampus. 5-LOX produces ▶leukotrienes from ▶arachidonic acid and this is likely to be involved in the regulation of ▶chromatin remodeling. Since increased levels of 5-LOX are linked to aging and neurodegeneration, it may be expected that the increase of 5-LOX expression may contribute to tumor cell aging and differentiation through chromatin remodeling. Valproic acid is likely to inhibit invasiveness of cancer cells at concentrations lower than those necessary for its anti-proliferative effect. Increased expression of genes that inhibit invasiveness of cancer cells was observed in response to treatment with relatively low doses of valproic acid (150 μmol/l). These genes included ▶signal transducer and activator of transcription 6 (STAT6), Ring1, RYBP and PCDHGC3. Valproic acid may affect the regulation of gene expression by ▶nuclear receptors which is critically

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influenced by ▶HDACs. Moreover, some members of this superfamily of genes may be affected by valproic acid more directly. PPARδ and PPARγ (but not PPARα) were activated by valproic acid in Chinese hamster ovary cells and F9 teratocarcinoma cells. At least in the case of PPARδ, this effect was not based on the binding and activation of the receptor by valproic acid as an agonistic ligand since it did not induce formation of heterodimers of PPARδ with retinoid X receptor on DNA response elements. Activated expression of PPARγ dependent genes may significantly contribute to the anticancer activity of valproic acid. PPARγ regulates the expression of the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) that is often mutated in cancers. ▶PTEN, a lipid phosphatase, dephosphorylates phosphatidylinositol (3,4,5)-triphosphate (PIP-3) to phosphatidylinositol (4,5)-diphosphate (PIP-2) and antagonizes the regulation by phosphatidylinositol-3 kinase (PI-3K).

References 1. Altucci L, Clarke N, Nebbioso A et al. (2005) Acute myeloid leukemia: therapeutic impact of epigenetic drugs. Int J Biochem Cell Biol 37:1752–1762 2. Blaheta RA, Michaelis M, Driever PH et al. (2005) Evolving anticancer drug valproic acid: insights into the mechanism and clinical studies. Med Res Rev 25:383–397 3. Kostrouchova M, Kostrouchova M, Kostrouch Z (2007) Valproic acid: a molecular lead to multiple regulatory pathways. Folia Biol (Praha) 53:37–49 4. Minucci S, Pelicci PG (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6:38–51

Vanadium M ALAY C HATTERJEE Department of Pharmaceutical Technology, Jadavpur University, Calcutta, West Bengal, India

Definition Vanadium, a member of group VB and in the 4th period of the periodic table is a first transition series, d-block, grayish metallic element with an atomic number of 23 and forms different oxidation states of –1, 0, +2, +3, +4 and +5. The oxidation states +3 (vanadic), +4 (vanadyl) and +5 (vanadate) being most common, the oxidation state +4 is most stable in biological systems. Vanadium is an endogenous constituent of most mammalian tissues and is further considered a ▶dietary micronutrient. It is an essential element for the proper growth and

development of mammals owing to its diverse physiological and biochemical functions.

Characteristics The uniqueness of vanadium is that it is included in the list of 40 essential micronutrients required in small amounts for normal cell metabolism. Accordingly, it has been incorporated into many multinational pharmaceutical preparations for maintenance of normal health. Although micronutrients never have had pharmacological potencies, they can prevent the minor wear and tear of the essential critical molecules of the cell; vanadium may thus have a role in DNA maintenance reactions and may prevent genomic instability leading to ▶cancers. The main source of vanadium intake for the general population is food, such as chicken, fish, grains, cereals, ▶liver, spinach, black pepper, parsley, fruits, and vegetables. The total body pool of vanadium in humans is about 100 μg, with the actual daily dietary intake estimated to be 10–60 μg, depending on individual human diet. Studies have established vanadium as a novel biological regulator and have confirmed the biphasic effect of this trace element in biological systems, that is, essentiality at low concentrations, and toxicity at higher concentrations. Vanadium compounds can influence the behavior of enzymes, mimic growth factor activities, regulate carbohydrate and lipid metabolisms, modulate gene expression and signal transduction pathways, and in particular exhibit anticarcinogenic/antineoplastic activities. The diverse biological action of vanadium results from its capacity to function as an oxyanion, oxycation, or prooxidant. Vanadium in Cancer Treatment Vanadium has a potential role in tumor growth inhibition and in prophylaxis against ▶carcinogenesis in different experimental cancer models namely ▶liver cancer, ▶colon cancer, ▶breast cancer and others and in various types of malignant cell lines. Vanadium compounds have been found potentially effective against murine ▶leukaemia, fluid and solid Ehrlich ▶ascites tumor, murine mammary ▶adenocarcinoma, HEp-2 human epidermoid carcinoma cells and human ▶lung cancer, ▶breast cancer, and ▶gastric cancer. Synthetic complexes of vanadium with amino acids, peptides, proteins, organometallic and inorganic ligands, and pharmacologically active moieties are an area of current interest in anticancer research. A cytostatic effect is observed with vanadium(III)–L-cysteine complex on chemically-induced ▶leiomyosarcoma bearing Wistar rats. [VIII (Hcys)3].2HCl.2.5H2O or compound 1 exhibits significantly greater total antioxidant capacity along with inhibition of neutral endopeptidase activity as potent as thiorphan. Moreover, compound 1 prevents lung

Vanadium

▶metastasis, thus proving its role as an antimetastatic agent. These beneficial effects of the above complexes, in combination with their low toxicity, provide evidence for their possible application in the treatment of human malignant diseases. A good candidate for the development of new vanadium derivatives with organic ligands is the flavonoid quercetin because of its own anticarcinogenic effect. The quercitin-vanadyl complex [VO(Quer)(2)EtOH](n) (QuerVO) stimulates ▶extracellular signal-regulated kinase (ERK) phosphorylation and this seems to be involved as one of the possible mechanisms for the biological effects of the complex. Organometallic vanadocene complexes have been found to be potent antiproliferative and antimitotic agents and block cell division at the G2/M phase of cell cycle in human cancer cells by disrupting bipolar spindle formation. Furthermore, vanadocenes as potent ▶apoptosis-inducing cytotoxic agents against human ▶testicular cancer cells in vitro as well as in experimental mice model in vivo may therefore have potential utility in the treatment of testicular seminomas in humans. Vanadyl complexes of 1,10-phenanthroline [VO(Phen)2+] and related derivatives possess strong antitumor chemopreventive activities against human nasopharyngeal carcinoma, and the observed effects are found to be superior to the chemotherapeutic drug, ▶cisplatin. Bis(4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium(IV) or Metvan [VO(SO4) (Me2-Phen)2] has been identified as the most promising multitargeted anticancer bisperoxovanadium (bpV) complex with apoptosis-inducing activity in human leukaemia cells, ▶multiple myeloma cells and solid tumor cells derived from ▶breast cancer, ▶glioblastoma, ▶ovarian cancer, ▶prostate cancer and ▶testicular cancer. It is highly effective against cisplatin-resistant ▶ovarian cancer and testicular cancer cell lines. Metvan inhibits the constitutive expressions of [▶Matrix metalloproteinases (MMPs)] -2 and 9 proteins and its gelatinolytic activity in HL-60 cells, and leukemic cells from patients, and also inhibits the leukemic ▶cell adhesion to the extracellular matrix proteins laminin, type IV collagen, vitronectin, and fibronectin and the invasion through Matrigel matrix. Metvan exhibits significant antitumor activity in severe combined immunodeficient mouse xenograft models of human malignant glioblastoma and breast cancer. The broad spectrum anticancer activity of Metvan together with favorable pharmacodynamic features and lack of toxicity of this oxovanadium compound may represent the first vanadium complex and a novel anticancer agent as an alternative to ▶platinumbased ▶chemotherapy. Chemopreventive/Anticancer Mechanisms Several mechanisms for the cancer chemopreventive role of vanadium have been proposed. The biochemical basis of the chemoprotective effect of vanadium may

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primarily be attributed to the substantial elevation of phase II conjugating enzymes, which may lead to a move and shift of the metabolic profile that may reduce the intracellular concentration of carcinogen-derived reactive intermediates. However, it is not yet clear whether the activity of specific ▶CYP isoforms is directly influenced/modulated by vanadium treatment. Ortho- and meta- vanadate have been found to drastically reduce the mutagenicity of metabolically activated carcinogens by modulation of protein phosphorylation through the inhibition of protein phosphotyrosine phosphatases (PTPases). This micronutrient is able to exert in vivo anticlastogenic effect through suppression of ▶micronucleus formation, sister-chromatid exchange, and structural, numerical and physiological chromosomal aberrations and may thereby prevent genomic instability. In aqueous solution, vanadium is found predominantly as oxo-anions (e.g., VO43–) and as such may exhibit nucleophilic character for the electrophilic agents to attack, thereby preventing DNA ▶alkylation damage as per the “carcinogen interception mechanism.” although at the moment we cannot say if such processes take place within cells. Experiments on various cell lines reveal that vanadate exerts its antitumor effects through activation of protein tyrosine kinases (PTKs) and/or inhibition of cellular PTPases leading to an accumulation of phosphotyrosine residues in cellular proteins. Both effects activate signal transduction pathways leading either to apoptosis and/or to activation of tumor suppressor genes. The stimulatory effect of vanadate on the cellular phosphorylation status of cytosolic proteins may be mediated in a dose-dependent manner probably via a ▶protein kinase C-dependent mechanism. Vanadyl state is readily converted to vanadate in presence of H2O2, and thus vanadate seems to be the species that inhibits specific PTPase, and correspondingly increases the steady states of phosphorylation and thereby activates cytosolic PTKs at relatively low concentrations. Vanadium compounds have been shown to inhibit cell proliferation by limiting the expressions of several potential marker proteins, such as proliferating cell nuclear antigen, Ki-67 nuclear antigen, etc. In vitro antiproliferative activity of a variety of vanadium compounds may be exerted by their ability to interfere with the molecular interactions between GATA binding protein 1 and ▶Nuclear Factor kappa B (NF-kappaB) transcription factors and target DNA elements. Vanadium complexes with a +5 oxidation state and their discrete anionic units appear essential for inhibition of tumor cell growth with induction of apoptosis, whereas a +4 oxidation state appears to be important in inhibiting transcription factors/DNA interactions. Vanadium-induced apoptogenic signals are mediated through upregulation and overexpression of ▶p53 tumor suppressor and proapoptotic Bax and by downregulation of Bcl2. Reactive oxygen species generated

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by Fenton-like reactions and/or during the intracellular one-electron reduction of V(V) to V(IV) by, mainly, NADPH participate in the majority of the vanadiuminduced intracellular events. ROS/H2O2 generated by vanadate triggers ▶DNA damage, and also activates ▶mitogen-activated protein kinases (MAPKs) signal transduction pathways leading to an increased p53 protein expression and p53 phosphorylation, respectively. This in turn induces p53 transactivation, which subsequently leads to cell apoptosis. p53 is also an important transrepressor of inducible ▶nitric oxidesynthase expression and thereby attenuates excessive nitric oxide production in a regulatory negative feedback loop. Future Direction Given what is known of vanadium effects on animal physiology, individual responses to vanadium treatment might be influenced by plasma- and cellular- binding proteins and genetic predisposition. Nonetheless, the potential therapeutic advantage of vanadium compounds and the available options for mitigating toxicities suggest that further development should yield safe and effective pharmacological formulations as antineoplastic agent.

References 1. Evangelou AM (2002) Vanadium in cancer treatment. Crit Rev Oncol Hematol 42:249–265 2. Chatterjee M, Bishayee A (1998) Vanadium – a new tool for cancer prevention. In: Nriagu JO (ed) Vanadium in the environment, part two, health effects. Wiley, New York, pp 347–390 3. Hamilton EE, Fanwick PE, Wilker JJ (2006) Alkylation of inorganic oxo compounds and insights on preventing DNA damage. J Am Chem Soc 128:3388–3395 4. Ray RS, Ghosh B, Rana A et al. (2006) Suppression of cell proliferation, induction of apoptosis and cell cycle arrest: chemopreventive activity of vanadium in vivo and in vitro. Int J Cancer 120:13–23 5. Chakraborty T, Chatterjee A, Rana A et al. (2007) Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcinogenesis in rats: chemopreventive role of vanadium on this process. Biochim Biophys Acta 1772:48–59

VANGL Definition Van Gogh-like proteins 1 and 2; four-transmembrane domain proteins involved in PCP signaling. VANGL2, also known as KITENIN, enhances tumor ▶metastasis. ▶Wnt Signaling

Variable Number Tandem Repeats Definition VNTRs. ▶Minisatellite

Variant Allele Definition Gene containing one or more single nucleotide ▶polymorphisms.

Variant Isoforms Definition Existence of more than one protein, generated from the same gene by alternative ▶splicing of the nuclear RNA. Unlike some years ago, nowadays it is anticipated that variant protein isoforms are not the exception, but rather the rule. ▶CD44

Vascular Disrupting Agents H OWARD W. S ALMON 1 , B ETH A. S ALMON 2 1

Department of Radiation Oncology, North Florida Radiation Oncology, Gainesville, FL, USA 2 Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA

Synonyms VDAs; Vascular targeting agents; Vascular targeted therapies

Definition Vascular Disrupting Agents (VDAs) disrupt tumor blood flow by specifically targeting vascular abnormalities associated with solid tumor development. The

Vascular Disrupting Agents

result is tumor vessel occlusion followed by extensive tumor necrosis.

Characteristics Tumor growth is limited by the diffusion of oxygen and nutrients from blood vessels into the surrounding tissue. In order to grow beyond a size of 1 mm3, tumors are dependent upon their ability to induce ▶angiogenesis (the formation of new blood vessels). As the tumor outgrows its blood supply, angiogenic factor (i.e. ▶VEGF and ▶bFGF) expression is induced, which stimulates the formation of new blood vessels. The growth rate of tumors is such that angiogenesis must be continuously induced to keep a steady supply of oxygen and nutrients to the tumor tissue, resulting in a state of endothelial proliferation not normally present in the adult body. The vessels produced by this elevated angiogenic state are also abnormal in structure: tortuous vessels that experience transient interruptions in blood flow, and are often leaky with reduced or non-existent ▶pericyte. The abnormality of the tumor vasculature provides a novel target for anticancer therapy, with the idea of interfering with tumor growth through the pharmaceutical disruption of the tumor blood supply (termed ▶vascular targeting). Several vascular targeting agents have since been identified and have begun entering clinical trials. The vascular targeting approach can be broadly divided into categories: the antiangiogenics and the Vascular Disrupting Agents (VDAs). The antiangiogenic agents prevent the formation of new blood vessels thereby slowing tumor growth, while VDAs target and disrupt the existing tumor vasculature, which results in acute destruction of tumor tissue. The angiogenic process is a multi-stage event including the upregulation of pro-angiogenic factors, increased endothelial cell proliferation, degradation of the vessel basement membrane, and endothelial cell migration and new tube formation. Each of these stages is a potential target for antiangiogenic agents, but agents inhibiting pro-angiogenic factors are perhaps the most common. The most studied angiogenic factor involved in tumor pathophysiology is VEGF, and several agents have been developed which either prevent the binding of VEGF to its receptors or inhibit activation of the receptor. The agent Bevacizumab is an anti-VEGF antibody that prevents VEGF from binding and activating its receptors and has had therapeutic success in clinical trials. The tyrosine kinase ZD6474 has been shown to inhibit angiogenesis by selectively preventing activation of the ▶VEGFR-2 receptor and is also undergoing testing in clinical trials. Vascular Disrupting Agents The VDAs can be further divided into two basic categories: biological agents and small molecules.

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Biologic approaches, such as antibodies and fusion proteins, utilize antigens specific to tumor endothelium to target and destroy these cells, while the small molecules rely upon physiological differences to selectively destroy tumor, not normal, endothelial tissue. The Biologic Approach The concept of using fusion proteins to target and disrupt tumor vasculature relies upon two necessary components: a ligand that selectively binds tumor endothelium and a conjugated toxin. The result is precise delivery of the toxic agent to only the tumor endothelium and not normal tissues. Two fusion protein VDAs that have produced positive preliminary results have utilized angiogenic proteins to specifically target the tumor endothelium. Testing of a VEGF121/ rGelonin fusion toxin in nude mice bearing ▶PC-3 tumors revealed specific localization of the conjugate to the tumor vasculature and resulted in tumor vessel thrombosis. The fusion complex of antibody against fibronectin conjugated to Tissue Factor was also shown to specifically target the tumor endothelium and treatment of tumor bearing mice with this agent resulted in disruption of tumor blood flow. Small Molecule VDAs ▶Flavonoids and ▶tubulin binding agents are two classes of small molecule VDAs that are currently being evaluated. The flavonoids are believed to induce endothelial cell death primarily through the induction of ▶inflammatory cytokines such as ▶TNF-α. The tubulin binding agents’ primary mechanism of action is the disrupting of the ▶microtubule network of the tumor endothelium. Flavonoids Agents Flavonoids such as flavone acetic acid (FAA) and its fused tricyclic analogue 5,6-dimethylxanthenone-4acetic acid (DMXAA) have been shown to induce extensive hemorrhagic necrosis in tumors as a result of vascular collapse. Mechanistically, the action of this class of agent is believed to be largely indirect, through the induction of cytokines, particularly TNFα. This view is supported by experimental evidence showing that antibodies to TNFα could inhibit FAA-induced vascular collapse. It is of interest to note that although both FAA and DMXAA selectively damage tumor blood vessels in pre clinical tumor models, only DMXAA induces TNFα in both human and mouse macrophages. Consequently DMXAA is currently considered to be the lead agent of this class of VDAs. Tubulin Binding Agents The tubulin binding agents ▶colchicine and the ▶vinca alkaloids were recognized to have anti-vascular

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properties as early as the 1930s. However, the high toxicity of these agents limited their usefulness as vascular targeting therapies. More recently, agents with much more favorable therapeutic indexes, such as Combretastatin-A4-Phosphate (CA4P) and ZD6126, have rekindled interest in the use of this group of VDAs. These agents have shorter half-lifes and bind tubulin in a reversible manner, which reduces their toxicity to normal tissues. Tubulin binding agents bind tubulin subunits and prevent their polymerization. The result is depolymerization of the microtubules, reorganization of the actin cytoskeleton, and increased cellular permeability, which has been shown to be particularly destructive to dividing endothelial cells. In vivo, VDA treatment induces rapid disruption of blood flow and collapse of the vascular network. Although the precise mechanism of tumor blood flow shut down and vessel disruption is not fully understood, in vitro experiments have demonstrated endothelial cell shape changes and interruption of vascular endothelial-cadherin (▶VE-cadherin) signaling as a result of tubulin binding agent treatment. In vivo endothelial cell shape changes could augment vessel occlusion, while disruption of VE-cadherin results in reduced endothelial cell adhesion and could lead to vessel disruption. The tumor cells downstream of the disrupted blood vessels die due to a lack of oxygen and nutrients and the build up of toxic biological byproducts. After VDA treatment, necrosis is induced throughout the central portion of the tumor. The tubulin binding VDAs should not be confused with other microtubule binding anticancer agents such as the ▶taxanes. These agents bind to ▶microtubules and prevent depolymerization, contrasting with the VDAs which bind the tubulin subunits to prevent microtubule elongation. Treatment with these agents has not been shown to result in tumor vascular collapse, and, consequently, anticancer therapies like taxanes are designed to directly target and destroy tumor cells, not the tumor vasculature. VDAs as Adjuvants to Conventional Therapy A common characteristic observed after VDA treatment is the presence of a so-called “▶viable rim” of tumor cell existing at the tumor periphery. This thin layer of viable tumor tissue remains at the tumor periphery, presumably because this tumor tissue is supported by the normal vasculature, which is not affected by VDA treatment. The cells of this region are able to survive regardless of VDA dose or treatment schedule. These tumor cells continue to divide and rapidly repopulate the tumor, limiting the success of VDAs as single agents. While VDA treatment does not entirely eradicate the tumor, a large proportion of the center of the tumor is killed. In an untreated tumor, the cells of this central region are typically hypoxic, slowly dividing, poorly

perfused, and located in a high interstitial fluid pressure and low pH environment, making them more resistant to conventional anticancer therapies. As VDAs destroy this typically resistant region, addition of VDAs to current treatment regimens could improve treatment outcome when used in combination with this therapies. Several VDA and conventional therapy combinations have been tested in the pre-clinical setting and demonstrated beneficial treatment outcome over either therapy alone. These studies found improved treatment outcome when the VDAs CA4P or ZD6126 were combined with radiation. VDA combination with radiation was also found to effectively treat large tumors successfully, more so than smaller tumors. The combination of ZD6126 with the antiangiogenic agent ZD6474 produced improved treatment outcome in the ▶HT29 and ▶OW1 tumor models over either treatment alone. A few combinations are currently being tested in various clinical trials. VDAs such as ZD6126 and CA4P have been shown to produce maximum effective at doses well below the maximum tolerated dose. Neither does treatment with these agents alone result in significant growth delay, presumably due to the rapidly dividing tumor cells at the tumor periphery which survive VDA treatment. Consequently, there have been difficulties evaluating VDA treatment efficacy in the clinical setting. Preclinical evaluations of efficacy centered on the percent induced tumor necrosis, but these measurements are not practical in the clinical setting as they require highly invasive procedures that are not applicable to tumors in all locations. The viable tissue that causes difficulties with the evaluation of treatment efficacy also limits the usefulness of VDAs as single agents. However, while the effects of these VDAs have been extensively studied for the tumor as whole, little data exists that describes how the characteristics of the tissue that survives VDA treatment change during the phase of VDA-induced damage. Changes induced in this region by VDA treatment have the potential to negatively impact the efficacy of agents used in combination with VDAs. By understanding the VDA effects upon the surviving tumor tissue, insight may be obtained into possible means of overcoming these limitations. For example, decreases in perfusion as a result of VDA treatment that do not result in tumor death may inhibit the delivery of chemotherapeutic agents to the affected region and decrease the ▶radiosensitivity of tumors cells in the affected area. A few studies have reported varying combination treatment efficacies with differing treatment schedules. Therefore, a better understanding of the effects of VDA treatment on the surviving tumor tissue could lead to more effective combination treatment strategies. ▶Vascular Targeting Agents

Vascular Endothelial Growth Factor

References 1. Horsman MR, Siemann DW (2006) Pathophysiologic effects of vascular-targeting agents and the implications for combination with conventional therapies. Cancer Res 66(24):11520–11539 2. Siemann DW, Chaplin DJ, Horsman MR (2004) Vasculartargeting therapies for treatment of malignant disease. Cancer 100(12):2491–2499 3. Thorpe PE (2004) Vascular targeting agents as cancer therapeutics. Clin Cancer Res 10(2):415–427 4. Tozer GM, Kanthou C, Baguley BC (2005) Disrupting tumour blood vessels. Nat Rev Cancer 5(6):423–435

Vascular Endothelial Growth Factor D IETER M ARME´ Tumor Biology Center, Institute of Molecular Oncology, Freiburg, Germany

Synonyms VEGF; vascular endothelial growth factor; VPF; vascular permeability factor

Characteristics VEGF Family Four VEGF family members have been described in mammals, VEGF-A through VEGF-D. VEGF-E, the fifth member of the family is coded by the Orf virus. An additional relative is the placenta-derived growth factor, PlGF. Whereas the VEGFs are potent growth promoting and vascular permeability enhancing factors, PlGF is incapable of inducing permeability. All members exert their biological functions as homodimers. VEGFs act almost exclusively on endothelial cells. VEGF is expressed by almost all cell types with the exception of endothelial cells which express only marginal amounts of the growth factor. Expression is controlled by a number of different mechanisms. Extracellular signals such as growth factors and cytokines are able to induce transcription of the VEGF gene. Activated oncogenes, such as ras, raf, or src, as well as inactivated tumor suppressor genes, such as ▶p53 or ▶von-Hippel-Lindau (VHL), contribute to enhanced transcription. The newly identified p53 analogue p73 in its wildtype form can cause repression of VEGF transcription. Hypoxia, a physiological signal in early embryonic development and a pathophysiological signal in many tumors, causes enhanced production of VEGF mRNA and also stabilizes the VEGF mRNA. The pattern of VEGF expression is strictly controlled by some of these factors in a time- and tissue-specific fashion during

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embryonic development and physiological angiogenesis. In pathological situations VEGF expression proceeds with no specific control, exceeds physiological concentration and occurs at wrong times and locations. VEGF-Receptor Family All VEGF family members mediate their signals through a family of distinct high affinity receptors; VEGF-R1 through VEGF-R3. All three VEGF receptors are tyrosine kinases that become stimulated upon ligand binding, with VEGF-R1 tyrosine kinase being much less activated than VEGF-R2 and VEGF-R3. VEGF-R1 is expressed as two distinct forms; the entire transmembrane receptor VEGF-R1 and a soluble variant sVEGF-R1 generated by alternatively spliced mRNA. VEGF-R1 and its soluble variant bind VEGF-A, VEGF-B and PlGF. VEGF-R2 has high affinity for VEGF-A, VEGF-C, VEGF-D and VEGF-E. VEGF-R3 binds VEGF-C and VEGF-D. All three VEGF receptors are exclusively expressed on the surface of endothelial cells. VEGF-R2 is the most important VEGF receptor mediating a proliferative response to endothelial cells. Upon transfection into non-endothelial cells VEGF-R2 becomes autophosphorylated in response to VEGF binding, but is no more mitogenic. This indicates the involvement of cell typespecific signaling mechanisms. The endothelial cell proliferation and survival in response to VEGF requires the association of cell surface adhesive molecules. Activated VEGF-R2 associates with integrins ανβ3. VE-cadherin colocalizes with VEGF-R2 and upon stimulation by VEGF becomes associated with VEGFR2, β-catenin and PI-3-kinase. This leads to activation of PKB/Akt and initiation of a survival signal. Disruption of VE-cadherin leads to prevention of VEGF-mediated cell survival. VEGF-receptor expression is under tight control in embryo development and normal physiology. In pathological situations, such as tumor angiogenesis, VEGF-R1 and VEGF-R2 are transcriptionally upregulated in response to VEGF and thus generate an amplification mechanism to enhance this fatal process. VEGF in Vasculogenesis and Physiological Angiogenesis VEGF is widely and abundantly expressed in many tissues during fetal development and has been implicated in the process of vasculogenesis, i.e. the de novo formation of the vascular system. Mice deficient for VEGF die at day 8.5–9.0 and show a delayed differentiation and an impairment of both vasculogenesis and angiogenesis, i.e. the sprouting of new capillary vessels from pre-existing vasculature. Similarly, the receptors VEGF-R1 and VEGF-R2 are strongly expressed in the developing embryo. In particular, VEGF-R2 is expressed in the hemangioblasts, the common precursor to both endothelial cells and hematopoetic lineages. VEGF-R1, being non-essential for endothelial development, is required

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at a later stage of the organization of the embryonic vasculature. Disruption of the VEGF-R2 gene interferes with endothelial cell development, leading to death of embryos at day 8.5–9.5. Disruption of the VEGF-R1 gene permits endothelial cell differentiation but results in thin walled vessels of larger than normal diameter, and the embryos die at day 9. In the adult organism, large amounts of VEGF are also found in the female reproductive tissues in association with hormonally regulated angiogenesis that takes place in the ovary and endometrium at specific stages of the menstrual cycle and in pregnancy. Strong expression of VEGF can be detected in several tissues of the adult in the absence of angiogenesis, particularly kidney, lung, adrenal gland and heart. However, some of these tissues express only low levels or no VEGF receptors which might explain the absence of angiogenesis. As VEGF has been shown to be a survival factor for vascular endothelial cells it might also be that this function, requiring only low levels of VEGF receptors, is important for maintaining vascular homeostasis without angiogenesis to occur. Clinical Relevance VEGF in Pathophysiological Angiogenesis VEGF, as well as its receptors VEGF-R1 and VEGFR2, are strongly overexpressed at both the mRNA and protein levels in almost all malignant tumors. Tumor metastases exhibit overexpression of VEGF similar to that found in the primary tumors from which they arose. Elevated VEGF levels have been found in the blood of tumor patients, correlating in many cases with poor clinical prognosis of the disease. Accordingly, the soluble variant of the VEGF-R1 is also found to be elevated in the blood of tumor-bearing patients indicating the presence of activated tumor endothelium in the diseased areas. Thus, VEGF and the soluble variants of VEGF-R1 could be regarded as surrogates for pathological tumor ▶angiogenesis. In addition to the intimate involvement of VEGF and its receptors in tumor angiogenesis, VEGF is also capable of increasing vascular permeability. VEGF-induced leakage of plasma from hyperpermeable microvessels results in fluid accumulation within tumors. This is also favored by the fact that tumors in general lack lymphatic vessels and hence are unable to drain extravasated proteinaceous fluid effectively. This is in particular obvious in ▶brain tumors, showing increased intracranial pressure and in tumors metastasizing to body cavities leading to substantial accumulation of fluid. VEGF and both of its receptors are also overexpressed in a number of pathological entities that involve angiogenesis, but are not associated with neoplasia. These include diabetic and other retinopathies,

rheumatoid arthritis and psoriasis. In all of these examples, overexpression of VEGF and its receptors is accompanied by increased microvascular hyperpermeability and pathological angiogenesis.

VEGF/VEGF Receptor System: Therapeutic Opportunities As VEGF and its receptors are intimately involved in the pathology of many diseases, such as cancer, rheumatoid arthritis, diabetic retinopathies, considerable efforts have been made to interfere therapeutically with this signaling system. Monoclonal antibodies against VEGF and the binding domain of its receptor VEGF-R2 have been generated. Animal experiments show efficacy against tumor vascularization, tumor growth and metastases formation. Both antibodies have been humanized (▶humanized antibodies) and are being evaluated in the clinic. Low molecular weight compounds were developed to inhibit the tyrosine kinase activities of VEGF-R1 and VEGF-R2. These compounds show considerable activity against growth of highly vascularized tumors and also inhibit metastasis formation. Some of these inhibitors also show activity against other non-VEGF receptor tyrosine kinases. Clinical evaluation is being carried out to demonstrate whether the additional non-VEGF receptor related activity is beneficial or not. Furthermore, combination of anti-VEGF strategies together with low dose cytotoxic strategies have revealed synergistic effects in animals. This indicates that anti-angiogenic therapy could be useful to enhance the therapeutic potential of conventional chemo-therapeutic drugs. ▶Vascular Endothelial Growth Factor

References 1. Veikkola T, Karkkainen M, Claesson-Welsh L et al. (2000) Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60:203–212 2. Carmeliet P (2000) Mechanisms of angiogenesis and arteriogenesis. Nat Med 6:389–395 3. Siemeister G, Martiny-Baron G, Marmé D (1998) The pivotal role of VEGF in tumor angiogenesis: molecular facts and therapeutic opportunities. Cancer Metastasis Rev 17:241–248

Vascular Maturation ▶Vascular Stabilization

Vascular Stabilization

Vascular Normalization Definition

Is the process whereby ▶antiangiogenesis prunes and remodels the abnormal tumor vessels, which become closer to normal tissue vasculature in terms of structure and function.

Vascular Permeability Factor ▶Vascular Endothelial Growth Factor

Vascular remodeling ▶Vascular Stabilization

Vascular Stabilization S U¨ LEYMAN E RGU¨ N 1 , D ERYA T ILKI 2 , N ERBIL K ILIC 3 1

Institute of Anatomy, University Hospital Essen, Essen, Germany 2 Department of Urology, University Hospital Groβhadern, Munich, Germany 3 Department of Hematology and Oncology, University Hospital Hamburg-Eppendorf, Germany

Synonyms Structural vascular stabilization; Functional vascular stabilization; Vascular maturation; Vascular remodeling

Definition Vascular stabilization stands for the basic steps of the morphogenetic processes, finally leading to vascular maturation such as establishment of inter-endothelial contacts, development of a regularly structured basement membrane enveloping both endothelial cells and pericytes, and integration of one layer of peri-endothelial cells (pericytes or smooth muscle cells) into the vascular wall.

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Characteristics Normal Vascular Hierarchy The adult vascular system is hierarchically organized in large, middle sized, and micro vessels at both the arterial and venous sides. The lumen of all blood vessels is lined by endothelial cells (EC). EC are of mesodermal/ mesenchymal origin and differentiate from hemangioblasts; they are probably the common precursor of endothelial and hematopoietic cells. The first hemangioblasts are visible in the blood islands of chorionallantoic membrane and the peripheral cells of these islands differentiate to endothelial cells, while the rest serve as the source for hematopoiesis. The primitive vascular network developed in the chorionallantoic membrane subsequently connects to the cardiac system and serves as the basis for a functioning blood circulation. However, this system comprising nascent blood vessels will undergo enormous remodeling processes containing several sequential steps which are precisely coordinated timely and spatially. The essential step of the vascular remodeling in the early phase of vascular development is structural stabilization, which involves the development of inter-endothelial contacts and basement membrane and integration of peri-endothelial cells into the vascular wall (Fig. 1). Accompanied by organogenesis and the demand on blood perfusion, further processes of vascular maturation will lead to the development of the vascular wall containing several layers of smooth muscle cells and connective tissue, depending on perfusion pressure and exchange processes between the vascular and interstitial compartments. The factors and mechanisms regulating these processes are also involved in the regulation of vascular permeability, which also depends on organ-specific requirements. The end of these processes is the construction of a vascular hierarchy comprised of large and mid-sized arteries and veins macroscopically visible, as well as arterioles, capillaries and venules making up the micro vascular part of the blood vessels (Fig. 1). As far as we can observe, the vascular stabilization and the final vascular maturation result in a decrease of vascular density as blood vessels which are not included in the organ perfusion by blood undergo regression and successively disappear (Fig. 1). Vascular Destabilization and Activation of Angiogenesis The initiation of ▶angiogenesis is marked by structural destabilization of the vascular wall, generally accompanied by an abnormal vascular permeability. This is also a common sign of the tumor vascular bed. Looking at the structure of tumor vessels, a structural instability marked by the loosening of pericytes/smooth muscle cells from the endothelial layer, opening of interendothelial junctions (▶Tight junctions), development of endothelial fenestration and/or transendothelial gaps

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Vascular Stabilization. Figure 1 From the primitive vascular plexus to the hierarchy of normal vascular system: the primitive dense vascular plexus is mainly composed of unstable nascent blood vessels of approximately equal state of vascular stabilization (a, left). Cross section shows that BM is not regularly structured and pericytes (red) are not tightly connected to the endothelial layer (green). Vascular stabilization during embryogenesis and fetal development have led to a decrease of vascular density (a, right). In cross section (a, right) a dense BM (gray) is visible, endothelial cells (green) build a closed layer and mural cells (red) are enclosed by the same BM and are tightly organized around the endothelial layer. These processes serve as the basis for the first step of vascular hierarchy in the adult as demonstrated in b.

and degradation of the vascular basement membrane are frequently observed alterations of the vascular wall in this initial phase of angiogenesis (Fig. 2). This initial destabilization is probably induced by a switch of the angiogenic balance towards the activation of angiogenic sprouting. The further duration of this process is mediated by pro-angiogenic factors such as VEGF (▶Vascular endothelial growth factor), FGF-2 (▶Fibroblast growth factors) and Ang2. It would lead to complete destabilization and disintegration of the endothelial layer. The detachment of endothelial cells from the basement membrane results in the migration and subsequent proliferation of these cells. Finally, new capillary sprouts formed by the new endothelial cells are nascent and structurally unstable. The maintenance of pro-angiogenic activation would hold these new vessels in structural instability and in an abnormal leaky state. This phenotype of vessels dominates in the tumor vascular bed. Particularly, the concerted action of VEGF and Ang2 at certain sites of tumor vasculature has been shown to be very effective in keeping the blood vessels unstable and angiogenesis ongoing. Vascular destabilization accompanied by abnormal vascular leakiness is also frequently observed during inflammation. It is well known that several cell types involved in the inflammatory process such as macrophages or lymphocytes produce high amounts of

pro-angiogenic factors including VEGF and bFGF. Also Ang2 which is involved in vascular destabilization has been detected in several inflammatory processes. Thus, inflammation seems to provide high amounts of VEGF and Ang2, and the concerted action of both is the main mediator of vascular destabilization. Vascular Stabilization While it has been assumed for several years that new vessels provided by angiogenesis are lined only by endothelial cells and would not be able to enter further steps of vascular maturation, detailed studies in the last decade have revealed without doubt that some of the new vessels developed by physiologic or pathologic angiogenesis as in tumors (▶cancer) do exhibit a basement membrane and mural cells such as pericytes and/or smooth muscle cells. As of now, only a few factors have been characterized as being involved in these processes. To give a systematic overview of the vascular stabilization, it makes sense to evaluate the following morphogenetic steps. 1. Inter-endothelial contacts: Like cell-cell contacts between epithelial cells, TJ or ▶zonula occludens, adherence junctions (AJ) or zonula adherens and ▶gap junctions (GJ) are the main cell-cell contact types between endothelial cells, but in contrast to


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Vascular Stabilization. Figure 2 Vascular destabilization and initiation of angiogenesis: a normal stabilized capillary with a dense BM (gray) enclosing both endothelial cells (green) and pericyte (red) (a) will be destabilized by the action of pro-angiogenic factors as shown by VEGF and Ang2 leading to endothelial fenestration (fn), opening of inter-endothelial contacts (iec), development of transendothelial gaps (gap), degradation of BM and finally detachment of pericytes from the endothelial layer (b). These morphogenetic events are accompanied by an abnormal vascular leakiness. The further duration of pro-angiogenic action would finally lead to the sprouting of new nascent and unstable blood vessels (c), a process defined as angiogenesis.

epithelial cells, ▶desmosomes are absent between endothelial cells. Also, in endothelial cells, TJ are localized at the boundary between the apical and basolateral sides of endothelial cells and thus, they are important for the barrier function. The members of the claudin family, occludin and junctional adhesion molecule-A (JAM-A) as well as their cytoplasmic interaction partners such as the members of the ZO (zonula occludens proteins) are the main players that govern the establishment of endothelial TJs. Also ESAM, the endothelial cellselective adhesion molecule, is localized at the TJ and regulates the paracellular permeability of the endothelial barrier. VE-cadherin (vascular endothelial cadherin) (▶Adherens junctions) is essential for the establishment of adherence zones between endothelial cells. The main intracellular partners of VE-cadherin are β- and γ-catenin. They link α-catenin, which anchors this complex to actin. A further intracellular partner of VE-cadherin is p120, a substrate of src. Of particular impact for vascular stabilization versus destabilization is the interaction of VE-cadherin with endothelial signaling proteins. VE-cadherin, VEGFR-2 (VEGF receptor type 2, KDR) and Src-kinase form a complex which is obviously essential for the maintenance of the endothelial barrier controlling the paracellular transport of molecules and cells through the endothelial layer. In contrast, the interaction of VE-cadherin with VE-PTP (vascular endothelial-specific receptorprotein tyrosin phosphatase) strengthens the AJ between endothelial cells. Gap junctions (GJ) represent intercellular channels mediating exchange of

ions and small molecules between neighboring cells by diffusion. The establishment of GJ is governed by ▶connexins; ~20 connexins (Cx) have been identified until now. Expression studies demonstrate the presence of Cx37, Cx40 and Cx43 between endothelial cells but the role of connexins in the inter-endothelial communication is not sufficiently understood. Considering the fact that the expression pattern of these connexins in the blood vessels depends on the vessel type and the position in the vascular tree a regulating role of the connexins in differentiation or maturation of blood vessels can be postulated. Although there are several excellent studies regarding the expression and the role of each factor involved in cell-cell contact between endothelial cells, it is not clarified at exactly which stage of vascular morphogenesis these contacts are established. Particularly the determination of temporary and spatial sequences of these processes needs to be evaluated. Based on recent studies it can be postulated that in the first step of vascular morphogenesis, where the nascent vessels are lined only by endothelial cells, the first provisional interendothelial contacts are mediated by ▶cell adhesion molecules like integrins, VCAM, ICAM and ▶CEACAM1. Particularly, CEACAM1 has been shown to be involved in vascular stabilization in a dual way: CEACAM1 overexpression in endothelial cells induces a signaling leading to vascular stabilization, while its down-regulation in epithelial cells as it occurs in several tumors leads to vascular destabilization. In a second step the special cell-cell contacts mentioned above will be established between

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endothelial cells serving the basis for the entering of nascent and still unstable vessels into the stabilization process. 2. Construction of the vascular ▶basement membrane (BM): The BM (or electron microscopically basal lamina) is an extracellular matrix structure of flexible thickness of 40–120 nm underlying all epithelial cell sheets and tubes formed by endothelial cells. In general, the assembly of BM is mainly provided by collagen type IV and laminin, which have the capacity to self-assembly, but several other molecules like perlecan, nidogens, and collagen type XVIII are identified components of BM and necessary for its regular construction. In transmission electron microscopic studies of the normal vasculature, the basal lamina (corresponding structure to basement membrane) shows an amorphous and dense structure. In small blood vessels BM encloses both endothelial cells and pericytes. The dense structure of BM is mostly not present in the basal lamina of tumor blood vessels, indicating a degradation or insufficient construction of the BM. This is one of the essential parameters leading to the loss of endothelial anchoring to the extracellular matrix and to detachment of pericytes from the endothelial layer with subsequent migration and proliferation of endothelial cells. The components of well structured BM signal an inhibitory effect on angiogenesis while the direct contact of endothelial cell to the “provisional” matrix components such as collagen type I, fibronectin and vitronectin accelerates the proliferation and migration of endothelial cells. BM collagens contain cryptic domains with antiangiogenic activity (▶Antiangiogenesis) if they are released from their mother substance as demonstrated by ▶endostatin, a fragment of collagen type 18, and tumstatin, a fragment of collagen type IV. Endostatin has been shown to stabilize newly formed endothelial tubes and to strengthen the endothelial barrier by protecting the basement membrane and interendothelial contacts in normal structure. Taken together, these data suggest that a well constructed vascular basement membrane stabilizes the newly formed endothelial tubes and reduces the angiogenic potency. Indeed, in experimental models and in preclinical treatment with different types of angiogenic inhibitors (▶Antiangiogenesis) ▶stabilization of the vascular wall and the decrease of vascular leakiness are frequently observed phenomena. 3. Integration of vascular mural/peri-endothelial cells into the vascular wall: The assembly of pericytes or smooth muscle cells to the vascular wall is an essential step of vascular stabilization. Factors involved in this process are Ang1 and Tie-2-system, TGFβ, PDGF and their receptors. Although the

vascular mural cells are of multiple origins, their presence and interaction with endothelial cells are crucial for vascular stabilization. Ang1 is not only involved in the assembly of pericytes into the vascular wall but it also mediates cell-cell and cellmatrix interaction, leading to a significant reduction of vascular leakiness. Ang1- and Tie-2 knock-out mice are lethal because they lack vascular plasticity and remodeling resulting in disorganization of the primitive vascular plexus and apoptosis of endothelial cells. In contrast, Ang1 overexpression in mice blocks the VEGF-induced vascular leakiness and increases the diameter of blood vessels. To reduce the role of pericytes in vascular morphogenesis only to a mechanical supportive function for the endothelial cells would be very simplifying and not appropriate. Via direct signaling facilitated by cellcell contacts between endothelial cells and pericyte processes through the basement membrane, pericytes influence the maturation and quiescence of endothelial cells. But these actions of pericytes on endothelial cells can only be effective when pericytes are well integrated into the vascular wall enclosed by the same basement membrane as endothelial cells (Fig. 3). Clinical Implications In several diseases the vascular dysfunction is either an accompanied clinical problem making therapeutic handling much more difficult such as in diabetic retinopathy and microangiopathy, or it is an essential prerequisite for the full development of diseases such as cardiovascular failure or tumor growth and metastasis. Since cardiovascular diseases rank first and tumor second in the list of fatal diseases world wide, the therapeutic managing of vascular morphogenesis is a big challenge in medicine. The role of vascular stabilization in the process of vascular morphogenesis, plasticity and remodeling is still not sufficiently understood because it was neglected for a long time. In the last few years, the mechanisms of vascular stabilization, particularly the interaction between endothelial and vascular mural cells, have received more attention in both cardiovascular and tumor angiogenesis research. While anti-angiogenesis targets the tumor vasculature to cut blood supply to the tumor tissue, the proangiogenic therapeutic strategies deal with the creation of new vessels, which should have the capacity, as far as possible, to achieve a structural stabilization and functional normalization. Therapeutic angiogenesis and vascular stabilization: “One is as old as one’s arteries” is a frequently cited statement from Virchow and underlines the impact of the arteries in determining the life span, and also the quality of life. Cardiovascular disorders such coronary artery disease or the sclerotic changes of the peripheral


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Vascular Stabilization. Figure 3 Vascular stabilization: the wall of a newly formed unstable blood vessel is constructed by endothelial cells (green) with provisional interendothelial contacts, provisional BM without the regular dense organization and mural cells (red) which are present but not regularly integrated into the vessel wall (a). Several endogenous factors as shown by Ang1, TGFβ, PDGF and endostatin, promote the structural stabilization and serve in a concerted interplay with other factors such as VE-cadherin, occludin, claudins, collagen IV, laminin and integrins as the basis for establishment of durable interendothelial contacts, a regular BM and mural cells tightly integrated into the vascular wall, a process named vascular stabilization.

arteries are mostly caused by degenerative alteration of the vascular wall at the arterial part of the vascular system. In all these disorders the formation of new vessels, for example the initiation of the growth of collateral vessels, is desirable, but it is difficult to bring the new vessels in a stable state, fulfilling the specific tissue demands on vascular perfusion and permeability. Although therapeutic angiogenesis in preclinical studies shows promise, no clear success has been achieved in controlled clinical phases II and III studies until now. These results demonstrate how complex the processes are which govern the morphogenesis of blood vessels, but they also show how difficult the way is from experimental models to human trials. Despite these ups and downs, the therapeutic angiogenesis still remains a fascinating perspective, highly promising for the treatment of cardiovascular and ischemic disorders. Anti-angiogenic tumor therapy and vascular stabilization: The destabilization of blood vessels accompanied by abnormal vascular leakiness is one of the earliest signs of angiogenic activation. This vascular phenotype dominates the tumor vascular bed. Although it was believed for a long time that tumor vessels lack BM and ▶pericytes, detailed studies in the last 5–6 years have revealed that in tumor vessels also, endothelial tubes are underlined by a BM and that pericytes are organized around these tubes. But in contrast to normal vasculature, the BM of tumor blood vessels is not well structured, the interendothelial contacts are not strong enough, and the pericytes are not tightly connected to the endothelial tubes. This vascular phenotype has a high plasticity, making it

highly susceptible to pro-angiogenic factors such as VEGF and Ang2. While the link between vascular destabilization and angiogenic activation, as well as tumor vascularization, has been known for several years, well studied and understood to a great extent, the impact of vascular stabilization on tumor vascularization, tumor growth and metastasis (cancer) was mostly neglected until a few years ago. Based on emerging data from anti-angiogenic therapy, a “normalization” of tumor vessels has been postulated. According to this hypothesis, the anti-angiogenic therapy would create a “therapeutic window,” enabling a more effective use of chemo- or radiation therapy for tumors. This suggests an indirect relation between vascular stabilization and tumor therapy, but what is the role of vascular stabilization per se on tumor angiogenesis and tumor growth and metastasis? The best known factor leading to vascular stabilization is Ang1. Ang1 overexpression blocks the VEGF-induced vascular leakiness by stabilization of blood vessels. In mice overexpressing Ang1, tumor vascularization was seen to be less active than those overexpressing Ang2 or tumor growth was suppressed. But there are also studies suggesting that vascular stabilization, for example by Ang1, results in the acceleration of tumor growth. These controversial findings demonstrate clearly that this aspect of tumor angiogenesis has not been studied sufficiently. Hypothetically, one would expect that vascular stabilization reverses the angiogenic phenotype of blood vessels to a more quiescent phenotype by reducing susceptibility of tumor vessels to pro-angiogenic factors. This in turn would cause a reduction of vascular density and part

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Vascular Targeted Therapies

References 1. Bazzoni G, Dejana E (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 84:869–901 2. Ergun S, Tilki D, Oliveira-Ferrer L et al (2005) Significance of vascular stabilization for tumor growth and metastasis. Cancer Lett 18:180–187 3. Jain RK (2005) Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 307:58–62 4. Kalluri R (2003) Angiogenesis: basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 3:422–433 5. Thurston G, Rudge JS, Ioffe E et al. (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 6:460–463

Vascular Stabilization. Figure 4 Vascular stabilization and tumor growth: Stabilization of tumor vessels occurs mostly in the tumor center but significantly extends to the marginal zone under anti-angiogenic therapy, e.g. with endostatin. This is accompanied by a dramatic regression of a part of the blood vessels, resulting in a significant reduction of vascular density. This process apparently changes the perfusion of tumor tissue. In the marginal tumor zone with unstable blood vessels, there is a nearly equal perfusion of the whole vascular bed as marked by arrows. In contrast, in the tumor center, the main route of blood flow is served by stabilized vessels while unstable blood vessels were successively cut of function and underwent regression. This may result in further necrosis of tumor tissue. Green: endothelial lining, grey: vascular basal lamina.

starvation of tumor tissue, resulting in tumor necrosis (Fig. 4). This has been observed in several experimental tumor models and experimental tumor treatments with different anti-angiogenic substances. It is imaginable that we principally need a two-step approach of antiangiogenic tumor therapy: the first step should aim to achieve a disruption of new nascent and unstable blood vessels and a suppression of endothelial proliferation, and the second would deal with the stabilization of tumor vessels switching tumor vasculature from the angiogenic to quiescent phenotype. Both these steps would serve the basis for a therapeutic “window” enabling the combination of angiogenesis inhibitors and chemotherapeutic drugs (▶Chemotherapy) and/or radiation (▶Radiation oncology) in the final step of tumor therapy. Taken together, emerging data recommend a stronger consideration of mechanisms that govern vascular stabilization in future anti-angiogenic tumor therapy strategies.

Vascular Targeted Therapies ▶Vascular Disrupting Agents ▶Vascular Targeting Agents

Vascular Targeting Agents M ICHAEL R. H ORSMAN 1 , D IETMAR W. S IEMANN 2 1

Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark 2 Department of Radiation Oncology, University of Florida, Gainesville, FL, USA

Synonyms VTAs; Vascular targeted therapies; Angiogenesis inhibiting agents; Vascular disrupting agents

Definition Vascular targeting agents (VTAs) are primarily cancer therapies that are specifically designed to target the vasculature of tumors and as a consequence will inhibit tumor growth and development. They may also be used to treat other pathophysiological conditions in which the tissue vasculature plays a role.

Characteristics Background In cancer, the vascular supply to tumors is critical. For most solid tumors to grow beyond a size of a few

Vascular Targeting Agents

millimeters it is necessary for them to develop their own functional blood supply, which they do from the already established normal tissue vasculature by a process called ▶angiogenesis. This process begins with the tumor cells secreting various angiogenic growth factors. These factors are upregulated by different environmental changes such as ▶hypoxia, loss of ▶tumor suppressor function or ▶oncogene activation. Of these angiogenic factors, the most potent and specific is ▶vascular endothelial growth factor (VEGF), which is not only crucial for endothelial cell proliferation and blood vessel formation, but also induces significant vascular permeability and plays a key role in endothelial cell survival signaling in newly formed vessels. These growth factors react with various receptor kinases on the endothelial cells and then put in motion a series of physical events that include destruction of the basement membrane of the normal endothelial cells, migration of endothelial cells into the extracellular matrix in the form of a sprout, division of endothelial cells away from the sprout tip, the formation of solid strands of endothelial cells in the extracellular matrix, the development of a lumen within the strands, fusion with other sprouts to form loops, and the formation of new sprouts and loops from these primary loops. All this ultimately results in the establishment of a functional vascular supply for the tumor. Once this occurs not only can the primary tumor begin to grow, but the tumor cells have a means of entering the systemic circulation and so move to other areas in the body and then ultimately form ▶metastases. This importance of the tumor vasculature makes it an excellent target for therapy and two major VTA approaches have now evolved. The first is based on controlling the development of the tumor blood vessels by inhibiting the angiogenesis process, while the second involves a disruption of the already established tumor blood vessels. It has been demonstrated that vascular effects are involved in the action of other therapies, including certain types of ▶chemotherapy, radiotherapy, and inhibitors of epidermal growth factor receptors or cyclooxygenase-2. But, in these situations the vasculature is far from being their principal target and as such it is technically incorrect to classify them as VTAs. Mechanisms Although angiogenesis inhibiting agents (AIAs) and ▶vascular disrupting agents (VDAs) both target the tumor vascular supply they are two distinct approaches. AIAs are designed to prevent further development of the tumor neovascular network. The complex process of tumor angiogenesis offers many possible targets for ▶antiangiogenesis strategies. These strategies vary from regulation of angiogenic factor expression in tumors to endogenous inhibitors of angiogenesis. Based

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on their biological activities, these strategies can be categorized into several broad classes. One class of agents specifically targets the angiogenic growth factors, which play the most significant role in neo-vascularization, especially VEGF. VEGF has been targeted by a variety of strategies, including inhibitors of endothelial cell receptor signaling that interfere with associated ▶receptor tyrosine kinase activities (e.g., SU5474, SU6668, ZD6474 and PTK787/ZK 222584), as well as monoclonal antibodies directed against pro-angiogenic growth factors (e.g., Bevacizumab/Avastin and DC101). Bevacizumab/Avastin, a recombinant humanized monoclonal antibody to VEGF, is the first antiangiogenic therapy to have demonstrated a survival advantage when given to patients with cancer. It is currently being investigated in a variety of tumor types. A second class of agents includes those designed to inhibit endothelial cell functions, basement membrane degradation, endothelial cell migration, proliferation, and tube formation. One example is ▶Endostatin, a naturally occurring fragment of collagen XVIII, which has been identified as a potent endogenous inhibitor of endothelial cell function. A third class consists of agents that target survival factors of neovascular blood supply, such as integrin antagonists. Integrins are heterodimeric transmembrane proteins that control cell motility, differentiation and proliferation via interactions with extracellular matrix molecules. The αvβ3 integrin is an attractive target for anti-angiogenic therapy because it is almost exclusively present on the cell surface of activated endothelial cells and is considered a survival factor for angiogenic vessels in tumors. Finally, nonspecific therapies that impact new vessel development are also being actively considered. ▶Thalidomide and its analogues are one such group of agents that inhibits angiogenesis, but the mechanism of action is poorly understood. VDAs are agents that cause direct damage to the already established tumor endothelium. These include physical treatments like hyperthermia or ▶photodynamic therapy, which have been well documented to induce direct tumor cell killing and an indirect effect through the induction of vascular damage. They also include biological response modifiers or cytokines like tumor necrosis factor (TNF) and interleukins; certain established chemotherapeutic drugs such as vinka alkaloids and arsenic trioxide; and various ligand-based approaches that use antibodies, peptides or growth factors that can selectively bind to tumor vessels. But, more commonly VDAs involve the use of small molecule drugs, of which there are two major classes of agents. The first includes flavone acetic acid (FAA) and its derivative DMXAA, which have a complex mechanism of action that is poorly understood, but their main effect on vascular endothelial cells is thought to involve a cascade of direct and indirect effects, the latter

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Vascular Targeting Agents

involving the induction of cytokines, especially TNF-α, leading to the induction of hemorrhagic ▶necrosis. A second group includes the tubulin-binding agents CA4P, ZD6126, AVE8062, NPI2358, MN-029, and OXi 4503. These tubulin depolymerizing agents are believed to selectively disrupt the cytoskeleton of proliferating endothelial cells, resulting in endothelial cell shape changes and subsequent thrombus formation and vascular collapse. Since they preferentially target dividing endothelial cells this accounts for their tumor specificity. Both types of small molecular drugs have been shown to have potent anti-vascular and anti-tumor efficacy in a wide variety of preclinical models and the lead agents are undergoing clinical evaluation. Since AIAs and VDAs induce vascular effects by very different mechanisms their anti-tumor activity and optimal application will be very different. Generally, AIAs are given as a chronic administration and essentially slow tumor development. There are examples where tumor growth can be completely inhibited or the treatment of established tumors can result in tumor regression, but these tend to be exceptions rather than the norm. As a result AIAs are probably best suited for early stage or metastatic disease. With VDAs the administration is of a more acute type to induce substantial vascular shut down. Anti-tumor effects should also be possible with lower doses given over a prolonged period, but that would probably increase the risk of normal tissue vessel damage and defeat the potential benefit. Following treatment with VDAs tumor shrinkage has been observed, but this appears to be tumor and drug dependent and although significant it is generally modest and thus tumor growth is only temporarily delayed. There is good evidence that VDAs have a superior effect on bulky disease. Given the key differences between AIAs and VDAs it should be clear from a therapeutic perspective that targeting the tumor vasculature with AIAs and VDAs is complimentary and not redundant. Such approaches are being actively pursued. Clinical Aspects It is clear that neither AIAs nor VDAs, whether given alone or even in combination, can induce tumor control. Therefore, their clinical potential as anti-cancer therapies requires that they must be combined with other cancer therapies, and numerous pre-clinical studies have demonstrated that when this is done significant improvements in tumor response are possible. For AIAs, this has been demonstrated when they have been combined with conventional treatments like radiation (▶ionizing radiation therapy) and chemotherapy, including ▶alkylating agents (e.g., ▶cisplatin, carboplatin, melphalan, cyclophosphamide and dacarbazine), nitrosoureas (e.g., BCNU), antimetabolites (e.g., ▶gemcitabine, 5-▶fluorouracil, and pemetrexed),

anthracyclines (e.g., doxorubicin), topoisomerase inhibitors (▶topoisomerase enzymes as drug targets) (e.g., ▶irinotecan, topotecan and etoposide), taxanes (e.g., ▶paclitaxel and ▶docetaxel), corticosteroid hormones (e.g., prednisone) and ▶bioreductive drugs (e.g., ▶mitomycin C). AIAs have to a lesser extent also been combined with hyperthermia and photodynamic therapy. With VDAs the combinations have also included radiation and various chemotherapy agents, such as alkylating agents (e.g., cisplatin, carboplatin, melphalan, cyclophosphamide and chlorambucil), antimetabolites (e.g., 5-fluorouracil), anthracyclines (e.g., doxorubicin), topoisomerase inhibitors (e.g., irinotecan and etoposide), taxanes (e.g., paclitaxel and docetaxel), vinka alkaloids (e.g., vincristine and vinblastine) and bioreductive drugs (e.g., mitomycin C, tirapazamine and AQ4N). Less conventional therapies combined with VDAs include hyperthermia, radioimmunotherapy, and antibody/clostridia directed enzyme prodrug therapy. One important issue here concerns the pathophysiological effects induced by VTAs. As a result of targeting tumor vasculature, VTAs modify the tumor pathophysiology and these include changes in vascular density, blood perfusion, oxygenation, metabolic activity, intracellular/extracellular pH and interstitial fluid pressure. Many of these pathophysiological changes can have a profound influence on the activity of the other combination therapy, and since the reported changes include both increases and decreases the effects can be in both a negative and positive fashion. For example, increasing tumor oxygenation status will enhance tumor radiation response, while decreasing oxygenation will reduce it. With the AIAs these pathophysiological changes can be highly variable between the different drug types. The same AIA can also produce completely opposite pathophysiological affects in different tumor types, even when administered using similar drug doses and treatment schedules. VDAs are more consistent in the pathophysiological changes induced. Essentially they all decrease tumor blood perfusion and as such will make the microenvironmental conditions, especially oxygenation and pH, worse. These effects suggest that timing and sequence between the different treatments must be considered in any clinical application, especially when combining VTAs with therapies that already have some beneficial effect in patients. Despite these potential limitations, preclinical studies with VTAs in combination with other therapies do show an enhanced tumor response without any significant increased damage in dose limiting normal tissues. Numerous VTAs are currently under clinical evaluation. With AIAs these include both specific and nonspecific inhibitors of angiogenesis, and the phase of testing ranges from Phase I to IV. The most popular agents in clinical testing are the anti-VEGF antibodies

Vasculogenic Mimicry

(e.g., Avastin), followed by receptor kinase inhibitors (e.g., Bay 43–9006, SU 11248, PTK 787/ZK 222584), and the non-specific inhibitor thalidomide. Far fewer trials are underway with the VDAs, and those agents that are being investigated are either in Phase I or II. The lead agent in this series is CA4P. Experimental studies strongly support the concept that the application of angiosuppressive and vascular disrupting strategies as adjuvants to standard anticancer therapy can improve treatment outcomes. Lead vascular targeting agents are now under active investigation in such settings in patients. Ultimately, it is possible to envisage future treatment protocols consisting not only of the current mainstays of cancer management, surgery, radiotherapy, and chemotherapy, but will also include a “vascular targeted therapy” consisting of a battery of tumor vessel directed agents. ▶Vascular Disrupting Agents

References 1. Folkman J (1976) The vascularisation of tumors. Sci Am 234:58–71 2. Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676 3. Kerbel R, Folkman J (2002) Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2:727–739 4. Siemann DW, Chaplin DJ, Horsman MR (2004) Vascular targeting therapies for treatment of malignant disease. Cancer 100:2491–2499 5. Horsman MR, Siemann DW (2006) Pathophysiological effects of vascular-targeting agents and the implications for combination with conventional therapies. Cancer Res 66:11529–11539

Vascular Targeting Agents ▶Vascular Disrupting Agents

Vasculogenesis Definition Is the reorganization of randomly distributed cells into a blood vessel network. ▶Vasculogenic Mimicry

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Vasculogenic Mimicry B AOCUN S UN , S HIWU Z HANG , DANFANG Z HANG Department of Pathology, Tianjin Cancer Hospital and Tianjin Cancer Institute, Tianjin, P.R of China

Definition The generation of microvascular channels by genetically deregulated, aggressive tumor cells was termed “vasculogenic mimicry” (VM) to emphasize their de novo generation without participation by endothelial cells. VM is thought to represent a vascular channel formation without the involvement of endothelial cells, in contrast to ▶angiogenesis. Vasculogenic mimicry refers to a blood supply pathway in tumors that is formed by tumor cells and that is independent of endothelial cell-lined blood vessels. Three factors are thought to govern the formation of functional and patterned microcirculation channels by VM: (i) plasticity of highly malignant tumor cells, (ii) remodeling of the ▶extracellular matrix (▶ECM), and (iii) connection of the VM channel with host blood vessels to acquire blood supply from the host tissue. Formation of VM in tumors may have substantial impact on clinical outcome of tumor patients. Tumor patients in the presence of VM have a poorer prognosis than those without VM, and ▶VM-targeted therapy is a perspective for tumors showing VM. At this point, VM is a new concept originally described for ▶melanoma that needs to be studied further in detail.

Characteristics Introduction Tumor angiogenesis is a key for tumor growth, ▶invasions, and metastasis. Tumor growth is ▶angiogenesis-dependent, and angiogenic switch is an essential step for a small and noninvasive tumor to transit into a tumor with invasive and metastatic ability. Blood vessels are assembled by two processes: (i) ▶vasculogenesis, the reorganization of randomly distributed cells into a blood vessel network, and (ii) ▶angiogenesis, the sprouting of new vessels from preexisting vasculature in response to external chemical stimulation. Current Status of Studies on VM in Tumors It is believed that VM consists of tumor cells and PASpositive ECM on the inner wall of channels. The constituents of PAS-positive ECM are ▶laminin, collagens IV and VI, mucopolysaccharide, and heparin sulfate glucoprotein (HSPG). Initially, PAS-positive ECM was considered as an absolutely indispensable element. However, VM channels in the absence of

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Vasculogenic Mimicry. Figure 1 Vasculogenic mimicry (VM). Melanoma cells form VM channels, and red blood cells (RBC) flow into the channel. Necrosis and inflammatory cells are not observed in tumors undergoing VM.

Vasculogenic Mimicry. Figure 2 The connection of VM channel and endothelium-dependent vessel shows VM is a functional microcirculation.

PAS-positive ECM were observed in a melanoma mouse model. VM is an independent blood supply pattern in tumors (Figs. 1–3). Compared with endothelium-dependent vessels, it has several characteristics as follows: (i) VM channels are lined by tumor cells but not endothelial cells. (ii) There is red blood cell (RBC) leakage into tumor tissue near to endothelium-dependent vessels, while leaking RBCs are lacking in tumors with VM. (iii) Necrosis and inflammatory cells are not observed in tumors undergoing VM.

Molecular Mechanisms Underlying VM Compared with less aggressive melanoma cells, highly aggressive melanoma cells express higher levels of ▶matrix metalloproteinases (▶MMP-1, 2, 9, and 14) and the 5γ2 chain of laminin. This increases expression of MMPs and presence of the laminin receptor on the surface of tumor cells help cells adhere to more laminin. The activated MMPs cleave laminin into several short chains and eventually promote the formation of VM. Phosphoinositide-3-kinase modulates the function of MMP-14 (MT1-MMP), which activates MMP-2 with the help of tissue inhibitor of MMP-2 (TIMP2), and the activated MMP-2 then cleaves 5γ2 chain into γ2′ and γ2x chains. The two chains facilitate the formation of VM. The cleavage fragments of 5γ2 can be secreted by highly malignant melanoma cells directly. VE-cadherin has been proved to be closely related to the formation of VM channels. Highly aggressive melanoma cells express VE-cadherin but less aggressive ones do not and inhibition of VM formation by downregulating the expression of the VE-cadherin gene.

Vasculogenic Mimicry. Figure 3 VM consists of tumor cells and PAS-positive ECM on the inner wall of channels. The PAS-positive patterns are lined by tumor cells, and there are red blood cells in the center of the pattern.

Dedifferentiation of Tumor Cells is the Key to Formation of VM Channels Much information on VM has come from studies of highly malignant melanomas. Tumor cells having the ability of VM formation show an embryonic phenotype. A ▶cDNA microarray study of 5,000 genes from a patient with poorly and highly aggressive melanoma cells revealed that there was a differential expression in 210 genes, including some genes associated with the phenotypes of endothelial and hematopoietic stem cells. Except for embryonic genotypes, cells of tumors with VM express various angiogenesis-related cytokines. Flt-1 and Tie-2 are expressed by tumor cells of naked


mice bearing human inflammatory breast carcinoma cells. Ovarian ▶cancer cells with high aggressivity express the vascular endothelial growth factor (VEGF) and other angiogenesis-related cytokines (e.g., Ang-1 and Ang-2), whereas those with low aggressivity express VEGF only. The expression of tyrosine kinase (an enzyme that catalyzes the phosphorylation of several signal transduction proteins) is upregulated in highly aggressive melanoma cells than in less aggressive melanoma cells. Aggressive melanoma cells show an increased activity of tyrosine kinase around VM channels. ▶Epithelial cell kinase (▶EphA2), a tyrosine kinase receptor, is specifically expressed in highly aggressive melanoma cells. Inhibitors of tyrosine kinase activity hinder VM channel formation, and a transient knockout of EphA2 shows reduced VM channel formation. Linearly Patterned Programmed Cell Necrosis and Three-Stage phenomenon ▶Linearly patterned programmed cell necroses (▶LPPCN) and ▶three-stage phenomenon are thought to play essential roles in the blood supply for melanoma. At the early stage of tumor generation, endothelium-dependent vessels do not sprout into tumor center. Under the pressure of hypoxia, some tumor cells activate ▶apoptosis-associated genes, and lacunas left by dissolving LPPCN cells connect with each other and form channel networks (Fig. 4). The channels coming from LPPCN cells face two opposite ends. If they connect with endothelium-dependent vessels, blood will flow into these channels lined by tumor cells and the channels will be a functional microcirculation. In contrast to this, the mass of tumor

Vasculogenic Mimicry. Figure 4 LPPCN. Cells undergoing LPPCN have spindle-like figure and dark blue nuclei. They connect with each other as lines and some cells enter the wall of VM channels.

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cells will undergo necrosis if these channels fail to link to endothelium-dependent vessels. Three microcirculation patterns – VM, ▶mosaic vessels, and endothelium-dependent vessels – coexist in melanoma tissue. Angiogenesis requires the recruitment of normal endothelial cells, which may not be efficient and/or sufficient enough for sustaining aggressive tumor growth at the initial stage of rapid growth. Some tumor cells dedifferentiate, connect with other tumor cells or endothelium, and finally line the wall of tube. They are vasculogenic mimicry and mosaic vessels. Mosaic vessel may be a transition between VM channel and endothelium-dependent vessel. The threestage phenomenon on tumor blood supply pattern assumes that there is a transformation among VM channels, mosaic vessels and endothelium-dependent blood vessels (Fig. 5). In the stage of rapid tumor growth, endothelium-dependent vessels sprout from normal tissue but are insufficient to support the rapid tumor growth. VM occurs on the base of LPPCN and acts as the major blood supply pattern for tumor growth. As tumor size becomes bigger, endothelial cells from peripheral blood home, proliferate on the wall of VM, and cover some tumor cells forming VM. Mosaic vessels appear and become the major microcirculation pattern in tumors. Finally, endothelium-dependent vessels take over the dominant role in tumor blood supply. The Effect of Local Tumor Microenvironment on the Formation of VM Channels Tumor growth and evolution are regulated by a good many factors and tumor cells display distinguished blood supply pattern and biological behavior to adapt different microenvironment. The environmental factors impacting VM channel formation include oxygen pressure, interstitial fluid pressure (IFP) in tumor tissue, pH, focal concentration of cytokines, and ECM. ▶Hypoxia is a two-edge sword for tumor generation and development. Hypoxia and ischemia induce tumor cells to necrosis and tumor suppression, whereas hypoxia actives metastasis-related genes to promote tumor invasion. The hypoxic condition enhances the formation of VM channels, which exerts its function though ▶HIF-1α and its downstream molecules. In the hypoxic environment, accumulated HIF-1α in tumor cells induces ▶MMP-2, MMP-9, and VEGF expression and activation. MMP-2 and MMP-9 proteinases degrade the ECM components and facilitate VM formation, tumor invasion, and metastasis. VEGF secreted by tumor cells results in permeability of blood vessels, resulting in an increase and leaking-out of many serum proteins. Such a milieu provides a temporary matrix for VM channel formation. Under the stimuli of hypoxia, LPPCN-associated genes can be triggered and some tumor cells undergo LPPCN. Interspaces left by

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Vasculogenic Mimicry. Figure 5 Three-stage phenomenon on tumor blood supply pattern. During rapid tumor growth, endothelium-dependent vessels sprouting from normal tissue can not satisfy the need for growth. VM occur on the base of LPPCN and acts as the major blood supply pattern for tumor growth. As endothelial cells from host microvessels migrate and endothelial progenitor cells from peripheral blood home into the wall of VM, endothelial cells cover some tumor cells forming VM. Mosaic vessels appear and become the major microcirculation pattern in tumors. Finally, endothelium-dependent vessels get the dominant role in tumor blood supply.

dissolving cells connect with each other as networks to provide the space basement for VM. Interstitial fluid pressure (IFP) is another important factor affecting tumor microcirculation patterns. Increased IFP is a characteristic of malignant tumors because of its rapid proliferation. It is similar to hypoxia and has double impact on tumor development. High IFP inhibits both endothelial cells of blood vessels and lymphatic vessels to migrate into the tumor center, with the result of tumor hypoxia. Elevated IFP stimulates tumor cells to secret invasion-associated proteins. High IFP is a barrier for endothelium-dependent vessels sprouting into tumor tissue, but hypoxia induced by it is an inducer for VM. Moreover, the expression of MMP-2, MMP-9, ▶integrin, ▶selectin, and ▶kinesin increase significantly in tumor cells growing in the ▶microenvironment with high IFP, which promote VM formation to provide sufficient nutrition and oxygen for tumor growth. Clinical Significance of VM VM has been observed in several human malignant tumor types, such as highly aggressive uveal ▶melanomas, ▶breast cancer, ▶liver cancer, ▶glioma, ▶ovarian cancer, ▶melanoma, ▶prostate cancer, malignant ▶astrocytoma, and ▶bidirectional differentiated malignant tumors. Tumor cells lining on the inner surface of VM channels are directly exposed to blood flow, may move into the bloodstream and metastasize to other organs. VM is associated with poor prognosis in patients. Tumors with VM have a higher rate of metastasis

compared with tumors without VM, and the patients have a lower 5-year survival rate. Routine ▶antiangiogenic drugs, such as angiostatin and ▶endostatin, which target endothelial cells, have not achieved a therapeutic effect on tumors that exhibit VM because of the absence of endothelium-dependent vessels (Fig. 6). Advances and Challenges VM and Lymphagenesis in Tumors VM, a new pattern of blood supply to the tumors, has attracted the attention of many researchers, but many phenomena unique to VM channel formation remain to be elucidated. As a functional tumor microcirculation, VM channels need to be studied with regard to their connection with endothelium-dependent vessels, their relationship with lymphatic tubes, and their dual function as vessels and lymphatic tubes. Uveal melanoma cells have a specific vortex vein but no lymphatic tube. Highly aggressive melanoma expresses lymphatic-vessel endothelial hyaluronan receptor1 (LYVE1) and VEGF-C, a lymphatic tube-related growth factor. VM-Targeted Therapy Given the important role of angiogenesis in tumor growth and metastasis, therapies aiming at endothelial cells represent promising antitumor strategies. As VM has a different structure from endothelium-dependent vessels, traditional antiangiogenic agents targeting at endothelial cells, such as anginex, TNP-470, and ▶endostatin, have no remarkable effects on malignant tumors with VM.

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Vasculogenic Mimicry. Figure 6 Comparison of overall survival time of patients with bidirectional differentiated malignant tumors. A Kaplan–Meier algorithm reveals that the survival time of melanoma, mesothelial sarcomas (MS), alveolar rhabdomyosarcomas (AS), and synovial sarcomas (SS) without VM are both significantly longer than that of patients with VM.

One of the distinguished features of tumors with VM is that cell adhesion molecules, tumor invasion-related proteinases and ECM synthesis and secretion-associated proteins are overexpressed by tumor cells. These molecules represent potential targets for anti-VM strategies of highly aggressive and blood metastatic tumors with VM. Suppressing tyrosine kinase activity, using a knockout ▶EphA2 gene, downregulating VE-cadherin, using antibodies against human MMPs and the laminin 5γ2 chain, and using anti-▶PI3K therapy are strategies that have been employed to inhibit VM.

References 1. Maniotis AJ, Hess FR, Seftor A et al. (1999) Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol 155(3):739–752

2. Zhang S, Guo H, Zhang D et al. (2006) Microcirculation patterns in different stages of melanoma growth. Oncol Rep 15(1):15–20 3. Hess AR, Gardner SE, Carles-Kinch LM et al. (2001) Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2). Cancer Res 61(8):3250–3255 4. Folberg R, Maniotis AJ (2004) Vasculogenic mimicry. APMIS 112(7–8):508–525

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Vasoactive Intestinal Contractor ▶Endothelins

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Vasoconstrictor

Vasoconstrictor

V(D)J Recombination

Definition

M ARKUS M U¨ SCHEN

Any factor that causes the constriction of blood vessels, which increases blood pressure.

Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA

▶Endothelins

Synonyms Somatic recombination of V, D and J segments; Immunoglobulin- or T cell receptor-gene rearrangement

Vasodilation Definition Is the regulated widening of blood vessels by a controlled relaxation of the surrounding smooth muscle cells, thus permitting a higher blood flow at a lower pressure. ▶Relaxin

Vasostatin Definition

Refers to the N-terminal domain of ▶calreticulin (CRT) (amino acids 1–180), is an endogenous inhibitor of ▶angiogenesis and tumor growth. The potency of vasostatin in mice is 4- to 10-fold that of ▶endostatin or ▶angiostatin.

VCAM-1 Definition

Is a ▶cell adhesion molecule that belongs to the immunoglobulin superfamily. It mediates the ▶adhesion of lymphocytes to endothelial cells, thereby being involved in various inflammatory diseases including ▶angiogenesis and atherosclerosis. ▶Minodronate

Definition Surface immunoglobulin (expressed on B lymphocytes) and T cell receptors (expressed on T lymphocytes) represent the central molecules for antigen recognition in adaptive immune responses. Variable (V), diversity (D) and joining (J) gene segments, which together encode immunoglobulin or T cell receptor variable chains, are present in every somatic cell. However, in order to acquire coding capacity, V, D and J gene segments need to be assembled in a functional configuration. The mechanism for the assembly of V, D and J gene segments, termed V(D)J recombination, represents a unique capacity of both B and T lymphocytes.

Characteristics More than 30 years ago, Hozumi and Tonegawa discovered that immunoglobulin genes in B lymphocytes as opposed to any other somatic cell type undergo a complex rearrangement process in order to assemble a functional immunoglobulin variable region. This process, later termed V(D)J recombination, is in fact unique for B and T lymphocytes and critical for the expression of immunoglobulins and T cell receptors, respectively. Immunoglobulin and T cell receptor genes represent gene families in mammalians that are arranged in segments and, hence require somatic recombination of individual variable (V), diversity (D) and joining (J) gene segments to assemble a coding immunoglobulin or T cell receptor gene. V(D)J-recombination defines an early step during B- or T-lymphocyte development within the bone marrow or the thymus, respectively. In humans, three immunoglobulin and four T cell receptor gene loci are known (see Table 1). Each locus carries gene segments that together encode one protein chain. Immunoglobulins or T cell receptors typically represent the assembly of two heterodimers: For instance, immunoglobulins expressed on the cell surface of B lymphocytes comprise of two identical heterodimers of each one immunoglobulin heavy chain and one immunoglobulin κ or λ light chain molecule.

V(D)J Recombination V(D)J Recombination. Table 1

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Immunoglobulin and T cell receptor gene loci in the human

Locus

V segments

D segments

J segments

Gene product

IGH, 14q32 IGK, 2p11.2 IGL, 22q11.2 TCRA, 14q11.2 TCRB, 7q34 TCRG, 7p14 TCRD, 14q11.2

123 76 74 49 64 14 8

27 None None None 2 None 3

6 5 9 61 13 5 4

Ig heavy chain Ig κ light chain Ig λ light chain TCRα chain TCRβ chain TCRγ chain TCRδ chain

Mechanism As opposed to meiotic recombination, V(D)J recombination represents a site-specific DNA recombination event in B or T lymphocytes. Site-specific recombination is conferred by recombination signal sequences (RSS) immediately flanking V, D and J gene segments. RSSs are composed of a conserved heptamer and nonamer and a non-conserved spacer of 12 or 23 bp in length. The length of the spacer determines the pairing of RSS-motifs, which only allows synapse formation between RSS motifs of different spacer-length. For the recombination process, RAG1 and RAG2 proteins (encoded by the recombination activating genes 1 and 2) are essential. RAG1 and RAG2 proteins are only expressed in B and T lymphocytes and their expression is tightly controlled during their development. RAG1 and RAG2 form a heterodimeric complex that only recognizes synapses between two RSS motifs of different spacer length, i.e. only 12/23 RSS pairs. The 12/23 rule for RSS pairing ensures that V, D and J segments are recombined in a coordinated manner to prevent mispairing of gene segments. The RAG1/RAG2 complex then introduces a DNA double-strand break exactly at the border of the heptamer element (Fig. 1). These DNA double strand-breaks are blunt ended and 5′-phosphorylated for signal ends, which facilitates self-ligation and formation of signal joints within so-called excision circles (Fig. 1). In contrast, the broken-ended DNA of gene segments (coding joint; Fig. 1) are protected against immediate re-joining by the formation of a closed hairpin-structure. Of note, even though the targeting by the RAG1/RAG2 complex is very precise, the sequence of coding joints, i.e. the junction between two rearranged gene segments, is extremely variable. This variability of coding joints is owed to further processing of the hairpin structure by the enzymes Artemis and DNA ligase IV. The diversity of coding joints is further increased by the activity of the lymphoid-specific terminal deoxynucleotidyl-transferase (TdT): TdT is able to introduce additional nucleotides, the so-called N-nucleotides, into the junction between two gene segments in a template-independent manner.

Classical RSS motifs containing the conserved heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences and a non-conserved 12 or 23 nucleotide spacer are exclusively found adjacent to immunoglobulin and T cell receptor gene segments. However, RSS-like motifs, so called cryptic RSS sites, have been identified in a number of genes outside the immunoglobulin and TCR loci. Functional assays have shown that these cryptic RSS motifs (cRSS, Fig. 1) can indeed be targeted by the V(D)J recombinase machinery. Given that many genes, among them tumor suppressor and proto-oncogenes, harbor cryptic RSS sites, targeting by the V(D)J recombinase may predispose early B and T lymphocyte precursors to malignant transformation. In this regard, it is important to note that the RAG1 and RAG2 enzymes also possess transposase activity. Thereby, 5′-phosphorylated signal ends carrying a complete RSS motif on each end may attack unrelated DNA, preferentially at cryptic RSS sites (cRSS; Fig. 1). If both RSS motifs of the excised signal ends participate in this attack, this may lead to the integration of the RSS-flanked DNA fragments at positions staggered by 3–5 bp, resulting in target site duplication at the integration site (Fig. 1, bottom). It is obvious that integration of DNA that was excised during V(D)J recombination into unrelated loci carries the risk of oncogenic transformation. Even though classical transposition events are rarely seen in lymphoid malignancies, chromosomal translocations and deletions bearing the hallmarks of illegitimate V(D) J recombination are frequent events during malignant transformation of lymphocytes. Clinical Aspects Malignant transformation of human B and T lymphocyte precursors towards ▶acute lymphoblastic leukemia (ALL) often coincides with the timing of activity of the V(D)J recombinase machinery. Therefore, it was an obvious hypothesis that aberrant or “illegitimate” V(D)J recombination may be the causative mechanism for many if not all subtypes of ALL. In many cases, ALL cells carry ▶chromosomal translocations. The breakpoints of these gene rearrangements, however, exhibit the hallmarks of

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V(D)J Recombination. Figure 1 Schematic representation of RSS pairing during D-J segment joining. A simplified model of the IGH locus is shown including a group of V, D and J gene segments. Each of these segments are flanked by either a recombination signal sequence (RSS) with a spacer length of 23 bp (V, light gray) or 12 bp (J, dark gray) or both types of RSS motifs (D). During the RAG1/RAG2-mediated recombination process, two types of break-intermediates are generated: a coding joint (top) with a junction between two rearranged gene segments and signal ends (bottom) containing a fragment of excised DNA between two RSS motifs. These signal ends are processed by self-ligation to a signal joint within a closed excision circle. These excision circles are very stable in lymphocytes but are not replicated during mitosis. On the other hand, broken ended RSS motifs may attack unrelated DNA, preferentially at cryptic RSS sites (cRSS). Thereby, RAG1 and RAG2 can act as transposases, in that they catalyze the integration of the intervening DNA between two signal ends into the attacked unrelated DNA.

V(D)J recombination in only a few cases (Table 2). For instance, targeting to immunoglobulin (IGH, IGK or IGL) or T cell receptor (TCRA, TCRB, TCRG, TCRD) loci would argue for involvement of V(D)J recombination. While BCL1 and BCL2 gene rearrangements typically target the IGH locus, rearrangements of the CCND2, HOX11, LMO2, TAL1, TAL2 and TTG1 gene target at least one of the four TCR loci (Table 2). In addition, one would expect that breakpoints reflecting illegitimate V(D)J recombination exhibit traces of site-specific targeting in that they precisely flank RSS or RSS-like motifs. Virtually all genes involved in an IGH- or TCR-specific gene rearrangement show targeting of an RSS or RSS-like motif at least for one of the two translocation partners. In addition, genetic abnormalities were found, in which RSS or RSS-like motifs were targeted even though no immunoglobulin or TCR locus was involved. This applies mainly to

intragenic/interstitial deletions. Such deletions often occur in T cell lineage ALL cells and deletions of the INK4 family genes CDKN2A, CDKN2B, CDKN2D and within the HPRT, NOTCH1 and SIL/SCL loci (Table 2) are examples for such a second type of genetic aberration induced by illegitimate V(D)J recombination. Finally, addition of N-nucleotides (i.e. a junction sequence that cannot be attributed to either of the two fusion partners) by enzymatic activity of TdT represents a unique feature of V(D)J recombination. Indeed, junctional diversity compatible with the introduction of N-nucleotide was found in many cases, in which a gene rearrangement was mediated by V(D)J recombination, but not in all. Applying these three criteria (involvement of IGH or TCR loci, site-specific targeting of RSS- or RSS-like motifs and presence of N-nucleotides) to B and T cell lineage ALL, it appears that T cell lineage as opposed to

V(D)J Recombination V(D)J Recombination. Table 2 recombination

Frequent genetic aberrations in lymphoid malignancies related to illegitimate V(D)J

Locus

References

BCL1 BCL2 CCND2

Tsujimoto et al. (1998) Van Drager et al. (2000) Clappier et al. (2006) Clappier et al. (2006) Kitagawa et al. (2002) Cayuela et al. (1997) Kitagawa et al. (2002) Cayuela et al. (1997) Wiemels et al. (2002) Kagan et al. (1989) Zutter et al. (1990) Finette et al. (1996) Chen et al. (1996) Champagne et al. (1989) Cheng et al. (1990) Tuji et al. (2004) Aplan et al. (1990) Raghavan et al. (2001) Finger et al. (1989) Chen et al. (1990) Tycko et al. (1998) Retière et al. (1998) Boehm et al. (1988) McGuire et al. (1989)

CDKN2A CDKN2B CDKN2D E2A HOX11 HPRT LMO2 NOTCH1 SIL/SCL TAL1 TAL2 TCRB/TCRG TTG1

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Genetic aberration

Target

Malignancy

t(11;14)(q13;q32) t(14;18)(q32;q21) t(7;12)(q34;p13) t(12;14)(p13;q11) 9p21 deletion

IGH IGH TCRB TCRA RSS

9p21 deletion 9p21 deletion t(1;19)(q23;p13) t(10;14)(q24;q11)

RSS RSS RSS TCRD

Mantle cell lymphoma (B cell) Follicular B cell lymphoma T cell lineage ALL T cell lineage ALL B- and T cell lineage ALL T cell lineage ALL B- and T cell lineage ALL T cell lineage ALL pre-B cell ALL T cell lineage ALL

del Xq26-q27

RSS

t(11;14)(p13;q11)

TCRD

Normal lymphocytes T cell lineage ALL T cell lineage ALL

del 9q34 del 1p32

RSS RSS

T cell lineage ALL T cell lineage ALL

t(1;14)(p34;q11)

TCRD

T cell lineage ALL

t(7;9)(q34;q32) inv(7) t(11;14)(p15;q11)

TCRB TCRB TCRD

T cell lineage ALL Normal T cells T cell lineage ALL

B cell lineage ALL frequently arises through genetic aberrations that were caused by aberrant V(D)J recombination. With the exception of the ▶E2APBX1 gene rearrangement, none of the classical chromosomal translocations in B cell lineage ALL are compatible with V(D)J recombination: ▶BCR-ABL1, MLL-AF4 and ▶TEL-AML1 gene rearrangements are not related to the IGH locus, do not show site-specific targeting at RSS-like motifs and do not carry intervening nucleotides within the junction between the two fusion partners. The case of E2A-PBX1 is complicated because only the breaks of the E2A gene are sitespecific and localized at RSS-like motifs, while the breaks within the PBX1 gene are scattered over a large genomic region. Unlike BCR-ABL1, MLL-AF4 and TEL-AML1 gene rearrangements, however, the E2APBX1 fusion carries N-nucleotides in almost all instances, which argues for a contribution of the V(D) J recombinase. ▶Chromosomal translocations affecting one of the immunoglobulin loci in mature B cell lymphomas may also be owed to somatic hypermutation and/or immunoglobulin class-switch recombination. These

two mechanisms are active in mature B cells during affinity maturation within germinal centers of tonsils, lymph nodes and spleen. Indeed, frequent recurrent gene rearrangements in germinal center-derived B cell lymphoma most likely result from somatic hypermutation and/or class-switch recombination and not from V(D)J recombination. V(D)J recombinase-related genetic aberrations indeed seem to have distinct mechanisms in B cell lineage (pre-B ALL and B cell lymphoma) and T cell lineage (T cell precursor ALL) malignancies. According to a recently proposed model, chromosomal translocations in T cell lineage ALL are more frequent and are mainly caused by illegitimate V(D)J recombination between a TCR locus and a proto-oncogene locus, i.e. both loci were targeted by V(D)J recombination. Conversely, for translocations in B cell lineage malignancy, involvement of the V(D)J recombinase machinery is rare. In these rare cases, in which aberrant V(D)J recombination contributes to B cell lineage malignany, V(D)J recombination typically targets only one of the two translocation partners: In t(1;19)((q23;p13) pre-B ALL, the E2A locus is targeted by the V(D)J recombinase but

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VE-Cadherin

not its fusion partner PBX1. In both ▶Mantle cell lymphoma and ▶Follicular lymphoma, the IGH locus is targeted by aberrant V(D)J recombinase activity but not the BCL1 and BCL2 genes on the respective partner chromosome.

References 1. Hozumi N, Tonegawa S (1976) Evidence for somatic rearrangement of immunoglobulin genes coding for variable and constant regions. Proc Natl Acad Sci USA 73:3628–3632 2. Schatz DG, Oettinger MA, Baltimore D (1989) The V(D)J recombination activating gene, RAG-1. Cell 59:1035– 1048 3. Schlissel M, Constantinescu A, Morrow T et al. (1993) Double-strand signal sequence breaks in V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev 7:2520–2532 4. Hiom K, Melek M, Gellert M (1998) DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations. Cell 94:463–470 5. Marculescu R, Le T, Simon P et al. (2002) V(D)J-mediated translocations in lymphoid neoplasms: a functional assessment of genomic instability by cryptic sites. J Exp Med 195:85–98

VEGF Definition

▶Vascular endothelial growth factor; Is a cytokine made by cells that stimulates new blood vessel formation, by mediating numerous functions of endothelial cells including proliferation, migration, invasion, survival, and permeability. VEGF is also known as vascular permeability factor. VEGF naturally occurs as a glycoprotein and is critical for ▶angiogenesis. ▶Vascular Endothelial Growth Factor

VEGF-C Definition

A member of the ▶VEGF growth factor family that stimulates lymphangiogenesis and is required for lymphatic vessel development.

VE-Cadherin


Definition

Vascular endothelial cadherin is a ▶adhesion molecule important in maintaining endothelial permeability. Inhibition of VE-cadherin by antibodies increases both permeability and neutrophil transmigration in vivo. ▶Vascular Disrupting Agents

VEGFR Definition

▶Vascular endothelial growth factor receptor; ▶VEGF.

Vector Definition

VEGFR-2

Genetic information encoding e.g. a therapeutic gene, plus those sequences required for gene expression and for integration into the host genome where applicable. In some situations, the term “vector” denotes the vector sequences in the context of a gene transfer vehicle (viral or non-viral).

Definition

▶Gene Therapy

▶Vascular Disrupting Agents

▶Vascular endothelial growth factor receptor 2 gene is transcriptionally regulated during ▶angiogenesis.

Vinblastine

VEGFR-3 Definition

A member of the ▶VEGF receptor family that recognizes the VEGF-C and VEGF-D isoform.

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V-Gene Definition

Variable regions of ▶immunoglobulin genes that encode light and heavy chains.


VHL Verapamil Definition Definition A calcium ion influx inhibitor (calcium entry blocker or calcium ion antagonist). Verapamil is used for reduction of blood pressure and for treatment of cardiac diseases.

Is the gene involved in von Hippel-Lindau disease (renal carcinoma). ▶von Hippel-Lindau Tumor Suppressor Gene

▶ABC-Transporters ▶Fluoxetine

Video-assisted Thoracoscopic Surgery Verner-Morrison Syndrome Definition Is a clinical syndrome characterized by a profuse watery diarrhea that results in massive loss of water, potassium, sodium, and bicarbonate, leading to dehydration, electrolyte deficiency, and metabolic acidosis. It is associated with endocrine pancreatic tumors that produce excessive amounts of the vasoactive intestinal polypeptide (VIPomas).

Definition VATS; Minimaly invasive surgical technique used to diagnose and treat problems in the chest. One or more small incisions are made in the chest and a fiberoptic camera called thoracoscope is inserted through one incision and surgical instruments are inserted through this or other small incisions. ▶Pleural Effusion

▶Neuroendocrine Carcinoma

Villin 2 Vesicle Trafficking

▶ERM Proteins

V

Definition The process through which membrane-bound molecules are routed to various cellular compartments. Cell surface receptors and their associated signaling complexes are routed to internal endosomal organelles through this process. Signal transduction and vesicle trafficking processes are coordinated and trafficking events can generate discrete cellular signals.

Vinblastine Definition

Vinblastine is a ▶chemotherapy drug of the group of ▶vinca alkaloids that is used as a treatment for some

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Vinca Alkaloids

types of cancer including ▶leukemia, ▶lymphoma, ▶breast cancer and ▶lung cancer. It binds ▶tubulin, thereby inhibiting the assembly of ▶microtubules. It is M phase ▶cell cycle specific since microtubules are a component of the ▶mitotic spindle and the ▶kinetochore.

Viral Oncogene Definition A viral gene that contributes to malignancies in vertebrate hosts. ▶Gastrointestinal Stromal Tumor

Vinca Alkaloids Definition A class of chemotherapeutic drugs that disrupts ▶mitosis by binding to ▶tubulin, thus preventing ▶microtubules from forming. These are also known as anti-mitotic, antimicrotubule agents, or mitosis inhibitors. Common vinca alkaloids include ▶vinblastine, ▶vincristine, ▶vindesine, and ▶vinorelbine.

Viral Oncology Epigenetics J AMES F LANAGAN Wolfson Institute for Biomedical Research, University College London, London, UK

Definition

Vincristine Definition

Chemotherapeutic agent; ▶vinca alkaloid; inhibits ▶tubulin polymerization.

Vinorelbine Definition

Chemotherapeutic agent; ▶vinca alkaloid; inhibits ▶tubulin polymerization.

VIPoma Definition

Is a functioning ▶islet cell tumor that produces excessive amounts of the vasoactive intestinal polypeptide (VIP) that can result in the ▶Verner-Morrison syndrome. ▶Neuroendocrine Carcinoma

▶Viral oncology epigenetics can represent the ▶epigenetic alterations that occur within the host cell genome as a result of viral infection or virally induced ▶carcinogenesis. Alternatively, viral oncology ▶epigenetics can refer to epigenetic alterations of the viral genome during latent or lytic infection or during carcinogenesis.

Characteristics One of the emerging concepts in cancer biology is that epigenetic alterations are important in the initiation and early progression of the majority of human cancers. However, differentiation of the early cancer causing epigenetic alterations from later consequences is difficult. Oncogenic viruses are typically very small and with very few genes and yet can induce transformation. Therefore, investigations into the epigenetic alterations that viruses make to the host cell genome may provide an indication of which epigenetic alterations are critical for early carcinogenesis. Oncogenic viruses include any virus that has been identified as the causative agent for cancer. They can induce cellular alterations that enable transformed cells to evade host responses, including the immune system and apoptosis. In vitro, oncogenic viruses induce cellular transformation creating cells capable of unrestrained proliferation and are often tumorigenic in athymic mice. Oncogenic viruses in humans include DNA viruses, such as ▶hepatitis B viruses (HBV), ▶human papillomavirus (HPV), polyomaviruses (BKV, JCV, and ▶SV40) and the gamma-herpesviruses ▶Kaposi sarcoma-associated herpesvirus (KSHV) and ▶Epstein-Barr virus (EBV) as well as ▶retroviruses, such as ▶human T cell lymphotrophic viruses 1 and 2

Viral Oncology Epigenetics

(▶HTLV1/HTLV2), the RNA flavivirus ▶hepatitis C virus (HCV). Epigenetics is a term used to describe the regulation of gene expression and genomic stability by heritable, but potentially reversible, changes in ▶DNA methylation and chromatin structure. DNA ▶methylation is controlled in the genome by various epigenetic regulators, including the DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), methylated DNA binding proteins (e.g. MECP2, MBD1-MBD4) and DNA demethylases (e.g. GADD45A, GADD45B). Chromatin structure is regulated by posttranslational modifications to the tails of the core histones that make up the ▶nucleosome including lysine acetylation, lysine and arginine methylation, lysine ubiquitination, serine phosphorylation and proline isomerization. The enzymes that catalyze these modifications (▶histone deacetylases, histone acetyltransferases, histone demethylases and histone methyltransferases) interact with other chromatin structure regulators, such as the polycomb group (repression) and trithorax group (activation) genes, ATP-dependent chromatin remodeling complexes and ▶microRNAs and the RNAi machinery to coordinate together the regulation of gene expression. In normal cells transcriptionally active genes typically contain unmethylated promoter ▶CpG islands, genewide histone hyperacetylation and a number of specific histone modifications, such as H3 lysine 4 (H3K4) methylation, H3K79 methylation, H3 arginine methylation, H3S10 phosphorylation and H2B ubiquitination. Transcriptionally repressed genes often contain methylated promoter CpG islands, histone hypoacetylation and H3K27 and H3K9 methylation. In cancer cells, however, there are marked differences in the epigenetic landscape of the genome. Genome-wide, there is an overall ▶hypomethylation associated with repetitive DNA in cancer cells, as well as promoter hypermethylation of specific tumor suppressor genes and hypomethylation of ▶oncogenes. In addition to altered DNA methylation, ▶chromatin remodeling in cancers is also considered a common epigenetic alteration, including loss of H4K16 acetylation fand H4K20 tri-methylation and gains in H3K4 di- and tri-methylation, H3K79 methylation and H3 and H4 hyperacetylation. Host Epigenetic Changes Due to Viruses and Virus-Associated Cancers Differentiating epigenetic causes of cancer from epigenetic consequences is one of the more challenging goals of current research in the epigenetics field. This conundrum can equally be applied to virally induced cancers. It is challenging to differentiate epigenetic changes that are directly due to viral infection, due to the anti-viral response of the host or due to downstream effects of the virally induced transformation. Important early epigenetic changes in cancer may be distinguished

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from consequences via the identification of specific interactions between epigenetic regulators and the viral proteins effecting those changes and then determining the downstream effects of these interactions. Kaposi Sarcoma Associated Herpesvirus ▶Kaposi sarcoma associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus identified as the causative agent of the endothelial tumor Kaposi sarcoma (KS) and associated with the lymphoproliferative disorders primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other gamma-herpesviruses KSHV has both a lytic and latent phase of its life cycle. In the majority of tumor cells the more restricted set of latent genes are often expressed. Recent evidence suggests that KSHV can reprogram the cellular gene expression profiles of both blood and lymphatic vessel endothelial cells, although the mechanisms behind this reprogramming remain unclear. A direct link between the KSHV protein latency associated nuclear antigen (LANA) and the de novo methyltransferase DNMT3a was examined in a study revealing recruitment of DNMT3a by LANA to the chromatin possibly via its interaction with histones H2A and H2B. This targeted repression of ~80 cellular genes, many of which are typical targets of epigenetic inactivation in numerous cancers. KSHV has been shown to hypermethylate the ▶CDKN2A gene promoter in the majority of PEL cell lines harboring KSHVand in primary PEL samples. This is not unexpected, given that CDKN2A is probably the gene most commonly hypermethylated in cancers. This suggests that KSHV has the ability to invoke DNA methyltransferase activity, via the LANA protein, and may inactivate numerous cellular genes by promoter hypermethylation. In addition, LANA has numerous other roles in epigenetic gene regulation via interactions with a methylated DNA binding protein MECP2, as well as the mSin3 repression complex and the SUV39H1 histone methyltransferase. Numerous studies have also proposed a direct interaction between the KSHV encoded interferon regulatory factors (viral IRF 1, IRF2 and IRF3) and the histone acetyltransferase complex ▶p300/CBP. The binding of CBP by the cellular IRF3 is thought to be a necessary interaction for transcriptional upregulation of the antiviral cytokine interferon-β (IFN-β)˙ In effect, the interactions of the viral IRFs with CBP inhibit histone acetyltransferase activity and promote histone hypoacetylation, altered chromatin structure and reduction of cytokine gene expression. Presumably other genes that are activated by p300/CBP would also be down regulated. Epstein Barr Virus Epstein Barr virus (EBV) is another gamma-herpesvirus that has been identified as the causative agent of

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numerous lymphoproliferative diseases such as ▶Burkitt lymphoma (BL), ▶Hodgkin Disease (HD) and posttransplant lymphoproliferative disease (PTLD). EBV is also involved in carcinogenesis of epithelial origin tumors, particularly ▶nasopharyngeal carcinomas (NPC) and some gastric cancers. The EBV latent membrane protein 1 (▶LMP1) increases DNA methyltransferase activity by upegulating the maintenance methyltransferase DNMT1 as well as both de novo methyltransferases, DNMT3b and DNMT3a. In EBV-related epithelial cancers this causes increased promoter methylation and reduction of e-cadherin expression, resulting in increasing cell migration, an important step during carcinogenesis. Other cellular genes that are methylated by EBV are yet to be identified. The EBV latent nuclear antigens EBNA 2 and 3c appear to alter histone acetylation via their interactions with either the p300/CBP complex or with histone deacetylases, respectively. One of the interesting observations regarding the EBV proteins that coordinate epigenetic regulation (EBNA2, EBNA3c, and LMP1) is that they are all latent genes that are not expressed in most Burkitt lymphoma, EBV-associated gastric cancer or nasopharyngeal carcinomas. This may lead one to conclude that the role of the virus induced host epigenetic alterations may be limited in these cancers. However, in these cancers and in other virally induced cancers it cannot be ruled out that the early cancer precursor cells may have been infected with the virus expressing these proteins that could alter the host epigenome. Epigenetic fingerprints such as histone modifications and DNA methylation are mitotically heritable and therefore even if the progeny cancer cells no longer express these latent genes, the epigenetic history of the cell may remain.

Hepatitis B Virus The hepatitis B virus (HBV) is a DNA virus from the hepadnavirus family and is the causative agent of viral hepatitis, a chronic inflammation of the liver, which can develop into ▶hepatocellular carcinoma. The hepatitis B virus oncogenic protein HBx has been shown to increase the activity of DNMT1, and similar to EBV related cancers, this results in increased DNA methylation of e-cadherin (CDH1) and an increased cell migration. Whether this can be attributed directly to increased activity of the maintenance methyltranserase, DNMT1, is not clear as EBV also activates the de novo methyltransferases, DNMT3a and DNMT3b which HBV does not. The tumor suppressor gene CDKN2A and the glutathionine-S-transferase (GSTP1) genes are both commonly hypermethylated in ▶hepatocellular carcinomas associated with HBV, however, there is currently no direct evidence that the virus causes the increased DNA methylation of these genes.

Human Papillomavirus The human papillomavirus (HPV) family is a large family of DNA viruses of which some subtypes (particularly HPV 16 and 18; ▶early genes of human papillomaviruses) are associated with ▶cervical cancer and rarer epithelial origin cancers. The two important HPVoncoproteins E6 and E7 have both been implicated in epigenetic alterations during carcinogenesis. HPV E7 protein increases the DNA methyltransferase enzymatic activity by direct interaction with DNMT1 and E6 can bind and inhibit the histone acetyltransferase activity of p300 and CBP similarly to KSHV. This transcriptional repression by HPV is supported by E7 protein interaction with the Nurd ATP-dependent chromatin remodeling complex and ▶histone deacetylase 1 which are both involved in transcriptional repression. Numerous cellular epigenetic alterations have been described in ▶cervical cancers including hypermethylation of tumor suppressor genes RB, CDKN2A, MLH1, VHL and CDH1. Whether the E7 mediated increase in DNA methyltransferase activity is responsible for methylation of these genes is yet to be established. Polyomaviruses (SV40, BK Virus and JC Virus) Polyomaviruses are very small DNA viruses (~5kb) encoding only six genes. In humans the BK virus is associated with some brain tumors and the JC virus has been associated with gliomas, medulloblastomas and a minority of colorectal carcinomas, however, neither virus has been implicated as direct causative agents in these tumors. The simian virus SV40 is not a causative agent of any human tumors, but has been found in some mesotheliomas. These three polyomaviruses encode the T-antigen oncoprotein which is involved in inducing DNA methylation alterations. The SV40 virus upregulates the de novo methyltransferase DNMT3b which results in increased DNA methylation and increased tumorigenicity in normal human bronchial cells. Abberrant methylation of RASSF1A, HPP1, CCND2, DCR1, TMS1, CRBP1, HIC1 and RRAD have all been detected in SV40 associated malignant mesotheliomas or SV40 infected human mesothelial cells. The BK virus increases transcription of DNMT1 through the pRB/E2F pathway in cells that have pRB inactivated, however, this has not yet been linked to increased methylation of any specific genes. The JC virus T antigen expression is associated with the methylator phenotype in colorectal cancer, however this link with methyltransferase is still rather speculative. Adenovirus The ▶adenovirus protein E1A can induce transformation in vitro and can induce tumors in animal models but the virus does not in itself cause any human cancers. E1A interacts with the p300/CBP complex and is likely to result in a loss of histone acetylation across the

Viral Vector-mediated Gene Transfer

genome. It has been proposed that this interaction could be one of the key events in E1A induced cellular transformation. Another important role could be its capability of increasing DNMT1 activity, although which cellular genes are hypermethylated as a result of this increased activity is yet to be determined.

Human T Cell Lymphotrophic Viruses Human T cell lymphotrophic viruses (▶HTLV1 and HTLV2) are single stranded RNA retroviruses that are causative agents of adult T-cell leukemias. While there is little data on host DNA methylation alterations directly mediated by this virus, the HTLV1 Tax protein does interact with the ▶p300/CBP complex to mediate transcriptional repression.

Epigenetic Alterations in the Virus Due to the Host The opposite side of viral oncology epigenetics is the epigenetic alterations that occur on the viral genome during the lytic and latent stages of infection and during carcinogenesis. In addition to regulation of their own genome, DNA methylation of viral genes has also been proposed as a mechanism for silencing potentially highly immunogenic proteins that would otherwise elicit an immune response. Due to spontaneous deamination of methylated cytosines to thymine there is a reduced rate of CpG dinucleotides (▶CpG island) in the genomes of organisms that use DNA methylation as a mechanism of gene silencing. This mechanism, known as CpG suppression, is seen in the human genome with an observed CpG dinucleotide rate of ~1% where the statically expected rate would be 1/16 (6%) of the total bp. The genomes of many gamma-herpesviruses, including EBV, show CpG suppression suggesting that their genomes have been methylated similarly to the DNA of the host cells in which they reside. In this way the epigenetic state of the viral genome can be considered due to the host in which they reside. KSHV on the other hand only shows CpG suppression at the lytic switch promoter, ORF50, suggesting that the rest of the viral genome is not extensively methylated. KSHV in fact controls its entry into the lytic phase by employing both demethylation and chromatin remodeling of the lytic switch gene Rta (ORF50) promoter and its latent cycle replication is controlled by hyperacetylation of the replication origin. The EBV C promoter which drives the expression of the latent gene transcripts is often methylated and silenced in most EBV associated tumors. This prevents the expression of highly immunogenic Epstein Barr nuclear antigen (EBNA) proteins that would elicit a cytotoxic T-cell response and provides a very good example of how viruses exploit epigenetic mechanisms to evade the immune system.

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Conclusions It is clear that oncogenic viruses increase the activity of DNA methyltransferases and have the ability to decrease ▶histone acetylation via ▶p300/CBP. These are both likely to be essential for the inactivation of tumor suppressor genes. That both of these events occur in many non-viral cancers as well suggests that they may be some of the earliest epigenetic alterations in carcinogenesis. The common pathways utilized by viruses to effect these epigenetic changes suggest that perhaps very few changes are required to initiate epigenetic misregulation in cancer. For example the BK virus increases transcription of DNMT1 through the pRB/E2F pathway in cells that have pRB inactivated, however this has not yet been linked to increased methylation of any specific genes. The HPV E7 protein, KSHV LANA, polyomavirus T antigen and adenovirus E1A are all known to inactivate pRB as well as increase DNA methyltransferase activity, suggesting that inactivation of pRB may be a critical step towards epigenetic alterations in carcinogenesis. It is also interesting to note that many of the viral proteins described here, LMP1, E6 and E7, LANA, E1A, large T antigen and Tax, are all often portrayed as the viral “oncoproteins” as they are often either essential components of viral transformation or oncogenic on their own. This is often used as evidence that the functions of these proteins, in this case the epigenetic interactions, are indeed essential for carcinogenesis induced by these viruses.

References 1. Fields BN, Knipe DM, Howley PM (2001) Fields’ virology, 4th edn, vol 2. Lippincott Williams & Wilkins, Philadelphia, PA, pp xix, 3087, I-72 2. Kouzarides T (2007) Chromatin modifications and their function. Cell 128(4):693–705. This Cell Special Review Issue has many excellent reviews on epigenetic control of transcription and chromatin organisation 3. Flanagan JM (2007) Host epigenetic modifications by oncogenic viruses. Br J Cancer 96(2):183–188

Viral Vector-mediated Gene Transfer Y UANAN LU 1 , LYNN S NIDERHAN 2 1

Department of Public Health Science, University of Hawaii, Honolulu, HI, USA 2 Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA

Synonyms Virus vector; Oncolytic virus; Virus vector-mediated gene transfer

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Definition Refers to the process by which virus vectors are used to deliver functional genes of interest into target cells and tissues, either in vitro or in vivo.

Characteristics Viruses have evolved natural mechanisms to efficiently transport their own genetic materials into host cells while also commandeering host cell machinery for replication. Therefore, the use of viruses for the introduction of therapeutic genes and stimulation of the immune system has become an attractive and promising method for the treatment of a variety of diseases, including cancer. The genomes of a wide array of viruses can be modified and used as a tool for the efficient transfer of exogenous genes into living cells or organisms. In broad terms, two types of virus vectors exist: replication-competent, and replication-defective. Replication- competent vectors are exemplified by liveattenuated strains of many common viruses, including both DNA viruses (such as ▶adenovirus, herpes simplex virus type-1 (HSV-1) and vaccinia virus) and RNA viruses (such as recombinant derivatives of vaccine strains of measles virus, poliovirus and vesicular stomatitis virus (VSV)). Replication-defective vectors have an intrinsically more favorable safety profile than replication-competent vectors, but can be more difficult to engineer and manufacture. Commonly used replication-defective vectors include both small (adeno-associated virus (AAV)) and large (adenovirus, HSV-1) DNA viruses, as well as RNA viruses (including alphaviruses such as Venezuelan equine encephalitis virus (VEE), lentiviruses and other retroviruses). In all cases, the vectors are designed such that one or more genes essential for viral replication or assembly have been deleted or otherwise rendered defective. Some viral vectors combine aspects of both replication-competent and replication-defective vectors. These include vectors that are fully competent for replication in cells of one type or species, but not in cells of a different type, or from a different host species. Widely used examples include baculovirus vectors, which are able to transduce mammalian cells but which can replicate only in insect cells, and certain poxviruses such as the Modified Vaccinia Ankara (MVA) strain of vaccinia virus, which replicates efficiently in chick embryo fibroblasts, but undergoes abortive infection in human cells. Conditionally replicating virus vectors, such as oncolytic viruses, are another example – and are especially attractive as potential therapeutic agents for cancer treatment, as will be discussed later in this essay. Finally, there is considerable interest in the development of artificial virus-like vectors, including virus-like

particles (VLPs). VLPs offer potential advantages for vaccine delivery or display of vaccine antigens due to the densely repetitive nature of the surface of many VLPs. These properties explain, in part, the tremendous effectiveness of the recently licensed VLP-based vaccines for oncogenic human papillomavirus (HPV) subtypes. Completely synthetic gene transfer vectors have particular appeal because of the potential to massproduce such agents using conventional chemical technologies, rather than the more complex and cumbersome biological production methods required to manufacture conventional virus vectors. However, the efficiency of gene transfer by completely synthetic vectors currently remains far inferior to that of conventional virus vectors. Detection of Viral Vector-Mediated Gene Expression and Vector Distribution. In order to analyze the success of viral vector-mediated gene transfer, it is important to be able to monitor both the distribution of the vector and the effectiveness of vector-mediated gene expression. This can be achieved by subcloning a reporter gene into the viral vector backbone. Several reporter genes are commonly used for this purpose including: fluorescent proteins of various colors (Including green fluorescent protein, GFP), E. coli β-galactosidase (LacZ), and various forms of luciferase (Luc). It is simple to detect and quantitate cells expressing these marker genes in vitro, using FACS analysis or fluorescence microscopy. Detection of vector-transduced cells in vivo often requires a different approach. Modified luciferase reporters such as the Gaussia luciferase can generate a very bright light emission that can be detected and localized to specific tissues by using highly sensitive light detection methods in combination with 3D tomography. For in vivo imaging applications, a number of marker genes are compatible with the use of currently available gamma cameras or ▶positron emission tomography (PET) instruments. These include the sodium iodide symporter (NIS), which has been used to target radioisotopes of iodine to cancer cells. This allows both in vivo imaging of the distribution of the vector, quantitation of gene expression, and also delivery of therapeutic radiation to cancer cells. The ▶HSV-1 thymidine kinase (TK) gene has also been a successful tool for imaging when coupled with PET tracers. Furthermore, in the case of cancer therapy, the TK gene can be combined with ▶prodrugs, such as ▶ganciclovir (GCV), which become activated by TK and exert cytotoxic effects on rapidly dividing cancer cells. Viral Vectors for Cancer ▶Gene Therapy. A number of vectors, including AAV, adenovirus, HSV, measles virus and ▶retroviruses have been developed to promote the selective elimination of tumor cells. Cancer gene therapy approaches include


immunotherapy and suicide gene therapy. In addition, naturally oncolytic viruses such as reovirus (exemplified by Reolysin) and oncolytic viral vectors that result in the destruction of transduced tumor cells are also being pursued. For reasons of space, we will focus the rest of this review on the latter. Virus platforms that are being used to develop oncolytic vectors include adenovirus, HSV-1, measles virus and vaccinia virus. The field of ▶oncolytic virotherapy has made rapid progress in the past decade, resulting in human clinical trials of multiple agents based on all of the above vector platforms. Clinical trials have included phase III studies, and in 2005, the field received a major boost when the Chinese government approved the first oncolytic virus therapy for cancer treatment. This is a significant landmark that likely presages the approval of other oncolytic virus therapies elsewhere in the world. There are several approaches that can be taken in the use of oncolytic vectors for cancer therapy. One approach simply utilizes the replicating virus itself as therapy. As the virus replicates within the tumor cells, the cells are lysed and destroyed. Considerable efforts are being made to improve tumor-selectivity, and to ensure that the ▶virotherapy spares surrounding healthy tissue. This can be accomplished by: (i) placing viral genes under the control of tumor-specific promoters (such as the PSA promoter in ▶prostate cancer); (ii) altering the receptor binding properties of the vector in order to target tumors; (iii) utilizing the oncolytic properties of the vector in conjunction with “arming” of the virus with therapeutic genes. The use of “armed” vectors is attractive due to the fact that it draws on multiple mechanisms to achieve the desired effect. For example, engineering oncolytic HSV vectors to deliver a therapeutic gene, such as an antiangiogenic factor, has been shown to enhance the therapeutic efficacy of the vector in small animal model systems. It is also possible to introduce a tumor suppressor gene, such as ▶p53, into cancer cells via an oncolytic vector in order to exert control over the cell cycle of the tumor cells while also subjecting the cells to the oncolytic activity of the virus itself. The success of oncolytic vectors as anti-cancer therapies can be further enhanced by combining the virotherapy with either chemotherapy or radiation treatment. Not only is it possible to use radiation-inducible promoters to direct the expression of essential viral genes, but also it has been shown that radiation and chemotherapy are able to enhance the replication of certain oncolytic vectors. For example, in the case of oncolytic HSV-1 vectors, the induction of DNA repair genes by chemotherapy actually augments viral replication. Also, the use of a reporter gene, which has therapeutic effects when combined with radiation, such

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as NIS, offers the promise of an effective synergistic approach to tumor treatment. Overall, the field of cancer gene therapy has made important advances within the past few years, and while oncolytic virotherapy may not offer a simple “magic bullet,” it has considerable potential to synergize with, and enhance the effectiveness of, other established therapies such as chemo- and radio-therapy. Viral Vector Cancer Gene Therapy – Pitfalls. While important advances have been made, concerns remain. First, oncolytic virotherapies may not be sufficiently effective to provide optimal therapeutic benefit as a stand-alone treatment. Therefore, the effectiveness of the vectors needs to be increased. In addition, the issue of pre-existing immunity to common viruses such as HSV-1 and adenovirus serotype 5 may need to be overcome, in order to make most effective use of oncolytic viruses based on these agents. Second, biosafety questions must be addressed. These include the potential for vector replication in normal tissues, such as rapidly dividing stem cells, and the inherent genetic instability of certain viruses. For example, it was recently found that an oncolytic HSV-1 vector being used in human clinical trials contained a previously unrecognized mutation (a truncation in the UL3 open reading frame). In conclusion, virus vector-mediated gene transfer holds significant promise as a potential therapeutic approach for many human diseases, including cancer. The recent approval of the world’s first oncolytic virus for human tumor therapy is indicative of the rapid and exciting progress in the field. Moreover, evolving success in combining oncolytic virotherapies with sophisticated tumor-targeting and gene transfer approaches, as well as conventional chemo- and radio-therapy, provide a compelling reason to anticipate new cancer treatments that more effectively exploit the potential of viral vector systems.

References 1. Advani SJ, Mezhir JJ, Roizman B et al. (2006) ReVOLT: radiation-enhanced viral oncolytic therapy. Int J Radiat Oncol Biol Phys 66:637–646 2. Dambach MJ, Trecki J, Martin N et al. (2006) Oncolytic viruses derived from the γ34.5-deleted herpes simplex virus recombinant R3616 encode a truncated UL3 protein. Mol Ther 13:891–898 3. Kirn DH (2006) The end of the beginning: oncolytic achieves clinical proof-of-concept. Mol Ther 13:237–238 4. Liu TC, Galanis E, Kirn D (2007) Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress. Nat Clin Pract Oncol 4(2):101–117 5. Young LS, Searle PF, Onion D et al. (2006) Viral gene therapy strategies: from basic science to clinical application. J Pathol 208:299–318

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Virilization Definition Refers to the process of normal male sexual development (puberty) regulated by the production of testosterone. In adrenocortical carcinoma, ▶virilization can occur in boys (precocious puberty) or girls (pseudoprecocious puberty) as a result of excessive amounts of androgens. ▶Childhood Adrenocortical Carcinoma

Virology PAUL G. M URRAY CRUK Institute for Cancer Studies, Molecular Pharmacology, Medical School, University of Birmingham, Edgbaston, Birmingham, UK

Definition

are usually insufficient to cause malignancy; virus infection is only one step in the multistep process leading to a cancer. Although virus infection may be linked to a particular cancer type, in order to establish a clear association between the virus and the development of a cancer it is usually necessary to detect the virus within the tumor cells. Immunosurveillance and Viral Oncogenesis Virus-associated cancers occur in both immunocompetent and immunodeficient patients. However, the latter group has a particularly high risk for the development of these tumors, suggesting that the immune system can prevent the development of virus-associated cancers. ▶Cytotoxic T cells (CTLs) are particularly important in the recognition and elimination of virus-infected tumor cells. CTLs recognize virus-derived peptides that are presented by the infected cell in association with MHC class I. The development of virus-associated cancers in immunocompetent individuals suggests that the virusinfected tumor cell or its progenitor has developed mechanisms to escape immune recognition. In some cases virus-specific CTLs have been used to treat virusassociated cancers (▶adoptive immunotherapy). In some cases gene therapy may be used to modify CTL function (▶CTL therapy).

Virology addresses the molecular nature of viruses, their genetic content, the pathway by which they enter cells and multiply using the molecular machinery of the host cell, and the mechanisms by which they elicit diseases. Tumor virology is a specialized discipline analyzing the association of particular virus types with cancers in animals and humans.

Tumor Viruses There are a number of different viruses that have been associated with the development of cancer. The acutely transforming ▶retroviruses cause cancer in animals but to date none have been associated with the development of human tumors. The following sections consider some of the important human tumor viruses.

Characteristics

Herpesviridae The major oncogenic herpesviruses are the EpsteinBarr virus (EBV) and the Kaposi sarcoma-herpesvirus (KSHV).

In 1964 the first human tumor virus, the ▶Epstein-Barr virus (EBV), was isolated from tumor samples of a patient with African ▶Burkitt lymphoma. Subsequently, EBV was linked to the development of other forms of cancer. In the 44 years since the discovery of EBV, other human tumor viruses have been identified (Table 1). Tumor Virus Epidemiology The development of cancer is an infrequent consequence of viral infection and often occurs many years after any initial infection. Therefore, tumor viruses often infect individuals without adverse effects. For example, approximately 95% of the World’s adult population are infected with EBV. The majority of these individuals carries the virus asymptomatically and will not develop cancer as a consequence of EBV infection. Likewise, tumors associated with the human T lymphotropic virus1 (HTLV1) arise infrequently in populations where the virus is endemic. Thus, presumably alone tumor viruses

Epstein-Barr Virus EBV (Epstein-Barr virus) is a double stranded DNA virus of the gamma herpesvirus family. EBV infects the majority of the World’s adult population and following primary infection the individual remains a lifelong carrier of the virus. In poorly developed countries, primary infection with EBV usually occurs during the first few years of life and is often either asymptomatic or produces only a mild febrile illness. However, in developed populations primary infection is frequently delayed until adolescence or adulthood, in many cases producing the characteristic clinical features of infectious mononucleosis (also known as glandular fever) including sore throat, fever, malaise, lymphadenopathy and mild hepatitis. EBV is often transmitted from one individual to another in saliva and the oropharynx is

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Virology. Table 1 Major oncogenic viruses Virus family Adenoviridae Polyomaviridae

Virus

Natural host

Human Adenoviruses Man A, B, D Polyoma Mouse

Host in which virus is oncogenic

Tumor

Major oncogenic protein (s)

Hamster, rat

Various

E1A, E1B

Mouse

Various

Middle T antigen, Large T antigen Large T antigen E6, E7

SV40 Papillomaviridae HPV 16,18

Monkey Hamster, rat Man Man

Herpesviridae

EBV

Man

Man

KSHV

Man

Man

Hepadnaviridae

Hepatitis-B virus

Man

Man

Flaviviridae

Hepatitis C virus

Man

Man

Retroviridae

HTLV1

Man

Man

believed to be both the primary site of infection and where virus replication occurs. Virus replication in the oropharynx ensures the production of new virions for transfer in saliva to other susceptible hosts. Because EBV is transmitted in this way infectious mononucleosis is often referred to as the ‘kissing disease’. Soon after primary infection EBV infects B-lymphocytes through an interaction of the viral envelope glycoproteins, gp350/220, with the cellular EBV receptor, CD21. EBV does not usually replicate in B-lymphocytes but instead establishes a latent infection during which no new virions are produced and only a subset of viral genes are expressed. As a consequence of the host immune response, the number of latently infected B-lymphocytes in the peripheral blood falls to approximately 1 in 106 during the months following primary EBV infection. This low number is maintained in healthy carriers by the continual elimination of proliferating EBV-transformed B-lymphocytes by virusspecific CTLs. The EBV genome usually exists in cells as extrachromosomal pieces of circular DNA, known as episomes. During latency only a limited number of viral genes are expressed; these include six nuclear proteins, referred to as the Epstein-Barr nuclear antigens (EBNAs), two latent membrane proteins (LMPs), two non-translated RNA molecules known as the EpsteinBarr encoded RNAs (EBERs 1 and 2), and transcripts from the BamH1A region of the viral genome (BamH1A

Various Skin cancer, cervical cancer, anal cancer Burkitt lymphoma, LMP1 nasopharyngeal carcinoma, Hodgkin lymphoma Kaposi sarcoma, multicentric Castleman disease, primary effusion lymphoma Hepatocellular carcinoma Hepatitis-B x-antigen Hepatocellular carcinoma HCV core, NS3, NS4B, NS5A Adult T cell leukaemia Tax

Virology. Table 2 Three major forms of viral latency are associated with EBV-positive malignancies Latency I II

III

Viral genes expressed EBERs, BARTs EBNA1 EBERs, BARTs EBNA1, LMP1, LMP2A, & LMP2B EBERs, BARTs EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C & EBNA Leader Protein (LP) LMP1, LMP2A, & LMP2B

During EBV latency only a limited number of viral genes are expressed; these include six nuclear proteins, referred to as the Epstein-Barr nuclear antigens (EBNAs 1, 2, 3A, 3B, 3C and EBNALP), two proteins found in the cell membrane of infected cells known as the latent membrane proteins (LMPs), two non-translated RNA molecules known as the Epstein-Barr virus encoded RNAs (EBERs) and the BARTs.

rightward transcripts; BARTs). Three major forms of viral latency exist in tumors (Table 2). The restricted forms of latency have evolved partly to prevent expression of the immunodominant EBNA proteins and are controlled by differential ▶methylation of viral promoters. Demethylating agents can induce

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expression of these EBNAs and in some cases can also induce lytic cycle; this has been proposed as an alternative treatment for some EBV-associated cancers (▶epigenetic therapy). Many viruses, including EBV, are able to subvert the cellular epigenetic machinery, not only to regulate virus gene expression, but also potentially to influence cell growth by silencing ▶tumor suppressor genes). LMP1 is the major transforming protein of EBV; it is a transmembrane protein which mimics a constitutively activated ▶tumor necrosis factor receptor (TNFR). In normal cells binding of the appropriate ligand to these receptors causes intracellular signaling and a number of effects including either proliferation or ▶apoptosis, depending upon the nature of the signal and the cell type involved. In contrast, LMP1 requires no ligand for its activation and delivers constitutive intracellular signals leading to cell proliferation and protection from apoptosis, which in turn accounts for the transforming properties of this protein. Signaling pathways activated by LMP1 include ▶NF-κB, JNK/AP-1, p38/MAPK, and JAK/STAT (▶signal transducers and activators of transcription). Several of these pathways are aberrantly activated in EBV-associated tumors, such as ▶Hodgkin lymphoma. LMP1 can decrease ▶ATM expression, and may represent one of a number of ways in which EBV can disable DNA repair. The malignant diseases associated with EBV include Burkitt lymphoma, ▶nasopharyngeal carcinoma, Hodgkin lymphoma and a variety of other cancer types. EBV-Encoded microRNAs ▶MicroRNAs (miRNA) are a class of small RNAs that are probably major regulators of gene expression. miRNA are complementary to their cognate mRNA sequences; their interaction in the RNA-induced silencing complex (RISC) results in the cleavage of the target mRNA or in some cases inhibits its translation. The first virus-encoded miRNAs that were discovered were those of EBV, from which five miRNAs were cloned. EBV miRNAs are clustered in two distinct regions; the first is located within the 5′ and 3′ UTR of the BHRF1 transcript and the other within the BART region. The precise functions of these and other viral miRNAs are yet to be defined but are likely to play key roles in the regulation of virus and cellular gene expression, with potential involvement in oncogenesis. Burkitt Lymphoma ▶Burkitt lymphoma (BL) is an aggressive tumor of B-lymphocytes. There are two main types of BL. The endemic (African) type occurs with high frequency (5–20 cases/100,000 children/year) in equatorial Africa and Papua New Guinea and with a distribution that matches that of holo-endemic malaria. Almost all cases of endemic BL are EBV-positive. On the other

hand, sporadic BL occurs world-wide with a much lower incidence and only around 15% of cases are EBV-positive. A third form of BL occurs in AIDS patients and approximately one-third of these tumors harbor EBV. The cells of EBV-infected BL tumors usually display a latency I phenotype – that is they only express one viral protein, EBNA1. More recently variant forms of BL have been described in which expression of some of the other EBNA genes is observed. These are known as Wp using BL since they use a distinct viral promoter (Wp) to drive EBNA expression. Although infected BL tumor cells could process peptides from the EBNA1 protein and present them to specific CTLs, the processing of endogenous EBNA1 through the Class I pathway is inhibited. This, together with the downregulation of MHC class I and ▶adhesion molecules that is a feature of BL cells, contributes to their ability to evade immunodetection. BL is characterized by reciprocal translocations that result in deregulation of the ▶myc gene. In endemic BL, EBV-driven proliferation of B-lymphocytes, together with a general polyclonal stimulation of B cells induced by malaria infection, is thought to increase the chances of one of these specific translocations occurring in the B lymphocytes that will eventually give rise to BL. Nasopharyngeal Carcinoma ▶Nasopharyngeal carcinoma (NPC) is an epithelial tumor of the nasopharynx that is rare in the West but endemic in China, South East Asia, and North Africa. There are three main types of NPC; undifferentiated, non-keratinizing and squamous NPC. EBV is strongly associated with the undifferentiated type (UNPC). UNPC is an aggressive tumor with ▶metastasis early to bone, liver, and lung and to the lymph nodes of the neck. The exact contribution of EBV to the pathogenesis of NPC has yet to be established, although recent studies suggest that EBV infection is preceded by genetic changes which include deletions of chromosome regions rich in tumor suppressor genes. Dietary factors, including nitrosamines from preserved fish, as well as EBV, are important risk factors for UNPC. Hodgkin Lymphoma ▶Hodgkin lymphoma (HL) is characterized by relatively low numbers of malignant, so-called ▶Hodgkin/ Reed–Sternberg cells (HRS cells) surrounded by a mass of ‘reactive’ non-malignant cells. HL is classified into four major subtypes on the basis of the relative proportions and morphology of the HRS cells, the nature of the reactive component, and the degree of fibrosis. In most cases the HRS cell is believed to derive from germinal centre (GC), or post-GC B-lymphocytes.

Virology

The frequency of the EBV association in HL is dependent upon a number of factors. EBV is most often associated with the mixed cellularity form and less commonly with the other subtypes. In North America and Europe, fewer (20–40%) tumors are EBV-associated, compared to developing countries where the association approaches 100%. Males also seem to be more at risk than females for EBV-positive HL. In EBVpositive cases, HRS cells express EBNA1, LMP1 and LMP2 (latency II pattern, Table 2). Epitopes from both LMP1 and LMP2 can be processed and presented to CTLs by infected cells. Thus, EBV-infected HRS cells should be recognized by EBV-specific CTLs; however, their survival in immunocompetent patients suggests that they, like BL cells, can escape the immune response. EBV-infected HRS cells express interleukin-10 (IL-10) which can inhibit EBV-specific CTL responses. Regulatory T cells have are also detected in the microenvironment of HL. It is likely that EBV and other persistent virus are able to recruit regulatory T cells to inhibit virus-specific T cell responses. Lymphoproliferative Disease in Immunosuppressed Patients In immunosuppressed patients the lack of EBV-specific CTLs can lead to an increase in the numbers of EBVinfected B-lymphocytes. In persistent ▶immunosuppression states, such as in post-transplant patients or in AIDS sufferers, EBV-infected B-lymphocytes can proliferate to produce tumor like masses. Later, the acquisition of other genomic changes, such as those that affect ▶p53, ▶MYC or ▶BCL-6 can lead to the formation of a classic ▶lymphoma. Therefore, these lymphoproliferative diseases constitute a spectrum of disorders ranging from relatively benign atypical lymphoproliferations which will regress if the immunosuppressive therapy is withdrawn through to highly aggressive lymphomas which do not respond to immune reconstitution.

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Other Tumors EBV is associated with a number of other tumors, including some T cell lymphomas, such as nasal T/natural killer cell lymphomas (▶NK cell lymphomas). EBV is also associated with some gastric cancers and with smooth muscle tumors that arise in some immunodeficient patients. Thus, EBV is apparently able to infect and contribute to neoplastic growth in a number of different cell types. Kaposi Sarcoma Herpesvirus ▶Kaposi sarcoma (KS), originally described in 1872, is a malignancy of endothelial cells that usually presents as a brown/purple skin tumor with more aggressive forms involving the lungs, lymph nodes, and gastrointestinal tract. Until the advent of the AIDS epidemic it was a relatively rare disease whose etiology remained obscure. KS occurs frequently in ▶HIV-positive individuals, particularly in homosexual/bisexual males. Suspicions that KS might be due to an infectious agent were confirmed when a new human herpesvirus, known as Kaposi Sarcoma herpesvirus or KSHV (also referred to as human herpesvirus-8, HHV8), was discovered in KS tumors from AIDS patients. In fact, viral sequences are present in all types of KS including KS that arises in HIV-negative individuals. Serological assays to detect antibodies to KSHV were developed and showed a higher prevalence of infection in those groups at high risk for the development of KS. KSHV is also associated with primary effusion lymphomas and a rare lymphoproliferative disease known as multicentric Castleman disease. KSHV is a double stranded DNA virus that is closely related to EBV. Its genome encodes many genes with homology to human genes (Table 3). Some of these are involved in the regulation of both innate and adaptive immune responses; many of them function to inhibit the immune responses to viral infection. For example, several virus-encoded ▶interferon (IFN)-regulatory

Virology. Table 3 KSHV encodes many homologues of cellular proteins, some of these viral proteins and their effects upon the infected cell are summarized Cellular gene Cyclin gene (cyclin D2) IL-6 IL-8R FLICE inhibitor protein (FLIP) Interferon regulatory proteins (IRFs) Bcl-2

KSHV gene

Effect of virus gene expression

v-Cyclin D v-IL-6 v-GPCR

Phosphorylates Rb and releases cell from cell cycle arrest Autocrine stimulation of cell growth Constitutive activation of phosphatidylinositol pathway leading to cell growth v-FLIP Inhibits CD95-mediated apoptosis v-IRFs 1–4 Inhibits interferon signaling v-Bcl-2

Protects infected cell from apoptosis

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factors (IRFs) negatively influence anti-viral interferon responses, while others protect cells from apoptosis (e.g. v-▶FLIP, v-▶Bcl-2). Polyomaviridae Polyomaviruses are DNA viruses with small circular genomes encoding only six proteins. They include the mouse polyoma virus, the ▶simian virus 40 (SV40), and the human viruses, ▶BK virus and ▶JC virus. With the exception of the mouse polyoma virus, these viruses do not cause cancer in their natural hosts but do induce tumors in newborn animals, including hamsters and rats. Polyomaviruses do not themselves encode replication proteins and must drive cells into S-phase where host DNA replication proteins can be utilized for virus replication. The large T (tumor) antigen (T-Ag) and the small t antigen (t-Ag) are major effectors of this. T-Ags bind to pRb encoded by the ▶RB gene and displace ▶E2F thereby promoting cell cycle progression; this is a major mechanism by which T-Ag promote the inappropriate cell proliferation leading to oncogenic transformation. The release of E2F from pRb activates p14ARF which can stabilize p53. However, SV40, JCV and BKV T-Ags can bind to and inactivate p53 and thus prevent inhibition of the cell cycle or apoptosis. Apart from their well established role in the binding and inactivation of p53 and pRb, T-Ag can influence other pathways leading to oncogenesis. Thus, JCV T-Ag can interact with insulin receptor substrate-1 (IRS-1); this is associated with transformation and might be involved in the development of childhood medulloblastomas. JCV T-Ag can also bind β-catenin, causing it to translocate to the nucleus where it can stimulate the expression of genes such as c-myc and cyclin D1. SV40 t-Ag binds and inhibits protein phosphatase 2A (PP2A), a major serine/threonine specific protein phosphatase; this leads to the activation of several pathways that promote cell proliferation, including the MAPK pathway. Although the polyomaviruses can transform cells in culture and under certain conditions are oncogenic in laboratory animals, their association with clinical human tumors has yet to be definitely proven (▶association of polyomaviruses with human cancers). Papillomaviridae ▶Human papillomaviruses (HPVs) are small DNA viruses that commonly infect epithelial tissues. At present there are over 100 known subtypes of HPV and the majority are responsible for benign lesions of the genital, upper respiratory and digestive tracts. However, some HPV subtypes are associated with malignant diseases of the skin and cervix (▶cervical cancers). Cutaneous HPV infection normally results in the appearance of benign warts, however in the rare but

lifelong skin disease, ▶epidermodysplasia verruciformis (EV), these multiple benign warts can progress to malignant squamous carcinoma when exposed to ▶ultraviolet light. The tumor cells often contain HPV 5 or 8. These two HPVs are also associated with the skin carcinoma observed in long-term immunosuppressed renal transplant patients. Virtually all squamous cancers of the cervix are HPVpositive; HPV16, followed by HPV18 are the commonest subtypes found in this disease. HPV18 is the type most strongly associated with adenocarcinoma of the cervix. HPV is sexually transmitted and the association of HPV with cervical cancer explains many of the risk factors for this disease including early age at first sexual encounter and multiple sexual partners. Although most women will have been infected with HPV at some time, very few will develop invasive cancer. In cancers, integration of HPV18 is almost universally observed, whereas integration of HPV16 is less common. Integration usually disrupts the E1 or E2 viral genes (▶early genes of human papillomaviruses); this results in the loss of negative feedback control of E6 and E7 expression by the viral regulatory E2 protein. Overexpression of E6 and E7 proteins is important in oncogenesis; E6 binds to and inactivates p53 and E7 binds to Rb. A bivalent HPV16/18, and a quadrivalent HPV 6/11/ 16/18 vaccine are being evaluated in phase III clinical trials. Preliminary data suggest that these prophylactic HPV virus-like particle vaccines are effective in preventing infections and also in reducing epithelial abnormalities (▶human papillomavirus vaccines). Hepadnaviridae Hepatitis B Virus ▶Hepatitis B virus (HBV) is associated with the development of ▶hepatocellular carcinoma (HCC) where integrated HBV DNA can been detected in the majority of tumors. The exact role of HBV in the development of HCC is yet to be established. However, the HBx protein is likely to be important since it can induce a number of cellular changes that contribute to transformation, including the activation of several intracellular signaling pathways, including NF-κB. Other factors in addition to HBV status contribute to the risk of developing HCC; these include smoking, dietary components such as ▶aflatoxin, and exposure to other hepatotoxic agents, including ▶hepatitis C virus. Flaviviridae Hepatitis C Virus ▶Hepatitis C virus is a ▶blood-borne RNA virus that can cause chronic hepatitis and later ▶cirrhosis and hepatocellular carcinoma. HCV is spread by

Virotherapy

blood-to-blood contact. Many people with HCV infection have no symptoms and are unaware of the need to seek treatment. An estimated 150–200 million people worldwide are infected with HCV. At least four HCV gene products, namely HCV core, NS3, NS4B and NS5A, have been shown to exhibit transformation potential in tissue culture. Both HCV core and NS5A induce the accumulation of wild-type β-catenin. Retroviridae Human T lymphotropic Virus-1 Human T lymphotropic virus-1 (HTLV1) (▶human Tcell leukaemia virus) infects ▶CD4-positive T-lymphocytes and is associated with the development of adult T-cell leukaemia/lymphoma (ATLL). Like other retroviruses, the HTLV1 genome consists of gag, pol, and env genes, which encode important structural and functional proteins, flanked by long terminal redundancies (LTRs). HTLV1 has an additional 3Υ region which encodes several proteins implicated in transformation; these include Tax, Rex, p12, p13, p30, and HBZ. ▶Tax has been shown to be necessary and sufficient for transformation by HTLV-1. Tax activates both the canonical and non-canonical NF-κB pathways, in turn leading to increased expression of many ▶cytokines and their receptors, including ▶interleukin-2 and the interleukin2 receptor, which leads to polyclonal proliferation of HTLV-1-infected cells by ▶autocrine and ▶paracrine mechanisms. Additional potentially transforming effects are contributed by some of the other HTLV1 genes.

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human immunodeficiency virus. These vesicles lack the functional viral nucleocapsid, including the viral genetic material, but can still be used to incorporate low molecular weight drugs, proteins, and nucleic acids. Virosomes can introduce these molecules into target cells by the same mechanism as viral infection because they retain the cell entry and membrane fusion properties of the parent virus. ▶Non-viral Vector for Cancer Therapy

Virotherapy ZENG B. ZHU1, B RUCE F. S MITH 2 , G ENE P. S IEGAL 3 DAVID T. C URIEL 1 1

Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology and the Gene Therapy Center, Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, USA 2 Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL, USA 3 Departments of Pathology, Cell Biology, and Surgery and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL, USA

Synonyms Oncolytic virotherapy

References 1. Tao Q, Young LS, Woodman CB et al. (2006) Epstein-Barr virus (EBV) and its associated human cancers–genetics, epigenetics, pathobiology and novel therapeutics. Front Biosci 11:2672–2713 2. Cullen BR (2006) Viruses and microRNAs. Nat Genet 38 (Suppl):S25–S30 3. Poulin DL, DeCaprio JA (2006) Is there a role for SV40 in human cancer? J Clin Oncol 24:4356–4365 4. Stanley MA (2006) Human papillomavirus vaccines. Rev Med Virol 16:139–149 5. Khan G (2006) Epstein-Barr virus, cytokines, and inflammation: a cocktail for the pathogenesis of Hodgkin’s lymphoma? Exp Hematol 34:399–406

Definition

▶Virotherapy utilizes ▶oncolytic viruses, which may occur naturally or more commonly be engineered, such as conditionally replicative ▶adenoviruses (▶CRAds), to selectively infect tumor cells and replicate within them, thus causing their demise while sparing surrounding normal cells in the host. ▶Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells whereby normal host cells are spared.

Characteristics

Virosomes Definition Are vesicles mimicking the envelopes of various viruses, such as influenza virus, Sendai virus, and

Virotherapy represents an exciting and novel interventional strategy for a range of neoplastic disorders. In this strategy a virus is rendered conditionally replicative for tumor cells whereby direct oncolytic target killing is achieved. A variety of viral species have been adapted as virotherapy agents with the majority of human clinical trials exploiting conditionally replicative ▶herpes simplex virus (HSV) and adenovirus. Of these, adenovirus has emerged as a promising model oncolytic

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vector to target tumor cells. Over 40 different human serotypes of adenoviruses have been identified with types 2 and 5 being the most extensively used in developing oncolytic constructs. Oncolytic Adenoviruses and Cancer Gene Therapy The concept of using conditionally replicative adenoviruses (CRAds) for the treatment of cancer, also known as ▶oncolytic virus therapeutics, originated in the 1950s. The knowledge that adenoviruses could eliminate cancer cells in vitro, as a consequence of their reproducitive cycle leading to cell lysis (“oncolysis”), resulted in clinical studies in which various wild-type adenoviral serotypes were examined for their effect on ▶cervical cancer patients. In these studies, no significant toxicity was reported after intratumoral injection (i.p.) or intravenous (i.v.) administration and a moderate tumor response was observed. The most studied CRAd is the one originally generated (dl1520) by Arnold Berk and used initially by Frank McCormick as a selective vector, named ONYX-015. This viral vector originally was believed to only replicate in p53defective cells (present in 50% of human tumors). It is now recognized that these agents have beneficial properties for cancer ▶gene therapy when compared with their nonreplicating counterparts, although this mechanism has subsequently been questioned. The adenovirus-based vectors have maintained their positions as the leading candidates for in vivo oncolytic virotherapy because they are safe, produced in high titers, do not integrate into the host chromosome, and have a wide tropism in neoplastic cells. While adenoviral transfection is efficient, the expression of the transferred gene is transient, as the viral genome remains episomal. To maximize CRAd mediated cell killing, one needs to achieve an amplification effect for transduction, via replication of the delivered viral vector post-infection, resulting in lateral spread of the progeny vector and cell killing via viral oncolysis. In addition, viral proteins expressed late in the course of infection are indirectly cytotoxic, including the E3 11.6 kDa adenovirus death protein, E4ORF4. CRAds in Human Clinical Trials and the Limitations Significant antitumor activity has been demonstrated using ONYX-015 both in vitro and in vivo using murine models. The preclinical potential of CRAds led to their rapid translation into human clinical trials, including those targeting recurrent head and neck, pancreatic, colorectal, ovarian, and ▶hepatobiliary cancer. The overall conclusion was that adenoviral virotherapy is a safe method when applied via various routes, thus validating the concept in vivo. In clinical practice, however, employing CRAd as a single

agent has demonstrated limited efficacy. This is due, in part, to relatively inefficient gene transfer to tumor cells. The replicative Ad system of ONYX-015, although demonstrating promise in preclinical studies, yielded no clinical effect in 16 patients with ▶ovarian cancer treated intraperitoneally with up to 1 × 1011 plaque-forming units of adenovirus daily for 5 days. Important in this regard, cancer cells have been specifically shown to be profoundly resistant to Ad infection. The results obtained in these studies have been helpful in determining the limitations of the current generation of CRAds. The critical problems that have been encountered involve two main limitations: (i) poor infectivity of cancer cells by adenovirus at the transductional level due to the lack of native adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR), on the surfaces of tumor cells; (ii) poor tumor selectivity of CRAd agents at the transcriptional level due to the lack of tumor specific promoters to selectively drive the viral replication in tumor cells. CAR-Deficiency on Tumor Cells Results in the Poor Infectivity of Ad Agents Cancer cells have been specifically shown to be profoundly resistant to Ad infection because of their lack of the primary receptor for viral entry, the coxsackievirus-adenovirus receptor (CAR). As these primary tumor cells often express relatively low levels of the CAR, this results in the poor infectivity of CRAd agents and the difficulty of lateral dispersion of virus in tumor tissue. This has been demonstrated by the fact that low CAR levels strongly reduced viral replication and oncolysis in monolayer cultures and murine models. On this basis, it was proposed that delivery be achieved via a heterologous entry pathway, or CAR-independent pathway to circumvent this key aspect of tumor biology. Infectivity Enhanced CRAd Agents-Retargeting CRAds to Tumor Cells Native adenoviral tropism is mediated by two capsid proteins, fiber and penton base. These proteins bind to the primary, high-affinity cellular receptor, CAR, and the integrins αvβ3 and αvβ5, respectively. Manipulation of these molecular interactions has been carried out to modify adenovirus tropism by routing entry through either a heterologous entry pathway or a CAR-independent pathway. Many approaches have been described to enhance the viral infectivity. These include: (i) Ad capsid modification for circumventing tumor cell CAR deficiency. Specifically, Dmitriev et al. (1998b) reported that construction of modified adenoviral vectors containing the RGD peptide in the HI loop region, which targets integrins αvβ3

Virotherapy

and αvβ5 instead of CAR, increased gene transfer to ovarian cancer cell lines (30–600 fold) and to primary ovarian cancer cells obtained from patients (2–3 fold). Many other approaches have been reported which include targeting Ad to the serotype 3 receptor with a chimeric fiber protein, such as F5/3, targeting Ad to tumor cells with the non-human canine Ad type 2 knob, targeting Ad to a heparin sulfate-containing receptor with a Ad fiber incorporating polylysine (pK7) and targeting Ad to the junction adhesion molecule 1 (JAM1) with an Ad fiber incorporating reovirus sigma 1 fiber. All these fiber modifications enhanced the viral infectivity of Ad vectors but with different levels of success dependent upon the tumor types. (ii) Transductional retargeting using heterologous targeting adapters. In adapter-mediated targeting, the tropism of the virus is modified by an extraneous targeting moiety, the ligand, which associates with an Ad virion either covalently or non-covalently. Zhu et al. described in 2004 that an adenoviral vector was successfully induced to transport itself across polarized epithelial monolayers by use of a fusion protein (sCARtransferrin), a bi-functional adapter, which bound to the knob of Ad through a sCAR (secretory CAR) domain and to the transferrin receptor on cell surfaces through a transferrin domain. In addition, sCAR-EGF, sCAR-SCF, and sCAR-scFv have all been reported as having successfully targeted EGF receptor-positive cells, c-Kit(+) and CAR(–) hematopoietic cells and ovarian carcinoma cells, respectively. The genetic fusion proteins of single-chain variable fragment (scFv) antibodies directed against the fiber knob and receptor on the surfaces of tumor cells have also been used in virotherapy. In this schema, an anti-fiber-knob Ab has been employed to attach to the cell recognition motif of the fiber knob and, importantly, ablate the native tropism determinants. An Ab, or Fab derivative, was then conjugated to a second moiety, which provided targeting specificity. To that end, receptor ligands such as folate and basic fibroblast growth factor-2 (FGF-2) have been used to successfully target tumor cells. Thus, the strategy of tropism modification either using bispecific conjugates or scFv molecules allowed dramatic augmentation in gene delivery to tumor targets, with a specificity that would predict an improved therapeutic index. Tumor Specific CRAd Agents – Using a Tumor Specific Promoter to Drive Specific Viral Replication Although viral replicative specificity is not the main limit of CRAd efficacy, our work and those of others have also sought to address this aspect to improve the overall therapeutic index. The method used to direct CRAd vector specificity is by regulation of viral replication via cellular promoters that are over

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expressed or reactivated in selected tumor cells, sometimes referred to as “tumor specific” promoters. A number of CRAd agents have been developed, which harbor the essential Ad E1A gene under the control of such promoters. They include the alpha-fetoprotein promoter, Cox-2 promoter, DF3/MUC1 promoter, midkine promoter, PSA/PSMA enhancer, secretory leukocyte protease inhibitor (SLPI) promoter, CXCR4 and survivin promoters, human telomerase reverse transcriptase (hTERT) promoter, and the tyrosinase promoter/ enhancer. Most of these agents have demonstrated remarkable preclinical results in eradicating tumors in xenograft mouse models. A Double Targeted CRAd for Ovarian Cancer Based on the foregoing considerations, we have developed a CRAd agent which addressed the limits of the current systems. As previously discussed, CAR deficiency in the context of the target tumor would clearly confound this process, thus undermining the overall efficacy of CRAd agents. Previous studies have demonstrated that incorporation of an Arg-Gly-Asp (▶RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. In the context of ovarian cancer, the RGD modified nonreplicative adenovirus mediates enhanced gene transfer in vitro in both established cell lines and freshly cultured ▶ovarian cancer cells. It exhibits preferential gene transfer to primary ovarian cancer cells when compared to non-transformed human mesothelial tissue, thus, indicating a degree of specificity for tumor cells. In addition, the RGD modified nonreplicative adenoviral vector demonstrated enhanced infectivity of primary ovarian tumor cells where infectivity was known to be inhibited by preformed neutralizing antibodies against adenovirus found in the ascites fluid in which the tumor cells float. Investigators at the University of Alabama at Birmingham (UAB) and the University of Texas-MD Anderson Cancer Center have constructed a novel infectivity enhanced CRAd, designated Ad5-Δ24RGD, that has a 24 bp deletion in the E1A gene and that incorporates the RGD modification in its fiber. The deletion in the E1A gene is from Ad5bp923 to 946 which corresponds to the amino acid sequence L122TCHEAGF129 of the E1A protein known to be necessary for host cell Rb protein binding. Adenoviruses having this partial deletion cannot induce resting cells to pass the ▶G2/M checkpoint and progress to mitosis and lysis. In contrast, most human tumor cells bypass the Rb/p16 pathway, thus allowing for selective replication of adenoviruses with this deletion. Preliminary experiments demonstrated that Ad5Δ24RGD propagated more efficiently than Ad5-Δ24 in

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A549 cells. Specifically, A549 cells transfected with Ad5-Δ24RGD yielded 43 fold times increase in viral progeny (3.75 × 109 pfu/ml) when compared to Ad5Δ24 infected cells (8.74 × 107 pfu/ml). Cell lysis in Ad5-Δ24RGD infected A549 and LNCap cells was 7 and 3.5 times higher, respectively, compared to that achieved with Ad5-Δ24. Validation of the potential benefits of incorporating infectivity enhancements into a CRAd provided the rational to translate this into clinical trials which has been recently approved by FDA.

Future Directions for Improvements CRAd human trials were historically limited by lack of useful information regarding the biologic basis of adenovirus efficacy barriers. Absent “surrogate endpoints,” imaging could potentially provide this information. We have thus developed an imaging system which addresses this problem, based upon fluorescently labeled adenovirus carrying EGFP (enhanced green fluorescent protein) on the pIX minor surface protein of Ad for vector detection. Furthermore, positron emission tomography (PET) scanning has been used to detect herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) which was fused to this same Ad protein IX (pIX). Other conventional imaging systems for virotherapy have been designed for the detection of transgene expression of reporters such as sodium iodide symporter, somatostatin receptor type 2 (SSTR-2), and luciferase. The combination of a noninvasive imaging modality with a genetic adenoviral labeling system for detection of viral replication and progeny localization has begun to provide a powerful means of real-time monitoring of CRAd function in vivo.

5. Zhu ZB, Makhija SK, LU B et al. (2004) Transport across a polarized monoloyes of caco-2 cells by transferring receptor-mediated adenovirus Transeytosis. Virol 325:116–128 6. Kruyt FA, Curiel DT (2002) Toward a new generation of conditionally replicating adenoviruses: pairing tumor selectivity with maximal oncolysis. Hum Gene Ther 13:485–495

Virtual High Throughput Screen Definition A procedure in which collections of computerized molecular structures are tested for their ability to activate, perturb, or modify a target or biological process of interest using digital models. ▶Small Molecule Screens

Virus-like Particles Definition Non-infectious assembly of viral proteins lacking a genome. Biotechnological virus-like particles elicit antiviral immunity without risk of infection and can be used to make vaccines. The term is also applied to some natural non-infectious mycoviruses with an RNA genome. ▶Immunoprevention

▶Oncolytic Virus

References 1. Heise C, Sampson-Johannes A, Williams A et al. (1997) ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med 3:639–645 2. Stojdl DF, Lichty B, Knowles S et al. (2000) Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 6: 821–825 3. Yu DC, Chen Y, Dilley J et al. (2001) Antitumor synergy of CV787, a prostate cancer-specific adenovirus, and paclitaxel and docetaxel. Cancer Res 61:517–525 4. Dmitriev I, Kransuyky V, Miller CR et al. (1998) An adevevirus vector with genetically modified fibres demonstrates expanded tropism via utilization of a coxsackie virus and adenovirus receptor-independent cell entry mechanism. J Virol 72:9706–9713

Virus Therapy of Cancer ▶Oncolytic Virotherapy

Virus Vector Definition Refers to genetically modified viruses or virus-derived particles that can be used to deliver a (non-viral) gene of interest to some specified target cell or tissue. ▶Viral Vector-mediated Gene Transfer

Vitamin D

Virus Vector-mediated Gene Transfer ▶Viral Vector-mediated Gene Transfer

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Vitamin D L AURA P. Z ANELLO Department of Biochemistry, University of CaliforniaRiverside, Riverside, CA, USA

Definition

Viruses Definition Viruses are pathogens composed of a nucleic acid genome enclosed in a protein coat. They can replicate only in a living cell, as they do not possess the metabolic machinery for independent life. ▶Virology

Vital Definition Crucial or extremely important.

Vitamin A Definition Is an essential human nutrient. It exists not as a single compound, but in several forms. In foods of animal origin, the major form of vitamin A is ▶retinol. ▶All-Trans Retinol ▶Retinoids

Vitamin C Definition A water-soluble vitamin found in fruits and vegetables and used as an ▶antioxidant. ▶Chemoprotectants

The biologically most active form of vitamin D, 1α,25dihydroxy-vitamin D3, and many synthetic analogs exert anti-proliferative actions on different cancer cell types, most typically breast, colon, and prostate. Recent clinical studies indicate a potentially important role for vitamin D in the prevention and treatment of cancer.

Characteristics

1α,25-dihydroxy-vitamin D3 (1,25D), or ▶calcitriol, the biologically most active form of vitamin D, is essential to bone and mineral metabolism. 1,25D deficiency has been traditionally associated with bone diseases characterized by decreased bone mass and reduced mineralization, such as rickets, osteomalacia, and osteoporosis. In addition to its well known actions on bone and calcium homeostasis, this ▶secosteroid hormone exerts a number of effects on many different target organs and systems. 1,25D actions include regulation of ▶cell cycle, cell proliferation and differentiation in breast, colon, and prostate, among others. 1,25D binds to the nuclear ▶vitamin D receptor (VDR), which acts as a ▶transcription factor for the regulation of gene expression. In addition, 1,25D stimulates signal transduction cascades implicated in non-genomic responses of target cells such as modulation of the electrical state of the plasma membrane, secretory activities, cell survival, and establishment of the apoptotic phenotype. Mechanisms In recent years, natural compounds and synthetic analogs of vitamin D have proved to have significant effects on cell cycle, differentiation, survival and ▶programmed cell death. More specifically, 1,25D has been shown to act as a powerful anti-proliferative agent on certain cancer cell types expressing the VDR, including breast, colon and prostate, both in vitro and in vivo. Typically, cell cycle arrest promoted by physiological doses of 1,25D is linked to cell differentiation, and therefore reduction in the probability of cells to become cancerous. Human trials on anti-cancer properties of calcitriol and synthetic analogs have increased in recent years, with ▶prostate cancer being the most studied one. Calcitriol and numerous analogs have shown potential to become therapeutic agents for the treatment of certain cancer types in the future. However, the molecular mechanisms by which 1,25D inhibits cancer cell proliferation remain only partially understood. To date, more

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than 1,000 vitamin D analogs have been synthesized, and many of these analogs have shown to have significant anti-proliferative effects. The earliest evidences on a relationship between vitamin D and cancer come from epidemiological studies conducted in the 1990s. An inverse relationship was discovered between sunlight exposure –which is lower as latitude increases– and ▶prostate cancer cases in the United States over 45 years of data (1950–1994). A main source of vitamin D in our bodies is by means of production of vitamin D3 or cholecalciferol, a hormone precursor, in the skin by ▶UV radiation. The second main source is vitamin D2 or ergocalciferol in the diet. However, these products are biologically inactive. They require two sequential hydroxylations, first in the liver (to produce 25-hydroxy-vitamin D3, or 25D), and then in the kidney (to render 1α,25-dihydroxy-vitamin D3). The major circulating source of the secosteroid hormone is 25D, therefore measurement of circulating concentrations is a good indicator of the overall vitamin D3 status of the individual. In addition, many tissues express 25-hydroxy vitamin D3 1α- hydroxylase, the enzyme that transforms 25D into 1,25D, which indicates that local production of the biologically active form may play an important role in ▶autocrine and ▶paracrine functions. Early epidemiological studies suggested that elevated circulating levels of 25D main protect against cancer, and that ingestion of vitamin D supplements may be helpful in the treatment of tumors. In addition to case studies, extensive in vitro work has demonstrated that high concentrations of 1,25D (10–9–10–7 M) inhibit the growth and induce differentiation of tumor cells, including malignant ▶melanoma, myeloid leukemia, ▶prostate cancer, ▶breast cancer, ▶glioma cancer, and ▶colon cancer. Clinical trials and treatment of cancer patients with high doses of 1,25D or synthetic analogs have been limited due to the fact that, at these high vitamin D3 doses, there is a significant risk to develop ▶hypercalcemia with fatal side effects. This has encouraged the pharmaceutical industry to develop new synthetic analogs with high potency for cell growth inhibition and low hypercalcemic effects (for example, analog EB1089). The highly conformationally flexible 1,25D molecule offers a broad source of possibilities for the design of chemical compounds with high affinity for the VDR. These are ideally capable of potentiating the genomic regulation of the cell cycle, while inducing only low calcium effects more likely to develop via non-genomic mechanisms. Recently, an alternative binding site for non-genomic analogs has been proposed on the basis of molecular modeling of the crystal structure of the VDR. This new hypothesis for the binding of ligands to the VDR brings an even higher potential for the design of analogs with more specific anti-proliferative actions and reduced hypercalcemic side effects.

Additional aspects that contribute to the tumor suppressive activity of vitamin D compounds include the ability of 1,25D and analogs to reduce ▶angiogenesis and ▶metastasis. While use of 1,25D and analogs with low calcemic effects for the treatment of cancer offers high promise, combination therapy with other anti-tumorigenic drugs may signify even higher beneficial effects. In vitro studies have demonstrated, for example, that 1,25D in combination with the antiestrogen ▶tamoxifen has greater efficacy in the inhibition of breast cancer cell lines. Different combinations of vitamin D3 and ▶retinoids, ▶cytokines, and a number of cytotoxic compounds including ▶adriamycin, ▶carboplatin, ▶cisplatin have proven to act synergistically on the suppression of cancer cell growth. The anti-cancer effects of vitamin D compounds and analogs can be explained on the basis of different aspects of cancer cell biology. More specifically, 1,25D effects on tumor cells include: 1. Regulation of the ▶cell cycle 2. Induction of ▶apoptosis or programmed cell death 3. Modulation of the expression of ▶oncogenes and ▶tumor suppressor genes 4. Induction of cell differentiation Studies performed with 1,25D and analogs on different cancer cell lines in vitro have shown that cells grown in the presence of the steroid significantly reduce their growth rate mostly by blockade of the transition from the G0/G1 phase (differentiated, non dividing cells) to the S phase (DNA synthesis). In addition to promoting cell cycle arrest and inhibition of cell division, 1,25D and related compounds have proved to induce apoptosis in glioma, breast, leukemia and colon cancer cells. It is not clear, however, how a cell follows its path to either cell cycle progression or apoptosis once it reaches early G1 phase. Vitamin D3 compounds appear to act at this point through different molecular mechanisms, depending on whether they induce cell death or stop cells from dividing. In human ▶osteosarcoma, for example, 1,25D treatment reduces cell proliferation in vitro by approximately 25% after 3 days. The mechanisms involve sustained activation of ▶MAP kinase/▶AP-1/p21(waf1) pathways. Upregulation of p21 gene expression led to control over the cell cycle and subsequent cell cycle arrest (Fig. 1). The modulation of the expression of certain oncogenes and tumor suppressor genes has been also shown to be affected by 1,25D treatment of cancer cell lines. Among genes that modulate cell growth and apoptosis, the ▶bcl-2, ▶c-myc, and retinoblastoma gene products are the most widely studied. The bcl-2 oncogene product protects against apoptosis, therefore inducing cell survival. In MCF-7 breast cancer cells, 1,25D and analogs KH1060 and EB1089 decreased bcl-2

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1,25D

H H

OH

Sustained

25(OH)

Transient

VDR HO

OH 1α p-JNK

Cytoplasm

MEK1 MEK2

1,25D / VDR

ERK1/2

p-c-Jun c-Fos

p-c-Jun/c-Fos DNA

AP-1site

Nucleus

1,25D-VDR/RXR VDRE

p21 gene

Cell cycle arrest

Osteosarcoma cell Vitamin D. Figure 1 1,25D reduction of human osteosarcoma cell proliferation occurs via sustained activation of mitogen activated protein kinases ▶JNK and MEK1/MEK2 downstream of non-genomic VDR signaling, leading to upregulation of a c-Jun/c-Fos (AP-1) transcription factor complex, which in turn modulates p21(waf1) gene expression. Transient refers to 1,25D treatment for 15 min; sustained implicates a treatment with hormone for 3 days. RXR: ▶retinoic acid receptor, VDRE: vitamin D responsive element, p: indicates phosphorylation of the protein.

expression, and cells progressed through apoptosis. The expression of the oncogene c-myc has also been shown to be reduced by 1,25D treatment of breast cancer cells, and resulted in reduction of cell proliferation. Finally, induction of cell differentiation by vitamin D3 compounds has been widely described in combination with its anti-proliferative effects. Typically, 1,25D inhibits cell proliferation and induces differentiation in the hematopoietic system, osteoblasts, and keratinocytes. Currently, the safe upper limits for vitamin D intake as supplements have been set in the US by The Institute of Medicine to be 1,000 IU/day for children under 1 year of age, and 2,000 IU/day for adults. Although rare, vitamin D intoxication may develop if intake of higher doses happens over significantly prolonged periods of time, and lead to hypercalcemia, hyperphosphatemia and hypercalciuria. A recent clinical trial conducted on patients with premalignant colon and rectal tissues showed that a treatment with 1,500 mg of calcium carbonate and 400 IU of vitamin D3 per day significantly decreased the levels of expression of markers of cell proliferation in polyp tissues, which is an indicator of

tumor size reduction. In Europe, fortification of milk and other food products with vitamin D has been prohibited because of an increase in hypercalcemia cases in infants after World War II. However, in consideration that vitamin D deficiency is widely recognized as a cause of impaired bone formation in children, and that there are clear beneficial effects of the hormone in cases of cancer and osteoporosis, many European countries currently fortify margarine and cereals with vitamin D. Conclusions In vitro and in vivo studies on anti-cancer effects of 1,25D and synthetic analogs have indicated that the steroid has high potential for therapeutic applications in a wide range of cancers. Of special interest is the development of future combination therapies with other anti-tumor drugs with synergistic effects on cell growth. However, it is imperative that the precise molecular mechanisms by which 1,25D modulates cell proliferation and death be investigated. In addition, more clinical studies are needed in order to establish whether 1,25D and analogs can be used as an effective treatment of cancer.

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Vitamin D Receptor

References 1. Holt RR, Bresalier RS, Ma CK et al. (2006) Calcium plus vitamin D alters preneoplastic features of colorectal adenomas and rectal mucosa. Cancer. 106:287–296 2. Schwartz GG, Hanchette CL (2006) UV, latitude, and spatial trends in prostate cancer mortality: all sunlight is not the same (United States). Cancer Causes Control 17:1091–1101 3. Schwartz GG, Skinner HG (2007) Vitamin D status and cancer: new insights. Curr Opin Clin Nutr Metab Care 10:6–11 4. Van Leeuwen JPTM, Pols HAP (1997) Vitamin D: anticancer and differentiation. In: Vitamin D, Feldman D, Glorieux FH, Pike JW (eds) Academic Press, New York, pp 1089–1105 5. Wu W, Zhang X, Zanello LP (2007) 1α,25-dihydroxyvitamin D3 antiproliferative actions involve vitamin D receptor-mediated activation of MAPK pathways and AP-1/p21(waf1) upregulation in human osteosarcoma. Cancer Lett 254:75–86

Vitamin D Receptor Definition Member of the family of nuclear receptors transcription factors. The vitamin D receptor is activated upon binding of the active form of vitamin D to its ligand binding site. Once activated, the ligand-bound VDR interacts with a vitamin D responsive element in the DNA to modulate the expression of specific genes in target cells. ▶Vitamin D

Vitamin E Definition A fat-soluble antioxidant that is known to benefit human health. ▶Chemoprotectants

Vitiligo

precise pathophysiology of vitiligo is complex and not fully understood. Some evidence suggests that it is caused by a combination of autoimmune, genetic, and environmental factors. ▶Melanoma Antigens

Vitrification Definition In cryosurgery, pure water that shifts into a solid state. ▶Cryosurgery in Bone Tumors

VLP Definition

▶Virus-like particles

VM-targeted Therapy Definition Vasculogenic mimicry-targeted therapy; A totally different structure from endothelium-dependent vessels and it reveals several molecules for antitumor therapy. Therapeutic strategies that target endothelial cells have no effect on tumor cells that engage in VM. VMtargeting strategies include suppressing tyrosine kinase activity using a knockout EphA2 gene, downregulating VE-cadherin using antibodies against human ▶matrix metalloproteinases (MMPs) and the laminin 5γ2 chain, and using anti-▶PI3K therapy. ▶Vasculogenic Mimicry

VNTR

Definition Is a chronic skin condition that causes loss of pigment, resulting in irregular depigmented skin patches. The

Definition

▶Variable Number Tandem Repeats

von Hippel-Lindau Tumor Suppressor Gene

Volume of Distribution Definition The theoretical volume required to distribute a drug at a defined concentration, as measured in plasma, throughout the body. ▶Lead Optimization

von Hippel-Lindau ▶von Hippel-Lindau Tumor Suppressor Gene

Von Hippel-Lindau Disease Definition

▶von Hippel-Lindau Tumor Suppressor Gene

von Hippel-Lindau Tumor Suppressor Gene J OCHEN D ECKER 1 , H ILTRUD B RAUCH 2 1

Bioscientia Institute Center for Human Genetics, Ingelheim, and University of Mainz, Germany 2 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, and University Tuebingen, Stuttgart, Germany

Synonyms von Hippel-Lindau; VHL; tumor suppressor gene

Definition

The von Hippel-Lindau ▶tumor suppressor gene (VHL) is a cellular gene that is required for normal development, differentiation and cellular stress response (hypoxia, lack of glucose). VHL was discovered in families with the hereditary von Hippel-Lindau (VHL) syndrome by virtue of its two-hit mechanism

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of inactivation, and identified in 1993 following a positional cloning strategy. The VHL gene may be subject to mutation either in the germline giving rise to VHL disease or in somatic renal epithelial cells giving rise to sporadic renal conventional (clear cell) carcinoma (CCRCC) functioning as a ▶gatekeeper. VHL mutation patterns suggested two functional domains within the protein pVHL. Current knowledge indicates similarities to the SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complex that targets proteins for degradation. Therefore, pVHL may function as a molecular adaptor in a similar proteolytic pathway. Today’s best understood function of pVHL is its role in the VHL/HIF (▶Hypoxia inducible factor 1 α) pathway.

Characteristics Molecular Features . Gene located at 3p25.3, single copy locus: NCBI GenBank, range: bp 10.158.319–10.168.762, as published in Nature 431(7011):931–945 (2004) (Fig. 1) . 639 Nucleotides in three exons (originally reported sequence contained 852 nucleotides with 213 untranslated base pairs at the 5′ end) . Exon 1 is a CpG island (G + C content is 70%, CpG/ GpC > 1) . TATA-less and CCAAT-less promoter . Two transcription initiation codons (amino acid 1 and amino acid 54) . Follows a two-hit mechanism of inactivation characteristic for tumor suppressor genes . Evolutionarily highly conserved . No homologies known . The full length protein pVHL contains 213 amino acids . Known isoforms result from tissue specific and developmentally selective alternative splicing (skipping of exon 2) . Differentially phosphorylated at serin 68 by casein kinase 2 or glykogen synthase kinase 3 . Expressed in a variety of adult and fetal tissues including those of VHL target organs . There are two protein binding domains that allow pVHL to function as an adaptor molecule in a proteolytic pathway . The gene may be subject to mutations at almost any nucleotide of the 470 bp COOH terminal sequence . Phenotypic variation may result from confounding effects of modifier genes . No imprinting reported . Posttranslational negative regulation by E2-EPF UCP (E2-EPF ▶ubiquitin carrier protein) Structure of the von Hippel Lindau gene (VHL), and protein, indicating the functional domains. The pattern of germ line mutations published so far, reflects the functional importance of the binding sites to the target as well as to the multimeric protein complex.

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von Hippel-Lindau Tumor Suppressor Gene. Figure 1 Structure, Function, and Germ Line Mutations of the VHL gene.

Role in Diseases – Clinical, Molecular and Cellular Characteristics von Hippel-Lindau (VHL) disease (OMIM 193300) is an ▶inherited tumor susceptibility syndrome predisposing gene carriers to a variety of benign and malignant tumors. VHL segregates in affected families as an autosomal dominant inherited trait. Phenotypic expression is highly variable. Clinical and Molecular Features Lesions Associated with von Hippel-Lindau Disease. (Fig. 2) VHL patients may present with a variety of tumors affecting eye, central nervous system, inner ear, adrenal gland, kidney, pancreas, epididymis. Most frequent tumors include retinal angiomas, and ▶hemangioblastoma of the cerebellum and of the spinal cord which are usually benign. Other benign lesions include ▶pheochromocytoma, renal and pancreatic cysts. Renal clear cell carcinomas are malignant. . Tumors and cysts are frequently bilateral and/or multiple in origin.

. All ethnic groups are involved, there is no sex bias. . Birth incidence is estimated 1/39,000 (Germany) to 1/53,000 (East Anglia). . Prevalence is 1/85,000–1/31,000. . Incidence of de novo mutations is about 5% (up to 20%). . Mean age at diagnosis is 26 years. . Penetrance by age of 65 years more than 90%. . Most severe complications are hemangioblastomas due to unrestricted growth in the confined space of skull or vertebral canal, and CCRCC due to metastasis. . Major cause of death are hemangioblastomas. Classical Clinical Definition . Known family history of retinal or cerebellar ▶hemangioblastoma: the presence of a single hemangioblastoma or a visceral manifestation, i.e. diagnosis of a CCRCC in a family member will define this patient as a disease gene carrier. . Isolated cases, possibly indicating a de novo mutation: two or more hemangioblastomas (spinal,


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von Hippel-Lindau Tumor Suppressor Gene. Figure 2 Lesions associated with von Hippel-Lindau disease.

cerebellar or retinal) or a single hemangioblastoma in association with a visceral manifestation, i.e. CCRCC) are sufficient to establish the diagnosis. It is possible in most cases to determine VHL carrier status by mutation testing of DNA derived from nuclear blood cells. This can either be performed to assist clinical diagnosis or to establish carrier status presymptomatically in at risk individuals. Based on the presence or absence of pheochromocytoma, VHL disease is phenotypically subclassified into VHL type 1 (without pheochromocytoma, applies to the majority of all families) and VHL type 2 (with pheochromocytoma, about 7–20% of all families). The VHL type 2 syndrome is further subdivided into type 2A (without CCRCC), type 2B (with CCRCC), and type 2C (pheochromocytomas as the sole manifestation). In VHL type 2C disease, it is especially important to carefully establish the molecular diagnosis in affected patients since pheochromocytoma is also a manifestation of other inherited syndromes such as multiple endocrine neoplasia type 2, neurofibromatosis type 1, and others. Germline Mutations Comprehensive germline mutation analysis allows the detection of more than 95% of VHL predisposing mutations in gene carriers (Fig. 1). As of today as many as 1,000 VHL mutations have been described in data bases, of which some 140 have been described as unique mutations. About 50–60% are missense mutations, 20–30% large intragenic deletions, 12–20 microdeletions, or insertions and 7–11% nonsense mutations. Most mutations can be readily identified by sequencing analysis. Large genomic and intragenic deletions may be identified by Southern blotting including quantitative Southern blotting, pulsed field gel electrophoresis (PFGE) or/and fluorescence in situ hybridization

(▶FISH), and more recently by Q-RT-PCR (quantitative real time polymerase chain reaction) and MLPA (multiplex ligase probe amplification). In those rare cases where germline mutations escape detection it is possible that an affected individual may be a mosaic, with some cells carrying the VHL mutation and others that don’t. Although it may be difficult to find a VHL mutation in a mosaic individual, offspring are at high risk to develop the disease. Once the mutation is passed on into the next generation, due to affected germ cells, it should be easily identified in the affected offspring who now will carry the germline mutation in all of the body cells. Most germline mutations cluster within exon 1 and exon 3 suggesting two functional protein domains. In particular, there is a mutational hotspot affecting codon 167, either changing an arginine to tryptophane or to glutamine. The affected amino acid is located within a 35 residue domain necessary for ElonginC binding for the formation of the ternary pVHL/ElonginC/ElonginB complex (VCB). Another frequently identified mutation causes a change of histidine to tryptophane at codon 98. The frequency of this mutation also known as Black Forest mutation is due to a founder effect. The affected amino acid is located within the HIF binding domain. Other commonly described mutations are delPhe76, Asn78Ser, Arg161Stop, and Leu178Pro. A VHL mutation data collection is available at http:// www.umd.necker.fr (Beroud C, INSERM). A unique hematological hereditary disease has been identified as a subtype of VHL germline mutations (Type Chuvash, OMIM 263400). Specific homozygous missense mutations C598T (Arg200Trp), and C571G (His191Asp) present exclusively with clinical features of polycythemia vera with elevated ▶erythropoietin level. Interestingly, so far no other typical VHL complications have been seen in any carrier of these homozygous

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von Hippel-Lindau Tumor Suppressor Gene. Table 1 Lesions associated with von-Hippel-Linder Disease Affected Organs Eyes Central nervous system Adrenal glands Kidneys Pancreas

Inner ear Epididymis Broad ligament Liver

Clinical Symptoms

Frequency

Angiomatosis retinae Cerebellar hemangioblastomas Spinal hemangioblastomas ▶Pheochromocytomas Renal cysts Conventional (clear cell) renal cell carcinomas (CCRCC) Pancreatic cysts Serous cystadenomas Islet cell tumors usually asymptomatic Endolymphatic sac tumors Cystadenomas Benign adnexal papillary tumors Cysts

50–57% 55–59% 13–14% 7–19% 76% 24–52% 22% Occasionally Infrequent 150 μm away from ambient oxygen. By studying human and animal cancer tissues, Warburg reached the profound conclusion that tumors prefer to convert glucose to lactate and speculated that “If the respiration of a growing cell is disturbed, as a rule the cell dies. If it does not die, a tumor cell results.” Through the glycolytic pathway, normal tissues convert glucose to pyruvate and generate ATP and NADH. Pyruvate is taken up and then further metabolized by mitochondria through the Krebs or tricarboxylic acid (TCA) cycle. From a single pyruvate molecule, the TCA cycle donates eight high-energy electrons to the mitochondrial transport chain, which creates a proton gradient across the inner mitochondrial membrane, with oxygen as the terminal electron acceptor. Dissipation of the proton gradient produces ATP through ▶oxidative phosphorylation. In the absence of oxygen, pyruvate is instead converted to lactate. Cancer cells, in contrast to normal cells, tend to convert glucose directly to lactate even when oxygen is available (Fig. 1). Mitochondrial DNA Mutations and the Warburg Effect Warburg’s postulation that mitochondrial dysfunction or disturbance contributes aerobic glycolysis is now partially supported by studies of cancer mitochondrial

DNA (mtDNA) mutations. Mitochondrial components are encoded by both nuclear and mtDNA, whose mutations can be heritable through maternal transmission, resulting in specific muscle diseases, blindness or deafness. Acquired somatic mtDNA mutations have been discovered in large fractions of human tumors; however, the functional consequences of these mutations are not firmly established. While many mtDNA mutations have unknown effects, some mutations may diminish components of the respiratory chain thereby cause disturbances in respiration. Cell fusion experiments support a functional role of defective mitochondria, which enhance tumorigenicity of prostate cancer cells. Tumor Suppressors and the Warburg Effect Since the mitochondrion plays a central and crucial role in programmed cell death or ▶apoptosis, it is possible that acute disturbances of respiration could result in cell death as a means to rid the organism of defective cells. However, as proposed by Warburg, if a growing cell with disturbance in respiration does not die presumably as the result of activation of anti-apoptotic pathways, then a tumor cell results. Intriguingly, the tumor suppressor protein ▶P53, which plays an intimate role in ▶apoptosis, was recently shown to directly activate genes that enhance cellular respiration, such that loss of P53 favors the conversion of glucose to lactate and cell survival. In particular, ▶P53 activates Synthesis of Cytochrome c Oxidase 2 (SCO2) which is critical for positively regulating the cytochrome c oxidase (COX) complex, a major site of oxygen consumption by the mitochondrion. Additionally, P53 activates TIGAR, which shares sequence similarity with the bisphosphatase FBPase-2 and diminishes fructose-2,6-bisphosphate

Warburg Effect. Figure 1 The Warburg Effect. In contrast to normal cells that utilizes glucose through mitochondrial oxidative phosphorylation in the presence of oxygen (normoxia) and undergo anaerobic glycolysis in hypoxia, Warburg hypothesized that tumor cells with disturbances in respiration, presumably due to defective mitochondria, have the propensity to convert glucose to lactate even in the presence of oxygen. In tumor cells oxygen and pyruvate are presumably inefficiently utilized (dashed arrows) by the mitochondria. Gluc, glucose; Pyr, pyruvate.

Warburg Effect

levels, thereby blocks glycolysis. Hence loss of the tumor suppressor P53 would be expected to diminish respiration and unblock glycolysis. Several ▶familial cancer syndromes have been linked to mutations of tumor suppressors that are enzymes in the TCA cycle enzymes or proteins involved in metabolism. Mutations of succinate dehydrogenase are associated with paragangliomas, while mutations of fumarate hydratase are linked with ▶renal carcinoma and uterine leiomyomatosis. These mutations result in high levels of succinate, which inhibit prolyl hydroxylase (PHD) function resulting in increased stability of the hypoxia inducible factor HIF-1 protein. PHDs hydroxylate HIF-1 and target it for recognition by the von Hippel-Lindau protein (vHL) and subsequent proteasomal degradation. Hence inactivating mutations of vHL also stabilize HIF, which is important for tumorigenesis through ▶transcriptional regulation and stimulation of glycolysis and angiogenesis. These observations indicate that mutations of tumor suppressors may not only decrease mitochondrial function, but may also activate glycolysis directly or through the stabilization of HIF-1. Mutations of the LKB1 tumor suppressor result in the rare autosomal dominant form of Peutz-Jeghers syndrome that is characterized by early age onset of gastrointestinal polyposis. LKB1 is a kinase that phosphorylates and activates AMP kinase (AMPK) in the presence of AMP when intracellular levels of ATP are low. AMPK, in turn, increases ATP production through stimulation of pathways including fatty acid oxidation. AMPK also activates two other tumor suppressors, TSC1 and TSC2, which are mutated in the familial syndrome ▶tuberous sclerosis complex characterized by benign tumors commonly affecting the brain, but also affecting the kidneys, heart, eyes, lungs, and skin. TSC1 and TSC2 form heterodimers that inhibit ▶mTOR, the ubiquitous kinase which stimulates cell growth and protein synthesis in response to growth factors. Hence, loss of LKB1, TSC1 or TSC2, activates the mTOR pathway. Hyperactivation of the mTOR pathway through loss of tumor suppressors also appears to increase translation of HIF-1α mRNA and thereby stimulates the Warburg effect. Oncogenic Activation of the Warburg Effect In vivo and in vitro studies have demonstrated that many tumors are severely hypoxic due to a disordered, inefficient neo-vascular system. Hypoxia results in the stabilization of HIF, which in turn transcriptionally activates genes encoding glucose transporters, glycolytic enzymes, and angiogenic factors. As a result, tumor uptake of glucose is avid and lactate production is robust. It is apparent however, that not all areas of a tumor that are highly glycolytic are necessarily hypoxic, suggesting that cell autonomous oncogenic

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alterations could also cause the Warburg effect. In one scenario, oncogenes could stabilize HIF itself or alternatively directly inhibit cellular respiration or activate glycolysis. Since HIF-1 has been found in non-hypoxic areas of tumors, it is likely that HIF-1 could be stabilized by non-hypoxic factors, such as loss of vHL. HIF-1 levels are increased by insulin-like growth factor stimulation, activated v-▶Src, ▶c-Src or ▶Ras oncogenes. Signaling through the EGF receptor, ▶HER2, or ▶PI3 kinase also promote increased synthesis of HIF-1 protein. Another view contends, however, that the PI3-kinase/ AKT and HIF1 are independent pathways. Hence, oncogenic activation of a number of pathways could result in the stabilization of HIF-1 under normoxic conditions. However, oncogenic activation of glycolysis also occur independent of HIF. Additional insights into the Warburg effect are provided through a novel HIF-1 target gene that actively inhibits mitochondrial function. Pyruvate dehydrogenase kinase 1 (PDK1) is one of four PDK family members and was identified as a direct transcriptional target of HIF-1. PDK1 inactivates the mitochondrial pyruvate dehydrogenase (PDH) complex by phosphorylating the PDH E1α catalytic subunit. Suppression of PDH inhibits the conversion of pyruvate to acetyl-CoA, thereby decreases mitochondrial respiration. As such, it is expected that the non-hypoxic oncogenic stabilization of HIF would increase PDK1 levels, which divert pyruvate from PDH and enhance lactate production. These studies indicate that PDK1 activation could be a key regulatory switch contributing to the Warburg effect. The ▶MYC oncogene is over-expressed in 20–70% of commonly occurring human cancers (www.myccancergene.org). Myc directly binds and transactivates the promoters of several key glycolytic genes (HK2, ENO1, and LDHA), which have highly evolutionarily conserved Myc binding sites, as well as many other glycolytic genes and glucose transporters. The activation of glycolytic genes by Myc could be physiological or pathological under non-hypoxic conditions. It is notable that Myc is also implicated in stimulation mitochondrial biogenesis and hence could normally play a role in driving glucose metabolism toward oxidative phosphorylation in non-transformed cells. In tumor cells with defective or suppressed respiration, however, constitutive activation of Myc is expected to contribute to aerobic glycolysis by directly activating glycolytic genes. Hence, in certain tumors Myc may contribute to a cell autonomous state of converting glucose to lactate even in non-hypoxic conditions. The ▶AKT oncogene, which encodes a protein serine-threonine kinase, also activates a cell autonomous state of aerobic glycolysis in a seeming HIFindependent fashion through mobilization of glucose

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Warburg Effect

transporters to the cell surface, and activation of hexokinase 2 and phosphofructokinase 2. Intriguingly, Akt increases glycolytic conversion of glucose to lactate without an increase in oxidative phosphorylation. Similar to Bcl-XL, a ▶Bcl-2 family member, Akt is anti-apoptotic such that its ability to increase aerobic glycolysis is coupled with suppression of cell death. However, unlike Bcl-XL, Akt is able to transform cells. Hence, activation of Akt appears to be sufficient for the Warburg effect and tumorigenesis under certain conditions. The Warburg effect has also intriguingly emerged in a model of ▶tumor progression triggered by serial transduction of normal human cells with immortalizing and oncogenic genes. By multidimensional metabolic profiling of primary human fibroblasts transformed step-wise by ▶hTERT, ▶SV40 large T antigen, small T antigen, and H-Ras, the highly tumorigenic cells were observed to display the Warburg effect with high rates of lactate production and low mitochondrial mass. Cells transformed by the other three genes but lacking H-Ras had high mitochondrial mass and a high oxygen consumption rate with lower lactate production. These observations suggest that a certain stage in tumorigenesis could be associated with increased respiration, but H-Ras appear to be associated with the final conversion of human fibroblasts to a highly tumorigenic phenotype associated with aerobic glycolysis. In fact, Ras is implicated in the regulation of PFKFB3, a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which diminishes the intracellular concentration of the activator, fructose-2,6-bisphosphate (F2,6BP). Paradoxically, while increased PFKFB3 appears to be necessary for Ras transformation of fibroblasts, the associated diminished levels of F2,6BP, which allosterically activates the rate-limiting phosphofructokinase 1 does not appear to correlate with cellular transformation. In this regard, the effects of Ras on PFKFB3 contrasts with the activation of TIGAR by P53 discussed above. In summary, many studies support the activation of glycolysis and lactate production as an adaptive response to hypoxia as well as a result of direct oncogenic activation. Although, distinction between these two possible mechanisms of activation of tumor aerobic glycolysis is necessary for our understanding of tumor metabolism, it is the cell autonomous oncogenic activation of glycolysis that appears to substantiate Warburg’s original observations. Does the Warburg Effect Contribute to Tumorigenicity? If aerobic glycolysis mediated by the Warburg Effect is much less efficient for ATP production as compared to oxidative phosphorylation, then what are the selective pressures for tumor aerobic glycolysis? Hypoxia is pervasive in tumors in vivo and likely to be a selective pressure on cancer cells. Although independence

from oxygen requirement through the Warburg effect could be advantageous to a cancer cell, there must be additional advantages since an adaptive hypoxic response of activated glycolysis should also confer the same advantage to normal cells. Yet, hypoxia diminishes the growth potential of normal cells, except endothelial cells. Several glycolytic enzyme genes, including glucose phosphate isomerase (GPI) and phosphoglucomutase, were found capable of immortalizing primary fibroblasts in an expression cloning study. The associated increases in glycolytic rates are hypothesized to suppress mitochondrial reactive oxygen species production, which has been implicated in cellular senescence and apoptosis. In this regard, ▶immortalization by glycolytic enzymes could be a step in tumorigenesis that is poised for additional oncogenic conversions. It should be noted that there are non-glycolytic functions, such as the roles of LDH and GAPDH in transcriptional regulation, of these enzymes that may participate in tumorigenesis. The multi-functional roles of glycolytic enzymes, which include the anti-apoptotic roles of glucokinase and hexokinase through direct interactions with the mitochondria, could contribute to tumorigenesis. In addition, GPI is also known as autocrine motility factor that was isolated as a factor capable of stimulating motility of cells in an ▶autocrine or ▶paracrine fashion. The increased motility could contribute to the malignant phenotype of metastasis. How these non-glycolytic functions directly contribute to tumorigenesis remains to be further studied. Clinical Aspects and Summary The characteristics of cancer cells including the avid consumption of glucose and robust lactate production were first described by Warburg about 80 years ago. Over the past two decades, the molecular basis for tumorigenesis unfolded in the discovery of oncogenes and tumor suppressor genes that are now linked to altered glucose metabolism. While the analogies of a defective automobile brake to loss of tumor suppressor and a stuck accelerator to oncogene activation are touted as central mechanisms in tumorigenesis, the fuel line or tumor energy metabolism has been an overlooked, yet critical aspect of tumorigenesis. In fact, the avid uptake of glucose by tumors, observed by Warburg decades ago, is now the foundation for the detection and monitoring of human cancers by 2-fluorodeoxyglucose ▶positron emission tomography (FDG-PET). Although Warburg suggested that cancer cells underwent aerobic glycolysis as a result of mitochondrial disturbances, more recent studies suggest that both cell autonomous genetic alterations as well as adaptation to hypoxia through the stabilization of HIF-1 contribute to this characteristic tumor metabolic adaptation. This common metabolic perturbation of aerobic glycolysis or the

T1-Weighted

Warburg effect in cancers appears attractive as a therapeutic target pathway, although the true therapeutic window remains to be established.

References 1. Brandon M, Baldi P, Wallace DC (2006) Mitochondrial mutations in cancer. Oncogene 25:4647–4662 2. Green DR, Chipuk JE (2006) P53 and metabolism: inside the TIGAR. Cell 126:30–32 3. Kim JW, Dang CV (2006) Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 66:8927–8930 4. Shaw RJ (2006) Glucose metabolism and cancer. Curr Opin Cell Biol 18:598–608 5. Warburg O (1956) On respiratory impairment in cancer cells. Science 124:269–270

Warts (wts) – Drosophila lats ▶Lats in Growth Regulation and Tumorigenesis

WARTS (WTS) – mammalian LATS1

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Many of the proteins containing WD repeats are subunits of protein complexes. WD proteins commonly have 4–16 repeating WD units. WD proteins are a large family found in all eukaryotes and are implicated in a variety of functions, such as signal transduction, regulation of transcription, cell cycle control, and apoptosis. The underlying function of WD proteins is the assembly of multimeric protein complexes with the repeating units serving as a rigid scaffold for protein interactions that are mediated by non-WD repeat regions. The specificity of the protein’s function is usually determined by its sequence that lies outside of the tandem repeats. ▶Molecular Chaperones

Wegener Granulomatosis Definition An uncommon disease occurring mainly in the fourth and fifth decades that is characterized by vasculitis of small vessels, granuloma formation in the respiratory tract, and glomerulonephritis, possibly caused by an immune disorder. ▶Rituximab

▶Lats in Growth Regulation and Tumorigenesis

T1- and T2-Weighted Images N-WASp Definition Definition Neuronal-▶Wiskott-Aldrich Syndrome protein is expressed in most tissues. It the main protein in cells that binds and activates the Arp2/3 complex to initiate actin polymerization and cell movement.

Two types of the images in the magnetic resonance imaging with different length of repetition time (TR) and echo time (TE). ▶Hepatic Epithelioid Hemangioendothelioma

▶Cortactin

T1-Weighted WD Repeats

Definition

Definition

Magnetic resonance imaging that predominantly reflects the longitudinal (spin – lattice) relaxation time of tissues.

A motif of approximately forty amino acids that commonly ends with a tryptophan and aspartic acid dipeptide. These motifs often occur as tandem repeats.

▶Dynamic Contrast-enhanced Magnetic Resonance Imaging

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T2-Weighted

T2-Weighted Definition Magnetic resonance imaging that predominantly reflects the effective transverse (spin – spin) relaxation time of tissues accounting for local magnetic field in homogeneity. ▶Dynamic Contrast-enhanced Magnetic Resonance Imaging

Whipple Triad Definition

Is the diagnostic hallmark of ▶insulinoma; which consists in symptoms of hypoglycemia (catecholamine release), low blood glucose level (40–50 mg/dL), and relief of symptoms after intravenous administration of glucose. The triad is not entirely diagnostic, because it may be emulated by factitious administration of hypoglycemic agents, by rare soft tissue tumors, or occasionally by reactive hypoglycemia. The clinical syndrome of hyperinsulinism may follow one of two patterns or sometimes a combination of both.

Well-differentiated Neurocytoma White Blood Cell ▶Neurocytoma

Definition

The two most common types are the ▶lymphocytes and ▶neutrophils.

Wermer syndrome

▶Leukocytes

▶Multiple Endocrine Neoplasia Type 1

WHO Definition

Wharton Jelly

World Health Organization.

Definition

Connective tissue surrounding the ▶umbilical cord.

WHO Grade IV Astrocytoma

▶Stem Cell Plasticity ▶Glioblastoma Multiforme

Whey Acidic Protein

Wide Excision

Definition

Definition

WAP; is the principal whey protein found in rodent milk.

Tumor surgery by which the excision takes place at 2 cm outside the pseudo capsule of the tumor.

▶Cripto-1

▶Cryosurgery in Bone Tumors

Wilms Tumor

Wild-type Definition Refers to the most prevalent gene variant (encoding a protein, or an active enzyme) in the population. If a variant is predominant in one population, but has a low frequency in another population, the terminology usually refers to the population in which the variant was first described. For this reason, it is advisable to refrain from using this terminology on its own because it may easily create confusion in a number of cases.

Wilms Tumor K ATHY P RITCHARD -J ONES Institute of Cancer Research/Royal Marsden Hospital, Sutton, Surrey, UK

Definition Wilms tumor (synonym nephroblastoma) is a childhood embryonal cancer of the kidney. It was named after a German physician, Max Wilms, who described the first large collection of cases of this tumor in a paper published in 1899. The term Wilms tumor used to be applied to other types of childhood kidney cancer (clear cell sarcoma of kidney and malignant rhabdoid tumor of kidney) but these are now recognized as distinct entities with different clinical behavior and requiring different treatments.

Characteristics How Common is Wilms Tumor? Wilms tumor affects 1 in 10,000 children before their fifteenth birthday, typically at around age 3–4 years. Ninety percent of cases will have been diagnosed before the age of 7 years. There is a degree of ethnic variation; it is commoner in blacks and relatively rare in Asians. Usually only one kidney is affected, but in 5–8% of cases there are tumors in both kidneys (bilateral disease). What Causes Wilms Tumor? Although the majority of cases of Wilms tumor are “sporadic” with no obvious cause, it is known that genetic predisposition to Wilms tumor can occur. One to two percent of affected children inherit a defective gene from a parent. There are at least four such “familial Wilms tumor genes,” only one of which has been identified to date. More commonly, in 5% of all cases,

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a child has a developmental defect associated with genetic predisposition to Wilms tumor and sometimes other forms of cancer. The first of these to be defined at a molecular level was the association of Wilms tumor with aniridia, a lack of development of the iris in the eye. Such children usually have other defects including abnormal genital development and variable mental retardation, and hence the acronym, WAGR syndrome (for Wilms tumor, Anividia, Genitourinary malformations, and mental retardations). This defect is due to a chromosomal deletion that deletes one copy of the ▶Wilms tumor gene, WT1, and the adjacent ▶PAX6 gene at 11p13. Loss of one allele of PAX6 is dominant, but development of the tumor requires loss or mutation of the remaining WT1 allele in one or more kidney cells (i.e. tumor development is recessive, hence WT1 belongs to the class of ▶tumor suppressor genes). Heritable mutations in WT1 are also responsible for the predisposition to Wilms tumor associated with abnormal kidney development seen in ▶Denys-Drash syndrome, where children lose large quantities of protein in their urine (nephrotic syndrome) and often have abnormal genital development. Thus, Wilms tumor provides a fascinating example of how normal development of an organ can be intimately linked to cancer predisposition in that organ. Indeed, Wilms tumor can bear an uncanny resemblance to cell types seen during normal embryonic kidney development, hence the term “embryonal tumor.” There are an increasing number of children recognized with nephrotic syndrome with underlying germline WT1 mutations who do not develop Wilms tumor. Hence, it is possible that inheriting a mutation in WT1 provides only a relatively weak stimulus to developing Wilms tumor (i.e. the gene is of low ▶penetrance). A second category of genetic predisposition to Wilms tumor occurs in the overgrowth syndromes of childhood, of which ▶Beckwith-Wiedemann syndrome (BWS) is the best recognized. The genetics of BWS are complex, involving several different genes within the 11p15.5 chromosomal locus and the phenomenon of ▶genomic imprinting, whereby expression of a gene depends from which parent it was inherited. The overall tumor risk is 10%, of which half are Wilms tumors. It appears that children with early nephromegaly (i.e. overgrowth of the kidneys) or asymmetrical overgrowth (hemihypertrophy) are at greatest risk. Finally, there is some evidence from case control studies that the risk of Wilms tumor may be somewhat increased by certain parental occupations or exposures. However, the relative risk to the child is usually small, of the order of two- to tenfold. Clinical Characteristics Wilms tumor is one of the most curable of childhood cancers, even when it has spread beyond the kidney to distant sites. The commonest site of such metastases

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Wilms Tumor

is the lung, followed by lymph nodes and liver. Wilms tumor rarely metastasizes to bone, bone marrow or brain. The treatment consists of chemotherapy with one to three different drugs (usually ▶vincristine, ▶actinomycin D+/– ▶adriamycin) together with surgical excision of the affected kidney. Radiotherapy is also used when there is residual or spilt tumor in the abdomen or metastasis to the lungs. With these regimens, ~90% of children with a tumor confined to the kidney (stages I and II) are cured, as are over twothirds of patients with metastatic disease (stage IV). There is a different philosophical approach to the organization of treatment between different national and international childhood cancer study groups. The National Wilms Tumor Study Group (▶NWTSG) of North America favors immediate surgical excision of the affected kidney, followed by chemotherapy with or without radiotherapy, according to the tumor extent found at the time of surgery. The approach of the International Society of Paediatric Oncology (▶SIOP) is to use pre-operative chemotherapy to shrink the tumor prior to surgery. This study showed a reduction in the risk of tumor rupture during operation and also reduction in the tumor stage, hence allowing less intensive post-operative treatment. The two groups have comparable cure rates, but the NWTSG approach uses slightly more radiotherapy whereas the SIOP approach uses more anthracyclines (adriamycin). Both these treatments have the potential for long term side effects on growth and fertility and on the heart muscle, respectively. The majority of children with Wilms tumor are cured without the need for either of these agents and are unlikely to suffer any long term sequelae. Is Wilms Tumor One Disease? As with most cancers, various “prognostic factors” can be recognized in Wilms tumor. The most obvious adverse factor is increasing tumor ▶stage. However, a distinct histological subtype called ▶Wilms tumor anaplasia carries a poor prognosis, especially when associated with advanced stage disease. Anaplasia is associated with mutations in the ▶p53 gene, which can occur focally as part of clonal evolution of a tumor. Other molecular characteristics that may be associated with worse outcome are allele loss or ▶loss of heterozygosity for markers on chromosome 16q, 1p and possibly 22q and 11q. Some of these are being tested prospectively in the current NWTS 5 clinical trial, which aims to use molecular characteristics of a tumor to better define risk groups. Molecular Characteristics of Wilm Tumor As soon as the WT1 gene was isolated in 1990 it became clear that it was not mutated in the majority of sporadic Wilms tumors. Among over 600 published cases analyzed for intragenic mutations, WT1 mutation

occurs in only 10%. In some tumors, mutation of both WT1 alleles appears to be sufficient for tumorigenesis, in accordance with ▶Knudson hypothesis of two hits. However, in other tumors, either only one WT1 allele is mutated or other genetic events are clearly interacting. The molecular biology of the WT1 protein is fascinating and has led to insights into both tumor and normal development and their inter-relationship. Although it is known that several different genetic loci exist for other Wilms tumor genes, their relative contribution to sporadic Wilms tumor is not yet known. A database of WT1 mutations is maintained (http:// www.umd.necker.fr). Treatment of Relapsed Wilms Tumor and Future Therapeutic Possibilities Although Wilms tumor is one of the most curable of childhood cancers at initial diagnosis, those cases that relapse carry a much worse prognosis, even with intensive retreatment. Less than a third of relapses are due to the anaplastic variant. Hence, one of the goals of current Wilms tumor clinical studies is to identify factors present at diagnosis that are predictive of outcome. Future treatment intensity could then be stratified according to predicted risk of relapse using molecular characteristics of individual tumors. Another potential avenue is to use knowledge of the biology of the Wilms tumor genes to devise novel therapeutic approaches. In the future this might also lead to preventative strategies for children at increased genetic risk of Wilms tumor. Heritability and Screening By clinical criteria it appears that 3–4 months. However, there are no definitive clinical trials to determine which screening method or interval is superior for detecting tumors at a low stage (I or II). In the future, if a larger proportion of children are shown to be carriers of one of the familial Wilms tumor genes, then more information should become available on the efficacy of screening. A further benefit of the application of molecular genetics is that children with germline WT1 mutations appear to be at risk of late renal failure and hence require appropriate monitoring. ▶Nephroblastoma

References 1. Hastie ND (1994) The genetics of Wilms’ tumor – a case of disrupted development. Annu Rev Genet 28:523–558 2. Pritchard-Jones K (1997) Molecular genetic pathways to Wilms tumour. Crit Rev Oncog 8:1–27 3. Rahman N, Arbour L, Houlston R et al. (2000) Penetrance of mutations in the familial Wilms tumour gene, FWT1. J Natl Cancer Inst 92:650–652 4. Hawkins MM, Winter DL, Burton HS et al. (1995) Heritability of Wilms’ tumour. J Natl Cancer Inst 87:1323– 1324 5. Tournade MF, Com-Nougue C, Voute PA et al. (1993) Results of the Sixth International Society of Paediatric Oncology Wilms Tumour Trial and Study: a risk-adapted therapeutic approach in Wilms tumour. J Clin Oncol 11:1014–1023 6. Green DM, Breslow NE, Beckwith JB et al. (1998) Effect of duration of treatment on treatment outcome and cost of treatment for Wilms tumour: a report from the National Wilms Tumour Study Group. J Clin Oncol 16:3744–3751

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marker of cellular resistance to therapy but not of increased tumor aggressiveness as anaplasia in stage I tumors has no adverse prognostic significance.

Wilms Tumor Suppressor Gene Definition WT1 gene; Is located at 11p13 and consists of ten exons spanning approximately 50 kb of genomic DNA. The gene encodes a zinc finger protein that has two alternative splice sites affecting the whole of exon 5 and a three amino acid insertion (KTS) between the third and fourth zinc fingers. The WT1 protein is multifunctional and the various isoforms can act as transcription factors or may be involved in RNA processing or splicing. WT1 is essential for formation of the kidney and gonad in the mouse. There is a genotype-phenotype correlation, with germline missense mutations as seen in ▶Denys-Drash syndrome having a more pronounced effect on genitourinary development than complete deletion of one allele, as seen in WAGR syndrome. Germline intronic mutations affecting splicing of the KTS linker also act in a dominant fashion on genitourinary development. ▶Wilms Tumor

WIN ▶Forkhead Box M1

Wilms Tumor Anaplasia Definition

The term “anaplasia” in ▶Wilms tumor is used to describe a histological pattern defined as the presence of all of the following three features: . Cells with a nuclear diameter at least three times of adjacent nuclei of the same cell type; . Marked hyperchromatism of these cells, indicative of increased chromosome numbers; . Abnormal mitotic figure. ▶Anaplasia may be focal or diffuse and is associated with mutation of the ▶p53 gene. Anaplasia is felt to be a

Wiskott-Aldrich Syndrome Definition

WAS; is a rare X-linked ▶recessive disease with variable expression, but commonly includes immunoglobulin M (IgM) deficiency. WAS always causes persistent ▶thrombocytopenia (low platelet counts), and, in its complete form, also causes small ▶platelets, atopy, cellular and humoral immunodeficiency, and an increased risk of ▶autoimmune disease and ▶hematologic malignancies. The exact function of the WAS protein is not fully elucidated, but it seems to function

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Withaferin A

as a bridge between signaling and movement of the actin filaments in the cytoskeleton. WAS is also sometimes called the eczema-thrombocytopenia-immunodeficiency syndrome.

other indigenous medical practices. A structural analysis of WA indicates that it is a highly oxygenated C-28 ergostane-type steroid with a 22, 26-lactone and a 1-oxo-group (Fig. 1).

Characteristics

Withaferin A C HENDIL DAMODARAN Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky, USA

Synonyms WA

Definition The natural product Withaferin A (WA) is an important bioactive component of Withania somnifera (WS), a medicinal plant of the Solanaceae family that is used in the Indian Ayurvedic medical system, as well as many

WA has been used in several ethnobotanical medical practices (▶Natural products) to treat a variety of ailments, including cancer, inflammatory conditions, and cardiac and neurological disorders (Fig. 1). With respect to cancer, seminal in vivo and in vitro studies have revealed that WA possesses chemopreventive, chemotherapeutic, and radiosensitizing character (▶Adjuvant therapy). The chemopreventive properties of WA were first identified in a Swiss albino mice tumor model, and were subsequently confirmed in mice using Withania spp. root extracts, as well as several withanolides isolated from these extracts. Similarly, early studies also revealed the chemotherapeutic utility of WA, as it potently inhibited mouse Ehrlich ascites carcinoma in vivo and induced G2/M arrest (▶G2/M Transition) of the cell cycle in Chinese Hamster V79 cells. This WA-mediated G2/M arrest has been shown to result in significant radiosensitization in cell culture

Withaferin A. Figure 1 The diverse spectrum of Withaferin-A activity.

Withaferin A

models, as well as in mouse fibrosarcoma and melanoma when administered prior to radiotherapy. Notably, when used in combination with radiotherapy in vivo, WA increased tumor cure and disease-free survival. Several reports substantiate these findings and support the activity of WA against various cancer models, including prostate, colon, breast, and lung cancer. The mechanistic details of WA anti-cancer activity have been extensively studied in prostate cancer (▶Prostate cancer, clinical oncology) models in vitro and in vivo. WA induces ▶apoptosis in androgen refractory prostate cancer cells, and causes regression of PC-3 xenografts in mouse models. These studies also reveal that WA alone fails to induce apoptosis in androgen-responsive prostate cancer, but the combination of WA and an anti-androgen induces apoptosis in androgen-responsive cells (▶Androgen receptor). Published reports from several labs confirm these selective effects of WA on prostate cancer. Given that androgen-responsive prostate cancer often progresses to an androgen-refractory phenotype, which is resistant to current therapeutic approaches, these findings are significant. WA targets multiple signaling pathways in prostate cancer cells to induce its apoptotic effects (Fig. 2). In

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prostate cancer cells, WA up-regulates the gene expression of prostate apoptosis response-4 (Par-4), which induces p53- and phosphatase and tensin homolog (PTEN)-independent cancer-selective apoptosis. The pro-apoptotic effects of WA are dependent on Par-4 expression and action. In normal and malignant cells, Par-4 is phosphorylated and inactivated by Akt (also known as Protein Kinase B), which is an evolutionarily conserved regulator of the cell cycle. However, as shown in prostate, breast, and lung cancer cells, WA inhibits Aktmediated pro-survival signaling (▶BCl-2) and initiates G2/M cell cycle arrest (unpublished data). WA also negatively regulates NFκB activity in variety of cancer cell lines. NFκB is a transcription factor, a regulator of key cellular processes, such as apoptosis, immunoresponse, differentiation, and proliferation, and its constitutive activation has been linked to cancer. Under normal conditions, NFκB activity is held in check in the cytoplasm by IκB. Upon phosphorylation of IκB by IκB kinase (IKK), NFκB translocates to the nucleus to effect gene transcription. Importantly, WA inhibits IκB kinase activity, thereby blocking NFκB nuclear translocation, and targets various cysteine residues of multiple kinases and phosphotases.

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Withaferin A. Figure 2 Withaferin-A mechanism of action in prostate cancer.

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mMEN1

As a consequence, WA alters the phosphorylation status of p38, MEK/ERK, JNK, and IKKβ, thereby inhibiting the pro-survival machinery and triggering apoptosis. Beyond WA effects on Par-4 and pro-survival signaling pathways (▶Apoptosis Signaling), it has been reported that WA “function both as a ▶proteasome” and ▶angiogenesis inhibitor. The proteasome is a proteinase complex that selectively degrades ubiquitinated proteins, and is considered to be a potential target for cancer chemotherapy. WA targets the proteasome β5 subunit, inhibiting cell survival in vitro and tumor growth in vivo. Structural modeling analysis indicates WA contains two conjugated ketone bonds that target the hydroxyl group of the N-terminal threonine of the proteasomal chymotrypsin subunit β5. With respect to angiogenesis, a recent study demonstrates that WA binds to annexin II to promote F-actin polymerization and growth inhibition in Prostate cancer cells. This observation complements a study that shows WA inhibits angiogenesis through its ability to negatively regulate NFκB signaling, in addition to ▶cyclin D1. Although preclinical studies reveal that WA impacts multiple signaling cascades associated with cancer cell proliferation and survival, careful analyses are needed to establish the clinical merit of WA in treating human cancers. The precise mechanism by which WA elicits such diverse biological effects remains unclear, thus additional studies are required to identify unknown targets of WA, and clarify the molecular nexus that directs this broad spectrum of WA activity.

References 1. Shohat B et al. (1976) The effect of withaferin A on human peripheral blood lymphocytes. An electron-microscope study. Cancer Lett 2(2):63–70 2. Srinivasan S et al. (2007) Par-4-dependent apoptosis by the dietary compound withaferin A in prostate cancer cells. Cancer Res 67(1):246–53 3. Kaileh M et al. (2007) Withaferin A strongly elicits I {kappa}B kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 282(7):4253–4264 4. Yang H, Shi G, Dou QP (2007) The tumor proteasome is a primary target for the natural anticancer compound Withaferin A isolated from “Indian winter cherry”. Mol Pharmacol 71(2):426–437

mMEN1 ▶Multiple Endocrine Neoplasia Type 1

Wnt Definition The name comes from the Drosophila gene wingless and the proto-oncogene int-1. The wnt/β-catenin pathway is highly conserved and regulates gene expression by ▶TCF/LEF transcription factors. The pathway is of importance in embryonic development, stem cell differentiation, cell death, and tumor progression. ▶Wnt Signaling

Wnt/beta-Catenin Pathway Definition In the canonical Wnt/beta-catenin pathway, Wnt ligand binding to its cell surface receptor triggers changes of cytoplasmic effector activities and stabilization of betacatenin protein. Beta-catenin then accumulates in the nucleus where it interacts with ▶high mobility group (HMG) box transcription factors to regulate gene expression. ▶APC/β-Catenin Pathway ▶Wnt Signaling

Wnt Signaling R OBERT M. K YPTA Cell Biology and Stem Cells Unit, CIC bioGUNE, Derio, Bilbao, Spain; Imperial College London, London, UK

Definition

The name ▶Wnt comes from wingless (Wg) and Int-1. Wingless is a Drosophila gene important during development and Int-1 is a gene into which the mouse mammary tumor virus (MMTV) integrated to cause tumors. Wg and Int-1 were subsequently found to be related in sequence. Wnt signaling is the result of binding of Wnt proteins to cell surface receptors. Wnt family members exist in all multicellular organisms.

Wnt Signaling

Characteristics Wnt Proteins Vertebrate Wnts comprise a family of 19 hydrophobic cysteine-rich secreted glycoproteins of ~350 amino acids in length. Their hydrophobicity stems from lipid modification – cysteine palmitoylation and serine palmitoleoylation – that is essential for signaling activity and secretion. Wnt proteins are quite insoluble and associate with ▶HSPGs (heparan sulfate proteoglycans) in the extracellular matrix, so they often signal only over short distances. Long-range Wnt signaling does take place, however, for example, when Wnts form gradients to pattern developing tissues. Long-range Wnt signaling is mediated by ▶lipoprotein particles and facilitated by the ▶retromer. Wnt signals can elicit a number of cellular responses including proliferation, differentiation and migration (▶Migration). The nature of the response is dictated by the responding cell and the specific Wnts and Wnt receptors present. Mutations of Wnt genes are rare and have not been found in human tumors. However, Wnt gene expression patterns are often altered in tumor cells and inhibition of Wnt activity using antibodies or siRNA (▶siRNA) induces apoptosis (▶Apoptosis) in some tumor cell types (see Table 1). Both increases and decreases in Wnt5a expression have been observed in cancer, perhaps because Wnt5a plays different roles in different tissues or at different stages during tumor progression. Wnt Signaling Pathways Wnt proteins normally bind to cell-surface receptors of the frizzled (▶FZD) family, which activate intracellular disheveled (▶DVL) family proteins. At this stage the Wnt signals bifurcate and can activate distinct pathways. The best understood of these is the Wnt/ ▶β-catenin pathway, also referred to as the canonical pathway (▶APC/β-catenin pathway) (Fig. 1). In this pathway, Wnt proteins also bind to ▶LRP5/6, which interact with the β-catenin-binding protein ▶Axin. In the absence of Wnts, a so-called β-catenin destruction complex controls the level of β-catenin in the

Wnt Signaling. Table 1 Wnt Wnt1

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cytoplasm. This complex, which includes Axin, glycogen synthase kinase-3(▶GSK-3), casein kinase-I (▶CKI) and adenomatous coli polyposis protein (APC) (▶APC in familial adenomatous polyposis), facilitates β-catenin phosphorylation, ubiquitination (▶Ubiquitination), and ultimately degradation in the ▶proteasome. Canonical Wnt signals disrupt this complex by eliciting changes in Axin localization and stability, resulting in the stabilization of β-catenin in the cytoplasm. Stabilized β-catenin enters the nucleus and binds to transcription factors, primarily of the ▶Tcf/ LEF family, thereby regulating gene expression. The Wnt/β-catenin pathway is permanently active in many tumor types, in particular in colon cancer (▶Colon cancer), as a result of mutations in APC, β-catenin or Axin. In rare cases, inactivation of Wnt/β-catenin signaling leads to tumor formation, for example, in sebaceous tumors with LEF-1 mutations (Table 2). Non-canonical Wnt signals do not stabilize β-catenin. Instead, they overlap with other known signaling pathways. The planar cell polarity (▶PCP) pathway is a noncanonical pathway that was first defined in Drosophila, where it controls the uniform orientation of hairs and bristles on the body. PCP signaling has also been studied extensively in frogs and zebrafish, where it regulates ▶convergent extension. In mammals, the best example of PCP is the uniform orientation of the hair cell stereociliary bundles within the cochlea. PCP pathway proteins include FZD, DVL and several proteins not involved in other Wnt signaling pathways; those relevant to cancer include ▶VANGL, which is overexpressed in tumors and promotes metastasis (▶Metastasis), and PTK7, a catalytically inactive transmembrane tyrosine kinase (▶Receptor tyrosine kinase) that is overexpressed in tumor-derived cell lines. The PCP pathway activates the small GTPases Rac and Rho (▶Rho family proteins) and the protein kinases c-Jun NH2-terminal kinase (▶JNK) and Rho-associated kinase (▶ROCK) to induce changes in the cytoskeleton. Whether Wnt ligands activate the PCP pathway is still unclear; there is no strong evidence for this in flies or humans, but mutations

Examples of Wnt genes with altered expression in cancer

Tumor type Several

Wnt2

Mesothelioma (mesothelioma), melanoma (melanoma) Wnt5a Gastric cancer, melanoma, leukemia Wnt16 Leukemia, basal cell carcinoma

Change in expression Increase Increase Increase (gastric cancer, melanoma) decrease (leukemia) Increase in expression of alternativelyspliced isoform b

Comments Anti-Wnt1 antibody induces apoptosis Anti-Wnt2 antibody and siRNA apoptosis Increase probably linked to metastasis Anti-Wnt16 antibody induces apoptosis

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Wnt Signaling

Wnt Signaling. Figure 1 A simplified view of Wnt/β-catenin signaling. In the absence of Wnt (left), a protein complex that includes Axin, APC, GSK-3 and CK-Iα promotes the phosphorylation, ubiquitination and subsequent degradation of β-catenin in the proteasome; Tcf/LEF proteins associate with Groucho to repress target gene expression. On the right, Wnt binds to FZD and LRP5/6 receptors resulting in DVL and LRP5/6 phosphorylation and Axin recruitment/degradation. This allows accumulation of β-catenin, which then enters the nucleus and associates with Tcf/LEF to activate gene expression. For simplicity several other components of the pathway are not shown.

Wnt Signaling. Table 2 Intracellular component APC β-catenin

Axin

LEF-1

Wnt/β-catenin pathway mutations in cancer Common tumor types Colon cancer Liver cancer Ovarian cancer Colon cancer Medulloblastoma Liver cancer Colon cancer Sebaceous tumors

in Wnt5 and Wnt11 disrupt convergent extension in zebrafish. In the Wnt-calcium (Ca2+) pathway, binding of Wnt to FZD leads to activation of ▶G proteins that then

Effect of mutations Stabilization of β-catenin Stabilization of β-catenin

Stabilization of β-catenin

Reduced Wnt/β-catenin signaling

activate phospholipase C and phosphodiesterase, leading to increased concentrations of free intracellular Ca2+. These events result in activation of ▶protein kinase C (PKC) and Ca2+/calmodulin-dependent

Wnt Signaling

protein kinase II (CaMKII) and the Ca2+-sensitive protein phosphatase ▶calcineurin. PKC and calcineurin directly and indirectly regulate various transcription factors such as nuclear factor of activated T cells (NFAT). The Wnt-Ca2+ pathway has been studied extensively in frogs and zebrafish, where it can be activated by Wnt4, Wnt5a and Wnt11 (Fig. 2). There is increasing evidence for the involvement of heterotrimeric ▶G-protein α subunits in canonical and noncanonical signaling pathways. For example, Gαo and Gαt2 participate in Wnt/Ca2+ signaling by activating a phosphodiesterase that lowers cGMP levels, thereby inactivating protein kinase G; Wnt3a signals through Gαq to activate PKCδ; and prostaglandin E2 receptors stimulate proliferation of colon cancer cells through the β-catenin pathway by association of Gαs with ▶Axin.

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The existence of Wnt-Ca2+ and Wnt/G protein signaling pathways in mammalian cells is largely supported by overexpression studies using cell lines and still requires confirmation using genetic approaches. Just how important non-canonical Wnt signaling is in cancer is still not clear. Wnt5a is probably the most studied non-canonical Wnt and its roles in cancer are complex. For example, Wnt5a expression is reduced in human leukemia and Wnt5a heterozygous mice (▶Haploinsufficiency) develop B-cell lymphoma. In contrast, the expression of Wnt5a is increased in gastric cancer (▶Gastric cancer), where it correlates with tumor aggressiveness. Wnt5a is likely to have more than one function, controlling cell proliferation and/or differentiation in some tissues and promoting cell migration in others. At the molecular level these differences might

W Wnt Signaling. Figure 2 A simplified view of Wnt/β-catenin-independent signaling. In the Wnt-Ca2+ pathway, Wnt binding to FZD activates G proteins, thereby activating a number of downstream signals. The PCP pathway involves activation of FZD and DVL, which together with several additional proteins including VANGL and PTK7, affect Rac/Rho signaling pathways. See text for more details. For simplicity several other components of the pathway are not shown.

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Wnt Signaling

Wnt Signaling. Table 3 Ligand

Receptor

Wnt ligands Wnt FZD (several) Wnt LRP5/6 (several) Wnt5a Ror2 Wnt Ryk (several) Non-Wnt ligands sFRP (FZD family family) DKK LRP5/6 family WIF1 – Norrin FZD4 Sclerostin LRP5/6 RLRP6, spondin FZD8

Examples of ligands and receptors involved in Wnt signaling Key events

FZD recruits DVL LRP5/6 recruits Axin Inhibits Wnt/β-catenin signal Ryk also binds FZD8 and DVL; activates Wnt/β-catenin signal

Examples of changes in cancer FZD7 overexpressed in hepatocellular carcinoma (▶hepatocellular carcinoma) – – Ryk overexpressed in ovarian cancer (▶ovarian cancer)

Inhibit Wnt signals by binding to Wnt Reduced expression (CpG methylation) (▶CpG islands) Inhibit Wnt/β-catenin signal

Reduced expression (CpG methylation)

Inhibits Wnt signals by binding to Wnt Activates Wnt/β-catenin signal Inhibits Wnt/β-catenin signal Activates Wnt/β-catenin signal

Reduced expression (CpG methylation) – – Reduced expression

be explained by the ability of Wnt5a to activate discrete signaling pathways through distinct receptors. For example, in transfected cells Wnt5a inhibits Wnt/βcatenin signaling by binding to the tyrosine kinase Ror2 and activates Wnt/β-catenin signaling by binding to FZD4. There are also examples of canonical Wnts that activate β-catenin-independent signals, such as Wnt3a activation of PKCδ. Moreover, although Wnt1 is best known as an activator of Wnt/β-catenin signaling, antiWnt1 antibodies can induce apoptosis in cells that do not express β-catenin, suggesting that Wnt1 noncanonical signals are important for cancer cell survival. Other Ligands and Receptors that Directly Regulate Wnt Signaling There are several other proteins that modulate Wnt signaling by binding to Wnts themselves, binding to Wnt receptors or by acting as Wnt receptors (Table 3). Most of the secreted proteins are so-called Wnt antagonists that normally inhibit Wnt signals. The secreted frizzled related protein (▶sFRP) family and Wnt inhibitory factor-1 (WIF1) have the potential to inhibit all Wnt signals, since they interact with Wnt proteins themselves. sFRP proteins also bind to FZD receptors and in some cases have been shown to augment rather than inhibit Wnt/β-catenin signaling. The expression of sFRP proteins and WIF1 is downregulated in many tumors as a result of promoter methylation. In contrast to sFRP proteins, dickkopf (▶DKK) family members and Sclerostin interact with LRP5/6 and so are thought to inhibit only Wnt/β-catenin signaling. The

expression of DKK family members is often reduced in tumors as a result of promoter methylation. However, DKK1 is highly expressed in myeloma (▶Multiple myeloma), where it contributes to osteolytic bone disease by inhibiting the differentiation of osteoblasts. Although DKK1 and DKK4 inhibit Wnt/β-catenin signaling, DKK2 can activate Wnt/β-catenin signaling, while DKK3 does not directly affect Wnt signaling at all since it cannot bind to LRP5/6. DKK1 is itself a β-catenin/Tcf target gene that provides a negative-feedback loop for Wnt/β-catenin signaling. Finally, Norrin and R-spondin are secreted proteins that activate Wnt/β-catenin signaling. Examples of proteins that act as Wnt receptors are Ryk, a catalytically inactive receptor tyrosine kinase that binds Wnt proteins using a domain related to WIF1, and Ror2, which binds to Wnt5a.

References 1. Barker N, Clevers H (2006) Mining the Wnt pathway for cancer therapeutics. Nat Rev Drug Discov 5:997–1014 2. Karner C, Wharton KA Jr, Carroll TJ (2006) Planar cell polarity and vertebrate organogenesis. Semin Cell Dev Biol 17:194–203 3. Kikuchi A, Yamamoto H (2008) Tumor formation due to abnormalities in the beta-catenin-independent pathway of Wnt signaling. Cancer Sci 99:202–208 4. Mikels AJ, Nusse R (2006) Wnts as ligands: processing, secretion and reception. Oncogene 57:7461–7468 5. Rubin JS, Barshishat-Kupper M, Feroze-Merzoug F et al. (2006) Secreted WNT antagonists as tumor suppressors: pro and con. Front Biosci 11:2093–2105

WTIP

Working Level Definition WL; Describes the concentration of radon and its progeny in the mining environment. A Working Level (WL) is defined as 1.3 × 105 MeV of potential alpha energy/l air.

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Wound Healing Assay Definition

Is an in vitro assay used to study cell ▶migration. A wound is created in a cell monolayer and the open gap is inspected microscopically over time as cells migrate and fill the damaged area.

▶Uranium Miners

Wox1 Working Level Month Definition WLM; equals the exposure to one working level for a working month (170 h). A working level is equivalent to any combination of radon progeny in 1 liter of air that will result in the ultimate emission of 1.3 × 105 MeV of potential alpha particle energy. ▶Uranium Miners

Working Level Months (WLM) Definition A miner’s exposure to radon and its progeny is given in Working Level Months (WLM). A Working Level Month equals an exposure to 1 WL for 170 h or any equal combination of WL and time. ▶Uranium Miners

Wound Healing Definition Refers to the predictable series of events that takes place after damage to dermal or epidermal tissue. ▶CXC Chemokines

▶WWOX

Wright-Giemsa Stain Definition Synonym wright stain; A specially prepared mixture of methylene blue and eosin in methanol, used to stain cells so that they can be seen microscopically. Most commonly used in hematology for peripheral blood smears and in cytopathology for fine needle aspirations and body fluids. ▶Fine Needle Aspiration ▶Pathology

WT1 Definition

▶Wilms tumor suppressor gene 1, a gene encoding a zinc-finger containing transcription factor that is mutated in a subset of ▶Wilms tumor.

WTIP Definition

WT1-interacting protein. Is a member of the ▶zyxin family of proteins. ▶Lipoma Preferred Partner ▶Wilms Tumor Suppressor Gene

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WW Domains

WW Domains Definition Named after two highly conserved tryptophan residues within the sequences of small protein modules (30 amino acids in length) that recognize and bind to proline-rich peptide motifs in interacting proteins. ▶WWOX

WWOX K AY H UEBNER , R AMI A QEILAN Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Comprehensive Cancer Center, Columbus, OH, USA

Synonyms FOR; Wox1

Definition Wwox, a 414 amino acid protein of 46 kDa, mapping to a common ▶fragile site at chromosome region 16q23.3, exhibits two N-terminal ▶WW domains and a central short chain oxidoreductase-like domain. Wwox is a cytoplasmic protein that binds other proteins containing PPxY-containing ligand proteins through the first Wwox WW domain. Through binding of its ligands, Wwox controls transcriptional activation and repression of nuclear genes and thus controls signaling pathways that affect aspects of cell growth and ▶apoptosis. Wwox expression is nearly ubiquitous in normal tissues but is lost or decreased in many cancers due to genomic or ▶epigenetic modification, and its restoration causes apoptosis of Wwox deficient

cancer cells; thus WWOX is the second known fragile ▶tumor suppressor gene.

Characteristics The Gene The WWOX gene spans a genomic locus of >600 kb and is composed of nine exons encoding an open reading frame of 1,245 bases; the protein sequence includes two WW domains and a short chain dehydrogenase/reductase domain that may be involved in sexsteroid metabolism, considering its sequence homology to 17β-hydroxysterol reductase 3 (Fig. 1). The WWOX gene also spans fragile site ▶FRA16D, encompasses a region involved in ▶loss of heterozygosity (LOH) in cancers, is associated with ▶homozygous deletions in cancer-derived cell lines, with chromosome translocations in ▶multiple myelomas, and its promoter region is frequently hypermethylated in cancers (Fig. 2). Most cancer cell lines with FRA16D homozygous deletions also exhibit deletions in ▶FRA3B and the ▶FHIT gene, consistent with the finding that common fragile sites or regions are highly susceptible to ▶DNA damage and recombination. The mouse ortholog, Wwox, at murine chromosome region 8E1, is also fragile and is highly homologous to the human locus. Internally deleted WWOX transcripts have been observed in human breast, ovarian and other tumor types, though bona fide point mutations have not been observed. The highest level of WWOX expression in normal tissues occurs in hormonally regulated tissues. The WWOX promoter is hypermethylated in many cancers in association with loss of Wwox expression, and detection of the ▶methylation status by ▶methylation-specific PCR (MSP) amplification may serve as a marker of cancer development or prevention. The Protein Wwox protein binds the proline-rich ligand PPxY and a number of proteins interacting with the first WW domain, WW1, have been identified. WW domains are grouped by binding preference for specific types of proline-rich ligands and the Wwox WW1 domain belongs to group I, that binds PPxY ligands; among

WWOX. Figure 1 Schematic of the Wwox protein showing the WW and short chain dehydrogenase/reductase domains. Proteins with a PPxY motif that interact with Wwox through its first WW domain are listed.

WWOX

WWOX. Figure 2 Schematic of the WWOX gene. The long arm of chromosome 16 is shown on the left. The dark boxes on the right represent WWOX exons. Many of the deletions detected in tumor cell lines are within WWOX intron 8.

these ligand-containing proteins are p73, Ap2α, Ap2γ, ErbB4, Jun and the SIMPLE protein; ▶p53 has also been reported to bind Wwox or Wox1, the murine ortholog, but other studies have not confirmed this ligand. The Cytogen Corporation has developed, through informatics and binding studies, a proprietary database that lists all potential Wwox ligands among known gene products, and the first three ligands listed above were selected from the database and confirmed as Wwoxinteracting proteins by in vitro analyses. The cytoplasmic Wwox protein binds its ligands and prevents the interacting proteins from entering the nucleus, where some have roles in transcriptional activation or repression. There are thus far unconfirmed reports of Wwox subcellular location also in mitochondria and nuclei of some cells. Biological Role In normal cells: human tissue samples of more than 30 organs have been analyzed by immunohistochemistry for expression of Wwox; Wwox is expressed in most organs but is expressed at highest levels in secretory epithelial cells such as those of breast, ovary, testes and prostate. Wwox is also expressed in various cells of neural origin. ▶Targeted deletion of the mouse Wwox gene revealed important roles of Wwox in tumorigenesis and metabolism. Using ▶homologous recombination, a mouse lacking exons 2, 3 and 4 of the mouse Wwox gene was generated. Progeny from Wwox heterozygous (Wwox+/–) intercrosses resulted in offspring of all three genotypes with ratios consistent with ▶Mendelian distribution. At birth, homozygous Wwox-deficient (Wwox–/–) pups were indistinguishable from wild type or heterozygous

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littermates up to 3 days postpartum; after 3 days, homozygous pups were easily identified by their smaller size. Wwox–/– pups continued to grow more slowly than littermates, and all homozygous knockout mice died by 4 weeks after birth. Serum chemistry analysis of Wwox–/– mice showed marked hypoproteinemia, hypoalbuminemia, hypoglycemia, hypocalcemia, hypotriglyceridemia and hypocholesterolemia, indicating that Wwox–/– pups suffered severe metabolic defects. Macroscopic and histological examination of the organs confirmed atrophy of many organs in Wwox–/– animals without significant microscopic lesions, though Wwox–/– mice are born with gonadal abnormalities and display bone growth retardation. Juvenile Wwox–/– mice displayed impaired steroidogenesis; levels of steroid biosynthesis enzymes, including Cyp11a1 (cytochrome p450 side-chain cleavage enzyme) and Hsd3b (3-β-hydroxysteroid dehydrogenase), were reduced in mutant testis and ovary compared to wild type and heterozygous testis. Radiography and highresolution microtomography (μCT) of limb bones showed that Wwox–/– mice develop less dense bones with slow growth rates. Analysis of the Wwox-mutant mice demonstrated that Wwox functions as a bona fide tumor suppressor. Spontaneous ▶osteosarcomas in juvenile Wwox–/– and lung papillary carcinoma in adult Wwox+/– mice were observed, and Wwox+/– mice developed significantly more ethyl nitrosourea (ENU)-induced lung tumors and ▶lymphomas in comparison to wild type littermates. These tumors still express Wwox protein, suggesting ▶haploinsufficiency of Wwox is cancer-predisposing. In cancer cells: Esophageal squamous cell carcinoma, ▶non-small cell lung cancer and ▶breast cancer showed high ▶LOH rates, low mutation rates and expression of aberrant transcripts of the WWOX gene. Wwox expression is reduced in 63% of invasive breast carcinomas and is correlated with ▶estrogen receptor alpha level, and prognostic features; Wwox and Fhit expression is coordinately lost in breast cancers. The WWOX gene is also inactivated in breast and lung cancers by regulatory region DNA methylation; promoter methylation was also detected in tissues adjacent to breast cancer, and methylation in WWOX exon 1 distinguished breast cancer DNA from DNA of adjacent and normal tissue. Wwox restoration in lung cancer cells in vitro, and in ▶xenografts, suppressed growth. Wwox-deficient breast cancer cells, by treatment with ▶5′-Aza-2′-deoxycytidine to demethylate the WWOX promoter, was associated with effective induction of apoptosis in vitro and suppressed breast cancer xenograft growth in vivo, without affecting the Wwox-sufficient cells or causing persistent changes in global methylation levels. ▶Tamoxifen is commonly used for treatment of ▶estrogen receptor alpha positive breast cancers, but

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WWOX

de novo or acquired tamoxifen resistance occurs frequently. Wwox protein, which binds and retains Ap2α and γ transcription factors in the cytoplasm, may mediate tamoxifen sensitivity in vitro; Wwox loss initiates tamoxifen resistance through release of Ap2 factors to the nucleus where Ap2γ up-regulates ErbB2 (▶Her2) expression. Restoration of Wwox in tamoxifen-resistant breast cancer-derived cells restored tamoxifen sensitivity and abrogated ErbB2 expression. Wwox expression was significantly ( p = 0.013) reduced in tamoxifen resistant breast cancers, and a reliable marker of tamoxifen resistance, especially in premenopausal and stage 3 patients. Thus, the Wwox signaling pathway may provide new targets for therapeutic intervention in antiestrogen-resistant breast cancers.

References 1. Ludes-Meyers JH, Bednarek AK, Popescu NC et al. (2003) WWOX, the common chromosomal fragile site, FRA16D, cancer gene. Cytogenet Genome Res 100:101–110 2. Matsuyama A, Croce CM, Huebner K (2004) Common fragile genes. Eur J Histochem 48:29–36 3. O’Keefe LV, Richards RI (2006) Common chromosomal fragile sites and cancer: focus on FRA16D. Cancer Lett 232:37–47 4. Gaudio E, Palamarchuk A, Palumbe T et al. (2006) Physical association with WWOX suppresses c-Jun transcriptional activity. Cancer Res 66:11585–11589 5. Guler G, Iliopoulos D, Guler N et al (2007) Wwox and Ap2γ expression levels predict tamoxifen response Chin Cancer Res 13:6115–6121

X

X Chromosome Inactivation Definition A gene dosage compensation mechanism by which one of the two X chromosomes in female cells becomes transcriptionally silenced early in embryonic development through a process understood to be controlled by an inactivation centre located on the X chromosome. The X-inactivation centre determines the X chromosome to be inactivated in each individual cell and produces a noncoding Xist transcript that coats the X chromosome in cis, triggering its silencing and further inducing a cascade of chromatin and ▶methylation changes completing the ▶epigenetic silencing process.

Xenobiotic Biotransformation ▶Detoxification

Xenobiotic Metabolism ▶Detoxification

Xenobiotic Receptor

▶LINE-1 Elements ▶Aryl Hydrocarbon Receptor

Xanthine Oxidase Definition

Is a ▶flavoprotein enzyme catalyzing the oxidation of certain purines; it is normally found in the liver of humans and it is released to blood during liver damage and can cause renal failure.

Xenobiotic Definition Chemicals not normally found in the body (including most carcinogens). ▶Carcinogen Metabolism

▶Polyphenols

Xenobiotics Xanthophylls Definition

Are ▶carotenoids where some of the double bonds have been oxidized, such as lutein and zeaxanthin.

PAVEL S OUCEK Group for Biotransformations, Center of Occupational Medicine, National Institute of Public Health, Prague, Czech Republic

Synonyms Foreign substances; Exobiotics

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Xenobiotics

Definition Xenobiotics are chemicals present in an organism or environment, which did not produce them. Some naturally occurring chemicals (endobiotics) become xenobiotics when present in an environment at excessive concentrations. The word “xeno” is derived from the Greek word “xenos,” meaning a guest, friend, or foreigner.

Characteristics Origin of Xenobiotics Xenobiotics are mostly produced by human activities and excite public awareness due to their ability to interact with the living environment. Some organisms may also form them as a part of their defense system, e.g., mycotoxins, bacterial, and herbal toxins, etc., and xenobiotics become harmful when entering the alimentary chain. Contemporary human exposure to xenobiotics is unavoidable, as xenobiotics are omnipresent. Human exposure to some xenobiotics is voluntary because of their anticipated beneficiary effects on human health (e.g., drugs, antibiotics, dietary supplements as antioxidants, etc.). Daily use of xenobiotics as food ingredients (dyes, stabilizers, emulsifiers, salt compounds, preservatives, etc.), cosmetics and personal care products (makeup, hair dyes, soaps, perfumes), and household products (chlorine bleach, bug sprays, cleaners, etc.) further increases exposure extent. Nevertheless, the positive role of xenobiotics as components of modern technologies enabling further progress of human civilization is inevitable. At present time, it is not possible to divide xenobiotics into straightforward categories as, e.g., good versus bad ones and simply eliminate the latter from living environment. Metabolism of Xenobiotics Metabolism or in other words biotransformation leads to conversion of xenobiotics in the organism. After entering the human body, blood vessels transport xenobiotics to the liver as the main ▶detoxification site. Hepatocytes (liver cells) contain xenobiotic-metabolizing enzymes (XMEs). The major task of XMEs is to convert xenobiotics soluble in fat into water-soluble products and thus facilitate their elimination from the cell and excretion from the body. XMEs are divided into three groups: ▶phase I enzymes (activation), ▶phase II enzymes (conjugation), and phase III (transport) enzymes (Table 1). ▶Cytochromes P450 (P450s) represent a major player in the field of phase I biotransformation. P450s and other phase I enzymes as NAD(P)H:quinone oxidoreductases (NQOs) usually perform activation reactions aiming at formation of reactive ▶intermediates. Such highly electrophilic intermediates efficiently conjugate with ▶nucleophiles, e.g., glutathione in reactions catalyzed by phase II enzymes as ▶glutathione

S-transferases (GSTs). The majority of conjugated xenobiotics undergo transport outside of the cell by ▶efflux pumps driven by ▶membrane ATP-binding casette transporters (ABC), e.g., ▶P-glycoprotein. Main routes of elimination of hydrophilic conjugates are urine, feces, sweat, or breath. Xenobiotics are often highly lipophilic and thus may accumulate in fat tissues and slowly drain into blood circulation long time after exposure event occurred. The typical example may be exposure to organic solvents as styrene or polychlorinated biphenyl ▶dioxin. Various physiological factors, e.g., age, gender, nutritional status (starving), or pathological factors, e.g., hypertension, diabetes mellitus, liver cirrhosis, renal failure, etc., significantly affect metabolism. An understanding of xenobiotic metabolism is critical for the pharmaceutical industry because it is responsible for the activity but also for the toxicity of drugs. Effects of Xenobiotics Common concern about xenobiotics is due to their toxicity toward living organisms and potential to cause environmental effects on global level. ▶Acid rains and production of glasshouse gases increase global warming. Sea pollution or soil contamination by agricultural chemicals facilitates incorporation of heavy metals into highly toxic organic compounds. High concentration of metropolitan transport and local heating enhances concentration of particulate matter containing ▶polycyclic aromatic hydrocarbons, ▶polychlorinated biphenyls, dioxins, etc. Toxicity classification concerns the grouping of chemicals into general categories according to their most important toxic effect. Such categories can include allergens, neurotoxins, carcinogens, mutagens, teratogens, ▶immunotoxins, etc. Xenobiotics act either directly as parent compounds or indirectly as intermediates or products of their metabolism. ▶Oxidative stress presents another secondary product of metabolism of xenobiotics. ▶Reactive oxygen species may then attack DNA, proteins, or stimulate ▶inflammation. In some cases, an inactive conjugated metabolite is reactivated, for example, by enzymatic cleavage and causes tissue-specific toxic effect, e.g., tumor promotion. Certain xenobiotics entering human body deregulate important cellular and organ signaling pathways because they mimic physiological substrates. Xenoestrogens or endocrine disruptors, e.g., phthalates or polychlorinated biphenyls (DDT, dioxin), etc., impair reproductive functions, disturb wildlife, and may cause sterility by decreasing sperm count in males. Another important issue is the widely discussed oncogenic potential of xenoestrogens. Evaluation of environmental exposure to xenobiotics complicated by the fact that combined exposures to several xenobiotics simultaneously occur rather than separate simple exposures. Chemicals acting via the same mechanisms produce ▶additive effects. However, interaction between chemicals may result in an

Xenobiotics Xenobiotics. Table 1

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Major xenobiotic-metabolizing enzymes Protein

Phase I Cytochrome P450 monooxygenases Flavin-containing monooxygenases Alcohol dehydrogenases Aldehyde dehydrogenases Monoamine oxidase NADPH-cytochrome P450 reductase Carbonyl reductases Aldo-keto reductases NAD(P)H-quinone oxidoreductase Epoxide hydrolases Carboxylesterases Deaminases Phase II Glutathione S-transferases UDP-Glucuronosyltransferases N-Acetyltransferases Sulfotransferases Phase III ATP-binding cassette transporter

Lung resistance-related protein

EC number

Gene

Reactions

P450

1.14.14.1

CYP

Oxidation, reduction, peroxidation

FMO

1.14.13.8

FMO

Oxidation

ADH ALDH MAO CPR

1.1.1.2 1.2.1.5 1.4.3.4 1.6.2.4

ADH ALDH MAO POR

Alcohol oxidation Aldehyde oxidation Oxidative deamination Reduction

CR ALR NQO EPHX CE CD

1.1.1.184 1.1.1.21 1.6.5.2 3.3.2.9 3.1.1.2 3.5.4.1

CBR AKR NQO EPHX PON CDA

Reduction Reduction Quinone reduction Epoxide hydrolysis Hydrolysis of ester-containing xenobiotics Hydrolytic deamination

GST UGT NAT SULT

2.5.1.18 2.4.1.17 2.3.1.5 2.8.2.3

GST UGT NAT SULT

Conjugation with glutathione Conjugation with glucuronide Acetylation Conjugation with sulfate

MDR MRP/MOAT BCRP LRP/VAULT1

3.6.3.44 3.6.3.44 3.6.3.44 3.6.3.44

ABCB ABCC ABCG MVP

Xenobiotic transport across cell membranes

inhibition (▶antagonism) or in ▶synergism, a more pronounced effect than would be expected by addition. Estimation of effects of combined mixtures by animal experiments and computer modeling belongs to the most difficult tasks of modern toxicology. Interactions Xenobiotics are able to enhance their own metabolism by ▶induction of XMEs. Induction of battery of XMEs through ▶arylhydrocarbon receptor (AhR) was the first proved example. Expression of XMEs is regulated also through other nuclear receptors, e.g., constitutive androstane receptor (CAR), ▶peroxisome proliferator-activated receptor (PPAR), pregnane X receptor (PXR), and others. Some xenobiotics cause inhibition of XMEs by covalent binding to active site or other functionally important part of enzyme or by competition for enzyme with physiological substrates or other xenobiotics. Interactions between xenobiotics and XMEs may cause severe health effects. Induction of P450 2E1 by ethanol consumption enhances metabolism of acetaminophen (paracetamol) by P450

Nucleo-cytoplasmic transport

2E1 to hepatotoxic intermediates leading to death of sensitive individuals. Inhibition of P450 3A4 by components of grapefruit juice may either decrease effect of P450 3A4-activated ▶prodrugs or/and prolong toxicity of P450 3A4-detoxified drugs. P450 3A4 metabolizes about 50% all currently prescribed drugs, e.g., amiodarone, budesonide, codeine, digitoxin, ▶irinotecan, lidocaine, midazolam, simvastatin, ▶tamoxifen. Drug–drug interactions may of course have similar consequences as interactions between drugs and nutrition. Individual Variability XMEs are highly genetically variable and contain a number of functionally relevant polymorphisms (▶metabolic polymorphisms). These inherited DNA variations may influence both level and activity of expressed XMEs. Altered enzyme then lacks critical detoxifying activity or gains activating properties, which may form excess of toxic metabolites and impair important physiological functions. Thus, genetic variability may explain why certain individuals are more susceptible to

X

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Xenogeneic

toxic effects of xenobiotics than others are. Various populations differ in frequencies of XMEs polymorphisms, e.g., GSTT1-null polymorphism conferring lack of enzyme activity affects 10–20% of Caucasian populations but 30–60% of Oriental ones. Thus, depending on exposure type and extent this polymorphism may be more important for Orientals. Polymorphisms in alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) converting ethanol to acetaldehyde cause unpleasant adverse effects in large group of Orientals and are suspected to modify acetaldehyde-induced ▶carcinogenesis. Polymorphisms in pharmacologically important P450 2D6 belong to the most studied ones and progress in science and technology enabled genotyping individuals to assess so-called poor metabolizer phenotype (persons with weak metabolizing ability), medium, and rapid phenotype (persons with high metabolizing ability). Such effort pays off both to society on the cost-benefit basis and to individual patient whom physician prescribes the most efficient drug with least adverse effects without blind tests of different medications with potential danger of longer periods of illness or even hospitalization. However, individual variability still greatly complicates assessment of drug–drug interactions and presents a challenge for contemporary pharmacology. Among possible solutions of this puzzle, design of new generation of drugs, which are not subjects to metabolism by polymorphic XMEs will soon play pivotal role. Xenobiotics and Cancer Various xenobiotics promote carcinogenesis or cause genotoxicity by interaction with genetic information. The majority of procarcinogens undergo metabolic conversion to become ultimate carcinogens. The extent of carcinogen production is dependent on ratio between activation and detoxification capacity. Thus, understanding the individual variability in XMEs may shed more light on the molecular basis of diseases apparently caused by interaction between genetic background and environment. Experts in molecular epidemiology now evaluate prognostic value of reported associations between genetic variants in genes coding XMEs and various diseases, especially cancer. Although the concept of association between metabolic capacity modified by genetic variation in XMEs and disease caused by metabolically activated procarcinogens seemed reasonable, ▶meta-analysis shows that the task will be extremely difficult. Association studies usually use very limited numbers of patients due to problems with recruiting. Moreover, each cancer type presents distinct disease with distinct etiology, so pooling of patients with different diagnoses is questionable. Quite often, the identification of individual chemicals and levels of exposure in studied individuals is very complicated by the long latency period from

exposure to onset of cancer (usually decades). Finally, interplay between different groups of low-penetrance genes (such as XMEs) most probably multiplies the effect of associations. The final goal of molecular epidemiology is to predict cancer susceptibility based on individual variability and prevent interacting exposures. In individuals already suffering from cancer, individualized therapy tailored to XMEs overexpressed in tumor cells will be available soon. Exploitation of Knowledge about Xenobiotics and XMEs Besides the use in pharmacology and ▶cancer epidemiology, the knowledge gained through intensive research on xenobiotics, their origin, and fate in living organisms over last decades suggested promising applications in many commercial areas. Genetically engineered XMEs are evolving tools for selective production of chemicals with valuable properties including drugs whose production by chemical synthesis is impossible or connected with overwhelming costs. Sewage is increasingly contaminated with xenobiotics of all sorts including human waste. Sorting of sewage is becoming extremely expensive and for its bioremediation treatment systems based on immobilized XMEs seem to be the most natural choice. After obtaining solution for some technical aspects broader applications of XMEs and their optimization for commercial needs will soon be performed.

References 1. Guengerich FP (2003) Cytochromes P450, drugs, and diseases. Mol Interv 3:194–204 2. Stanley LA, Horsburgh BC, Ross J et al. (2006) PXR and CAR: nuclear receptors which play a pivotal role in drug disposition and chemical toxicity. Drug Metabol Rev 38:515–597 3. Ekroos M, Sjogren T (2006) Structural basis for ligand promiscuity in cytochrome P450 3A4. Proc Natl Acad Sci USA 103:13682–13687 4. Gillam EM (2005) Exploring the potential of xenobioticmetabolising enzymes as biocatalysts: evolving designer catalysts from polyfunctional cytochrome P450 enzymes. Clin Exp Pharmacol Physiol 32:147–152 5. Urlacher VB, Eiben S (2006) Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol 24:324–330

Xenogeneic Definition Derived or obtained from an organism of different species and, therefore, immunologically incompatible.

Xeroderma Pigmentosum

Xenograft Definition The engraftment of cells or tissue originating from one species into the body of a different, often immunesuppressed, host species. In the field of oncology, a commonly used term to describe small rodent tumor cell implantation models. For example, human breast cancer cells injected into nude mice. ▶Bioluminescence Imaging ▶Mouse Models

Xenograft Models ▶Mouse Models

Xeroderma Pigmentosum R OB J. W. B ERG University Medical Center Utrecht, Utrecht, The Netherlands

Definition Xeroderma pigmentosum (XP) is a genetic disease with clinical and cellular hypersensitivity to ▶ultraviolet radiation and defective ▶repair of DNA. XP patients display a marked increase in the frequency of skin malignancies. XP, thus, serves as a model disease to study the relationship between defects in DNA repair and (skin) cancer. It is a skin cancer prone disorder, due to defective ▶nucleotide excision repair (NER).

Characteristics Xeroderma Pigmentosum (XP) Subgroups The DNA repair pathway that deals with the removal of DNA damage induced by UV radiation is NER (nucleotide excision repair). In 1972 it was demonstrated by using cell fusion techniques that fibroblasts from one patient were able to compensate the repair defect in fibroblasts from another patient. Heterokaryons (cells with nuclei from different donors in a common cytoplasm) were found to exhibit mutual correction of the defective removal of UV radiationinduced DNA damage. Such cells were said to be

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in different ▶complementation groups. If both nuclei in a heterokaryon have the same genetic defect, then the heterokaryons show defective repair of DNA damage and the patients are in the same complementation group. Such studies have revealed the existence of at least seven complementation groups in XP (XP-A through XP-G). Each complementation group may represent a gene that, if mutated and in homozygous condition, causes XP. In addition, a subgroup exists with the clinical symptoms of XP but with normal NER of UVinduced DNA damage. Patients in this subgroup have a defect in an alternative repair process, viz., in postreplication repair. Such patients are called XP-V (XP variants). Clinical Aspects XP is clinically characterized by photosensitivity, pigmentary changes, premature skin ageing and a high incidence of ▶skin cancer. In the majority of cases the first symptoms are noticed between 6 months and 3 years after birth. Freckling, sensitivity to sunburn and an increased dryness on sun-exposed skin are usually the earliest manifestations. The first malignant tumors may develop as early as the third or fourth year. ▶Basal cell carcinoma, ▶squamous cell carcinoma, and ▶melanoma, are common and may be multiple. Besides the skin, the eyes and the nervous system may be affected. In the large majority of patients, photophobia and conjunctivitis are early symptoms. Neurological abnormalities occur in ~20% of the cases, which are predominantly patients in the XP-A and XP-D complementations groups. These abnormalities may comprise mental retardation, spacticity, ataxia, dysphasia and areflexia. Two-thirds of XP patients die before 20 years of age from metastases, neurological complications or infections, to which they are also abnormally susceptible. Table 1 summarizes the clinical features of the different XP complementation groups. Cellular Parameters The two major types of DNA damage induced by UV radiation are CPD (cyclobutane pyrimidine dimers) and [6–4]PP (6-pyrimidine-4-pyrimidone photoproducts). Both CPD and [6–4]PP are formed between two adjacent pyrimidines (cytosine and/or thymine) on a DNA strand. Both lesions lead to a considerable distortion of the three-dimensional structure of the double helix. CPD and [6–4]PP are removed from the genome by nucleotide excision repair (NER), which comprises two subpathways: . GGR (global genome repair) . TCR (▶transcription-coupled repair) The GGR subpathway is responsible for the removal of lesions from the transcriptionally inactive DNA and from the non-transcribed strand of active genes.

X

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Xeroderma Pigmentosum

Xeroderma Pigmentosum. Table 1

Clinical properties of the separate XP complementation groups

Group

Skin cancer

Neurological abnormalities

Relative frequency

XP-A XP-B XP-C XP-D XP-E XP-F XP-G XP-V

+ ± + + ± ± ± +

++ ++ – + – – ++ –

High Very rare High Intermediate Rare Rare Rare High

In the TCR subpathway, the repair machinery is directed preferentially to the transcribed strands of active genes to avoid unrepaired lesions interfering with transcription. Generally, the transcribed strand is corrected up to 5–10 times as fast as the nontranscribed strand. As different mutations in the same gene can lead to different levels of impairment, patients within the same complementation group can vary quantitatively in their residual GGR and/or TCR capacity. All NERdefective XP complementation groups are more or less defective in both GGR and TCR, with the exception of XP-C and XP-E, which are defective in GGR only. Molecular Parameters ▶NER is the process in which damaged DNA is removed and replaced with new DNA using the intact strand as a template. This complex system involves the concerted action of multiple proteins. The first step in mammalian NER is damage recognition. The XP-C and the XP-E protein play a role in recognition of UV-damaged DNA in nontranscribed DNA. In transcriptionally active DNA, the arrest of transcription at the site of a DNA lesion serves as the damagerecognition signal. Subsequently, the XP-A protein and a protein complex called ▶TFIIH are recruited to the lesion, and the double helix is opened around the site of damage. It is assumed that in both transcription and NER the function of TFIIH is the unwinding of the double-stranded DNA helix. Both the XP-B and the XP-D protein are part of the TFIIH complex. After damage recognition and partial unwinding of the double helix the actual removal of DNA damage is identical for the GGR and TCR subpathways. The XP-F and XP-G proteins play a role in cutting the DNA on either site of the damage, thereby releasing a 24- to 32-residue oligonucleotide. Subsequently, the gap is filled in by a DNA polymerase and sealed by a DNA ligase. XP-V cells have normal removal of UV-induced DNA damage from both transcribed and nontranscribed DNA. Cells can tolerate unrepaired damage in their

genome and removal of damage does not have to be complete before DNA replication takes place. As CPD and [6–4]PP are effective blocks to the progression of replicative DNA polymerases, cells have developed specialized DNA polymerases that can bypass DNA damage and extend replication forks through damaged sites. One of these DNA polymerase can bypass CPD at thymine-thymine sites and usually correctly inserts two A residues opposite the lesion. In XP-V cells this specialized polymerase is defective. Consequently, in XP-V cells lesions are bypassed by polymerases that insert incorrect residues, leading to a high level of mutations in XP-V cells. Animal Models Experimental studies on the relationship between NER defects and clinical phenotype are difficult to carry out as XP is a rare disease, which consists of at least seven NER-defective subgroups. In addition, experimental studies with UV radiation in XP patients can be considered questionable for obvious ethical reasons. Therefore, mouse models for XP have been developed, which are well suited to study the relationship between deficiencies in NER and susceptibility to skin cancer. The first viable animal models for XP were XP-Adeficient and XP-C-deficient transgenic mice (XP-A and XP-C knockouts). These animals develop normally, are fertile and do not show signs of a NER-disorder. However, after exposure to UV radiation both animal models strongly mimic the phenotype of humans with XP, i.e., they show a severely increased susceptibility to skin cancer. Neurological disorders, common in XP-A patients, were not found in XP-A knockout mice. A direct comparison in skin cancer susceptibility between XP-A and XP-C ▶knockout mice has shown that XP-A knockouts are more cancer prone than XP-C knockouts. Hence, skin cancer susceptibility is determined by both GGR and TCR, and defective GGR contributes more prominently to skin cancer development than defective TCR. However, acute UV effects

Xiphophorus

appear to be related primarily to TCR; the minimal dose required to induce a slight sunburn is strongly reduced in XP-A knockouts but not in XP-C knockouts. Probably blockage of RNA synthesis during transcription, by persistent CPD or [6–4]PP, triggers the influx of pro-inflammatory molecules leading to sunburn. As parents of XP patients usually are clinically normal, inheritance of XP is considered to be autosomally recessive. Whether carriers of XP genes (heterozygotes) have a subtle increase in skin cancer risk can easily be addressed by XP mouse models. Heterozygous XP-A animals did not show a higher skin cancer susceptibility than wildtypes, whereas heterozygous XPC animals have been reported to have a higher susceptibility to UV carcinogenesis than their wildtype litter mate controls. The reason for this difference has not yet been elucidated. Other NER-Related Syndromes In addition to XP, two other rare genetic diseases have been associated with a defect in NER. The first is CS (Cockayne syndrome), which comprises at least two complementation groups (CS-A and CS-B). CS patients have a defect in TCR and they exhibit a severe clinical phenotype. CS patients show growth failure, progressive neurological degeneration, retinal degeneration, photosensitive skin and deafness, and most CS patients die at an early age. The second NER-related disease is a photosensitive form of ▶trichothiodystrophy called PIBIDS. This is an acronym for photosensitivity, ichthyosis, brittle hair and nails, intellectual impairment, decreased fertility and short stature. Patients with PIBIDS have mutations in the XP-B or XP-D gene, which are both components of the ▶TFIIH complex. It has been suggested that specific mutations that preserve the transcription function of TFIIH leads to XP, whereas mutations that also modify the transcription function lead to PIPIDS.

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Xerophthalmia ▶Sjögren Syndrome

Xerostomia ▶Sjögren Syndrome

XIAP Definition A member of the IAP (▶inhibitors of apoptosis) family of proteins containing one or more characteristic BIR domains. These proteins bind and inhibit ▶caspases. ▶APAF-1 Signaling

Xiphophorus M ANFRED S CHARTL Biozentrum, Universität Würzburg, Würzburg, Germany

Synonyms Gordon-Kosswig melanoma system; Platyfish-swordtail melanoma

References

Definition

1. Kraemer KH, Lee MM, Scotto LJ (1987) Xeroderma pigmentosum. Cutaneous, ocular, and neurological abnormalities in 830 published cases. Arch Dermatol 123:241–250 2. Bootsma D (1993) The genetic defect in DNA repair deficiency syndromes. Eur J Cancer 29A:1482–1488 3. Van Steeg H, Kraemer KH (1999) Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer. Mol Med Today 5(2):86–94 4. Lindahl T, Wood RD (1999) Quality control by DNA repair. Science 286:1897–1905 5. Berg RJW, Rebel H, Van der Horst GTJ et al. (2000) Impact of global genome repair versus transcriptioncoupled repair on ultraviolet carcinogenesis in hairless mice. Cancer Res 60:2858–2863

Small aquarium fishes of the genus Xiphophorus are known by the common name of platyfish (X. maculatus) and swordtail (X. hellerii). Introgressive hybridization results in offspring that develop melanoma according to Mendelian principles. This represents the first animal model, described in 1927, systematically employed for studies of genetic factors in cancer and to show induction of melanoma by ultraviolet light (▶UV-A).

Characteristics Genetics of Melanoma Formation The genetic basis of melanoma formation after introgressive hybridization is explained by the independent

X

3222

Xiphophorus

segregation of a pigmentation locus (Sd), which contains a dominantly-acting ▶oncogene, designated Tu, and a trans-acting regulatory gene R (also termed Diff, MelSev or RDiff) that suppresses the oncogenic activity of Tu (Fig. 1). Independent segregation is possible because Tu and R reside on different chromosomes. Crossing and backcrossing the fish carrying both Tu and R (platyfish) to the swordtail results in the progressive replacement of platyfish chromosomes bearing the R by swordtail chromosomes lacking R. The stepwise elimination of R from the hybrid genome allows expression of the Tu phenotype, leading to a benign hyperpigmentation in cases where one functional allele of R is still present (F1 and 25% of backcross) or to malignant melanoma in cases where R is completely absent (25% of backcross). The Xmrk Oncogene The melanoma oncogene from the Tu locus is referred to as Xmrk (Xiphophorus melanoma receptor tyrosine kinase, also termed Xmrk-2). It encodes a transmembrane ▶receptor tyrosine kinase (RTK) that is an oncogenic version of the epidermal growth factor (EGF) receptor. During evolution, Xmrk was generated by gene duplication from its corresponding proto-oncogene. The new copy was fused to the 5′ non-coding region of another (anonymous) sequence. This process generated a novel promoter for the oncogenic version (called Xmrk to

distinguish it from the proto-oncogenic copy Xegfrb) of the gene. As a consequence of this rearrangement, the protooncogene and the oncogene are subject to different transcriptional regulation. Specific overexpression of the Xmrk oncogene in the pigment cell lineage of hybrid fish is responsible for melanoma formation. An R locus-dependent transcriptional control of the oncogene promoter allows high levels of expression exclusively in pigment cells of certain hybrid genotypes but not in non-hybrids. This explains why the dominantly-acting Xmrk oncogene is ineffective in the pure bred parental Xiphophorus fish and is a nonhazardous constituent of the genome in natural populations for many generations. This is reminiscent of the situation found for the ▶RET and ▶MET oncogenes in humans, where dominantly-acting mutations are transmitted through the germ line and elicit ▶multiple endocrine neoplasia type 2 (MEN2A, MEN2B) and hereditary papillary renal-cell carcinomas (HPRCC), respectively. Transcriptional control of Xmrk by R involves the suppression of an Sp1 transcription factormediated constitutive promoter activity in non-melanoma cells and a specific hypomethylation of the oncogene promoter, compared to the proto-oncogene promoter. The Xmrk oncogene is necessary for melanoma formation since transposon inactivation of the oncogene in one

Xiphophorus. Figure 1 Genetic tumors in Xiphophorus. The classical cross-breeding experiment: a female platyfish (X. maculatus), which is homozygous for the X-chromosomal locus Sd, encoding the pigment pattern “spotted dorsal” (small black spots in the dorsal fin composed of a specific type of pigment cells – so-called macromelanophores) is mated to a swordtail (X. hellerii), which does not have the corresponding locus. The F1 hybrids show enhancement of the Sd phenotype. Backcrossing of F1 hybrids to X. hellerii results in offspring that segregate. Fifty percent do not inherit the Sd locus and are phenotypically like the X. hellerii parental strain. The other 50% carry the Sd locus and develop melanoma. Here, in approximately half of the fish, the severity of melanoma ranges from very benign (phenotype like the F1 hybrids) to extremely malignant in the others. Highly malignant melanomas become invasive and exophytic, and are fatal to the individual. These melanomas grow progressively, following transplantation into thymus-aplastic “nude” mice. Symbols below the fish describe their genotypes in respect to the R and Tu-loci.

XRF

mutant strain results in loss of melanoma formation. However, overexpression of the receptor is not the only reason for tumor induction. Mutations in the extracellular domain that create covalently linked receptor dimers with constitutive activity and a cell type-specific signal transduction machinery downstream of Xmrk are necessary for tumor formation in transgenic fish and for transformation of cells in tissue culture. Signal transduction by Xmrk affects all cellular events needed for a full transformation. Some, though not all of the pathways induced by the oncogene are also shared by mammalian EGFR. It starts with the parallel activation of the ▶Ras/Raf/MAP kinase pathway, the ▶PI3 kinase pathway, the ▶signal transducer and activator of transcription STAT5, the small src kinase fyn and the ▶focal adhesion kinase. These events result in the transcription of numerous target genes belonging to different functional categories (secreted proteins, anti-apoptotic effectors, cell cycle regulatory proteins etc.). Finally, an induction of cellular proliferation, protection from ▶apoptosis, ▶migration and contact-independent survival takes place. In addition, transcriptional and posttranslational regulation of the differentiation and survival factor MITF impairs the final differentiation of the pigment cell. Consequently, Xmrk alone is able to elicit all the processes that are necessary for establishing the full malignant neoplastic phenotype of a melanoma cell.

References

UV-Induced Tumors Melanoma in certain hybrid crosses can be induced by UV-B and photoreactivation can reverse this melanoma incidence to background levels. When young backcross hybrids were irradiated with wavelengths from 365–436 nm, melanomas could be induced. This led to the appreciation that UV-A, and perhaps the visible light spectrum as well, are important in the etiology of human melanoma. Research on UV-induced melanoma uncovered CDKN2 a/b as a candidate gene for R.

Definition

Carcinogen-Induced Tumors Besides the propensity to develop melanoma on a hereditary basis, certain backcross strains have a susceptibility to develop cancer after exposure to carcinogens. While fish of wild type strains resist the development of tumors after exposure to N-methyl-Nnitrosourea (MNU) or to X-rays, certain hybrids are highly sensitive and respond to treatment by developing a large spectrum of tumors.

1. Schwab M (1987) Oncogenes and tumor suppressor genes in Xiphophorus. Trends Genet 3:38–42 2. Vielkind JR, Kallman KD, Morizot DC (1989) Genetics of melanomas in Xiphophorus. J Aquat Anim Health 1:69–77 3. Anders F (1991) Contributions of the Gordon-Kosswig melanoma system to the present concept of neoplasia. Pigment Cell Res 3:7–29 4. Nairn RS, Morizot DC, Kazianis S et al. (1996) Nonmammalian models for sunlight carcinogenesis: Genetic analysis of melanoma formation in Xiphophorus hybrid fish. Photochem Photobiol 64:440–448 5. Meierjohann S, Schartl M (2006) From Mendelian to molecular genetics: the Xiphophorus melanoma model. Trends Genet 22:654–661

XME Definition

Xenobiotics Metabolizing Enzymes; ▶Aryl Hydrocarbon Receptor.

X-Ray Crystallography

A method that uses x-ray diffraction from a crystallized molecule to determine its three-dimensional structure. ▶Structural Biology

X-ray Induced Cancer ▶Radiation Carcinogenesis

XRF Definition X-ray Fluorescence.

Further Information http://www.xiphophorus.org/

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▶Lead Exposure

X

Y

YAC

YKL-40

Definition

Definition

Acronym of yeast artificial chromosomes. A vector system used to clone large DNA fragments.

Is a member of the mammalian chitinase-like proteins. Elevated serum values of YKL-40 have been reported in a variety of cancers, including breast, colon/rectum, ovary, lung, kidney, glioblastoma, and melanoma. YKL-40 levels may also be increased in other diseases with an inflammatory component.

▶ArrayCGH

Yin and Yang

▶Serum Biomarkers

Definition Is a philosophical concept developed in ancient China. According to the theory of yin and yang, things and phenomena in the natural world oppose and complement each other. Yin and yang not only means two things opposing each other, but they also means two opposite components in the same thing. The contradiction movement between unity and opposite of yin and yang exists universally within everything and its consequence is the occurrence, development, and changes of all things in the universe. The theory of yin and yang has been widely used in Chinese medicine to refer to various antitheses in anatomy, physiology, pathology, diagnosis, and treatment, such as feminine, interior, cold, and hypofunction being yin while masculine, exterior, heat, and hyperfunction being yang.

YM-529 ▶Minodronate

YM-529/ONO-5920 ▶Minodronate

▶Chinese versus Western Medicine

Yinxing ▶Ginkgo Biloba

Yondelis ▶Trabectedin

Z

Z´ factor ▶Z-Factor

Zeolite Definition Are inorganic, porous, lattice-like structures in which the principal bases are aluminum, calcium, and magnesium. Zeolites often are found where volcanic rock has been immersed in water with resulting leaching of internal components. ▶Asbestos

Z-Factor J I -H U Z HANG 1 , K EVIN R. O LDENBURG 2 1

Lead Discovery Center, Novartis Institute for Biomedical Research, Cambridge, MA, USA 2 MatriCal, Inc., Spokane, WA, USA

Synonyms Z′ factor

Definition The data quality of an assay can be estimated by the Z′-factor formulae: Z 0 ¼ 1 ð3cþ þ 3c Þ=ðjcþ c jÞ; where (σc+) and (σc−) is the data standard deviation for the high reference control and low reference control, respectively, and |μc+–μc−| is the absolute value of the

difference of the two control signal means and it defines the usable dynamic range (usually the linear range) of the assay.

Characteristics Z factor (Z′ factor) is a statistical data quality indicator for a bioassay, particularly that used in the field of high throughput screening (HTS). In most HTS programs in drug discovery, each compound from a chemical library is only evaluated in a single test during primary screening. A high degree of accuracy and sensitivity in the assay is therefore critical for identifying active compounds (or “hits”). Due to various unavoidable sources of errors, the measurements from any assay will contain a certain degree of variability. Yet hits need to be identified in the presence of such variations in signal measurement. Reducing the measurement variation will give higher fidelity of the screening data. Therefore, in the design and validation of HTS assays, assessment of the screening data variability, by metrics such as the standard deviation (SD) and coefficient of variation (CV) of the replicate data set, is critical in determining whether an assay can identify hits with high confidence. The quality of an assay has also been loosely expressed as signal-to-noise ratio (S/N) and/or signalto-background ratio (S/B). Both ratios reflect the assay signal strength and therefore are useful parameters in assay quality assessment. In HTS, the compound activity is normally expressed as a percentage measured against some selected reference control signals, usually a high reference control and a low reference control. However, both S/N and S/B ratios fail to simultaneously take into account all of the data variability information of the sample and reference controls, as well as the assay dynamic range. To overcome some of the pitfalls of S/N or S/B ratios, Zhang and Oldenburg (1999) proposed a new, simple statistics, the Z factor (Z′ factor) as defined above, for use in evaluation of assay performance and assay signal robustness in HTS. The Z′-factor contains data variability information of both the high and low reference controls (the same high and low reference controls usually used for normalization of the measured raw data to a percentage activity) as well as the dynamic range information. Therefore, compared to CV, S/B or S/N, the Z′-factor is more

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Zinc-Finger Proteins

appropriate for evaluating overall assay quality. It is a useful tool for the evaluation, comparison, and validation of any assays in general and has been widely used for quality control (QC) of HTS assay quality. As originally proposed, the Z-factor refers to the specific Z′-factor when the mean and standard deviation of one of the corresponding reference controls are replaced by those of the testing samples. Therefore, the Z-factor is affected by the screening conditions such as the compound concentration. Z-factor has been used as a screening quality assurance parameter for assay plates owing to its sensitivity toward signal strength and data variability of test samples on the plate as well as the hit rates on the plate. Generally, a assay with Z′-factor value of close to 1 is an ideal assay for HTS, with Z′ values between 0.5 and 1 are considered good quality, and with values between 0.5 and 0 are considered to have a moderate to poor quality. An assay with Z′10-fold, the Z′-factor values can become less effective in indicating the assay data quality. Another limitation is that, due to its dependence on two reference control states of the assay, sometimes the Z′-factor becomes a less defined measure of assay quality for stimulator or agonist screens when there is no known stimulator/agonist reference control available. When there is a strong or natural agonist/ stimulator available as reference control, the Z′-factor can be rightfully defined. Connections to Other Assay Data Quality Indicators Besides the commonly used Z′-factor, S/B and S/N ratios, there are several other assay performance statistical parameters used in the HTS literature. A “signal window” (SW) ratio had been reported to evaluate the performance of HTS assays. The so called assay variation ratio (AVR) was also used to evaluate assay quality. AVR and Z′-factor are inter-related and inter-changeable to one another, except that the original recommended values for the acceptable and unacceptable assay for screening were somewhat discrepant.

Some simulation studies compared the performance of the Z′-factor with both the SW and AVR and recommended the Z′-factor as a preferred assay performance measure. The power analysis is another approach for assay quality indication. However, the power analysis is usually based on one reference state of the assay signal distribution, and may not reflect the quality of the assay over the entire usable signal dynamic range. Compared to the power analysis, the Z′-factor is a much simpler statistical parameter to use. Z′-factor can be used in conjunction with CV, S/B or S/N to give a more comprehensive assessment of the assay variation and performance. Z´-factor and Lower Limit of Detection of an Assay The assay sensitivity can be evaluated by the lower limit of detection (LLD) of the assay measurement. The LLD as assay sensitivity limits are evaluated in terms of signal strength versus variability. The S/N ratio is a good indicator for strength of signal. The concentration of an analyte that gives a S/N = 3 is conventionally regarded as the LLD of the assay measurement with regard to that analyte. Similarly, one can use an analyte concentration that gives Z′ = (−1) as LLD of the assay.

References 1. Buxser S, Vroegop S (2005) Calculating the probability of detection for inhibitors in enzymatic or binding reactions in high-throughput screening. Anal Biochem 340:1–13 2. Iversen PW, Eastwood BJ, Sittampalam GS et al. (2006) A comparison of assay performance measures in screening assays: signal window, Z′ factor, and assay variability ratio. J Biomol Screen 11:247–252 3. Sittampalam GS, Iversen PW et al. (1997) Design of signal windows in high throughput screening assays for drug discovery. J Biomol Screen 2:159–169 4. Talor PB, Stewart FP Dunnington DJ et al. (2000) Automated assay optimization with integrated statistics and smart robotics. J Biomol Screen 5:213–225 5. Zhang JH, Chung TYD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73

Zinc-Finger Proteins Definition A zinc finger is a protein domain that can interact with DNA. Folding of the polypeptide chain, into finger-like projections that can intercalate with the DNA helix, is stabilized through interactions with a zinc atom. Many

Zoledronic Acid

transcription factors and other regulatory proteins that interact with DNA contain zinc finger domains. ▶Mineral Nutrients

Zinc Transporters Definition Proteins that coordinate zinc ions and transport them to different intracellular compartments. ▶Snail Transcription Factors

ZO-1 Definition Zonula occludens-1 was the first tight junction protein identified. It serves to link the actin cytoskeleton to proteins that control cell-cell contacts and paracellular permeability in ▶tight junctions. ▶Cortactin ▶Zonula Occludens Protein-1

Zoledronic Acid D OMINIQUE H EYMANN Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, University of Nantes, Nantes, France

Definition Zoledronic acid is a stable synthetic derivative of the naturally occurring endogenous pyrophosphate, which acts as a powerful anti-bone degradation drug and exhibits anticancer activities.

Characteristics Structural Characteristics Zoledronic acid belongs to a large drug family named ▶bisphosphonate derived from endogenous ▶pyrophosphate (Fig. 1). In contrast to pyrophosphate, bisphosphonates have a carbon atom replacing the central oxygen which is associated with two additional

3229

substituents. There are three main generations of bisphosphonates: (i) the first generation, including compounds called clodronate and etidronate, possess simple substituents attached to the central carbon; (ii) the second generation such as pamidronate, alendronate, and ibandronate are characterized by aliphatic side chain containing a single nitrogen; (iii) the third generation such as risedronate and zoledronic acid is composed by a heterocycle side chain containing one or two nitrogen atoms. Thus, zoledronic acid is a nitrogen bisphosphonate composed by two nitrogen atoms in an imidazole ring (Fig. 1). Zoledronic acid, like the other bisphosphonates, possesses a high affinity for calcified matrix such as bone tissue via its phosphate groups. In vitro Activities of Zoledronic Acid and Mechanisms of Action Due to the high tropism of bisphosphonates for ▶hydroxyapatite crystals in bone and the ability of ▶osteoclasts to release bone-bound bisphosphonates, osteoclasts represent the main target of zoledronic acid. Thus, after administration, zoledronic acid selectively concentrates on the bone surface at the interface with the active osteoclasts where bone tissue is most exposed. Zoledronic acid is one of the most potent bisphosphonates in terms of anti-bone resorptive activity. While the first generation of bisphosphonates has a relative potency of 1–10, the potency of the second generation reaches 10–10,000, and zoledronic acid has a relative potency more than 10,000. To exert its activities, zoledronic acid must be internalized by the cells. Although the internalization of zoledronic acid is still controversial, two mechanisms have been proposed to explain its entry into the cells: in the first case, the cellular uptake of zoledronic acid would require fluidphase endocytosis, and in the second case, ▶integrins located at the cell membrane would represent the binding site of zoledronic acid which could explain why zoledronic acid is able to inhibit cell adhesion. The main targets of nitrogen-bisphosphonates are the two enzymes involved in the ▶mevalonate pathway: farnesyl diphosphate synthase (FPP) and geranylgeranyl diphosphate synthase (GGPP). FPP and GGPP are required for the ▶prenylation of small GTPases (i.e., Ras, Rho, and Rac), a biochemical reaction essential for the anchorage of small GTPases to cell membranes and to protein–protein interactions. In fine, their inhibition by zoledronic acid results essentially in the blockade of osteoclast function and also osteoclastogenesis decreasing the proliferation, viability, and recruitment of preosteoclasts. In addition to their powerful anti-▶bone resorption effects, recent in vitro studies evidenced a direct antitumor activity exerted by zoledronic acid on several cancer cells including breast and prostate ▶carcinomas, ▶osteosarcoma, ▶neuroblastoma, ▶multiple myeloma.

Z

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Zoledronic Acid

Zoledronic Acid. Figure 1 Zoledronic acid structure compared with pyrophosphate and basic bisphosphonate structure, characterized by a central carbon substituted by two phosphate groups and two other substituents R1 and R2.

Similar to its effects on osteoclasts, zoledronic acid treatment impairs protein geranyl-geranylation in these cancer cells. The main biological effects of zoledronic acid on malignant tumor cells include the inhibition of cell adhesion (associated with an alteration of cytoskeleton organization), of cell proliferation, and induction of cell death. Indeed, zoledronic acid treatment results in the cell cycle arrest in S and G2/M phases through the control of the intra-S DNA checkpoint and induces cell death independently of ▶caspase activation but via the mitochondrial pathway, in particular, the apoptosisinducing factor and endonuclease G translocation. Furthermore, zoledronic acid exerts its antitumor activity independently of the ▶p53 and ▶retinoblastoma (Rb) protein status of the cells. This point is of paramount importance, because several mutations or inactivations of the ▶antioncogenes p53 and Rb are detected in high percentage of patients suffering from malignant pathologies. Zoledronic acid also exerts synergistic effects in vitro with conventional ▶chemotherapy (carboplatin, cisplatin, ΥDFUR, docetaxel, epirubicin, fluvastatin, gemcitabine, imatinib, paclitaxel, trastuzumab, or vinorelbine, etc) on cancer cells. Antitumor and Bone Resorption Activity of Zoledronic Acid in Preclinical Models Although nitrogen-bisphosphonate has been widely used to treat ▶osteoporosis and to limit the decrease of bone mineral density, zoledronic acid has been successfully used to reduce the skeletal complications (▶osteolysis, hypercalcemia) associated with bone ▶metastases. According to the in vitro studies, several animal models demonstrated that zoledronic acid can reduce skeletal-tumor burden in prostate, breast, and renal carcinomas, multiple myeloma, osteosarcoma, chondrosarcoma. For instance, zoledronic acid inhibits the development of ▶osteoblastic bone lesions, as well as

osteolysis in a murine model of ▶prostate cancer. The efficacy of zoledronic acid can be explained by several mechanisms. First, a “vicious cycle” has been described in osteolytic tumors consisting in the activation of osteoclasts by mediators produced by tumor cells, which in turn release osteoclastic factors and/or bone-stocked factors favorable to the proliferation of cancer cells. In this context, zoledronic acid inhibits osteoclastogenesis and bone degradation and then reduces tumor growth. Second, zoledronic acid can affect directly the growth of tumor cells (induction of tumor-cell apoptosis and/or inhibition of tumor-cell proliferation and/or inhibition of tumor-cell spreading and invasion). Indeed, such direct activity can be underlined in nonosseous tumor models such as lung metastases. Thus, zoledronic acid diminishes osteosarcoma-induced lung metastasis in a murine model, thereby prolonging the animal survival. Third, zoledronic acid exerts an antiangiogenic activity and inhibition of tumor cell bone invasiveness by a transient reduction of circulating levels of several growth factors and matrix metalloproteinase. Moreover, it also sensitizes endothelial cells to cytokine-induced, caspaseindependent programmed cell death. Fourthly, zoledronic acid exerts a variety of immunomodulatory effects that might contribute to its antitumor activities. It stimulates the proliferation of a specific γδ T-cell subpopulation which exhibits cytotoxicity against numerous tumor cells. Taken together, the published studies demonstrated that zoledronic acid must be considered as a multifunctional molecule exerting both antiresorption and tumor activities. Therapeutic combinations of zoledronic acid and chemotherapeutic agents have also been assessed in animal models. Thus, in murine models of bone metastases, zoledronic acid combined with UFT (a combination drug of tegafur and uracil) decreases not only bone metastases but also lung metastases and

Zollinger-Ellison Syndrome

visceral metastases. In a rat osteosarcoma model, zoledronic acid associated with ifosfamide enhances tumor regression and tissue repair and increases animal survival by inhibiting lung metastases. Similarly, zoledronic acid associated with ▶Glivec increases the survival of the leukemia animals. In light of these preclinical data, the main benefit of combined treatment with zoledronic acid and anticancer drugs is to prolong significantly the life of cancer-bearing animals. Clinical Aspects On the basis of results obtained from randomized phase III clinical trials, zoledronic acid was approved in the United States for the treatment of patients with documented bone metastasis from solid tumors in conjunction with conventional chemotherapy and patients suffering from multiple myeloma. Zoledronic acid was also approved in Europe for the prevention of ▶skeletalrelated events (hypercalcemia, osteolysis, bone pain, fractures, etc) in patients suffering from advanced malignancies implicating bone. Thus, zoledronic acid as the other bisphosphonates has became the standard treatment for metastatic disease spread to the bone from prostate, breast, lung, and renal cancers and for multiple myeloma. Zoledronic acid therapy is generally well-tolerated, is safe for long period, and provides durable therapeutic benefits but can be associated with an upmodulation in serum creatinine. In this context, patients must be adequately hydrated, monitoring of renal function being required for all patients receiving zoledronic acid treatment. Caution must also be exercised for patients receiving other potentially nephrotoxic treatment. The current treatment guidelines recommend intravenous administration of 4 mg zoledronic acid diluted in calcium-free solution (approximately 100 ml of saline or 5% dextrose) and infused over 15 min, every 3–4 weeks. This recommended 15 min infusion time is significantly shorter than other i.v. bisphosphonate such as pamidronate (90 mg infusion for 2 h). As the optimal duration of the zoledronic acid treatment is not really documented, the treatment should be continued as far as it is well-tolerated or until significant effects on pathologic bone tissues are observed. Plasmatic concentrations of zoledronic acid quickly increase and reach their maxima at the end of infusion. Thereafter, these concentrations rapidly decrease (

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