Advances in Cancer Research

ADVANCES IN CANCER RESEARCH VOLUME 15

Contributors to This Volume

L.

Renato Baserga

J.

Ernest Borek

J. Paul

J. S. Butel

V. S. Shapot

Philip J. Fialkow

Gary Stein

J. H. C. Ho

Harold M. Swartz

Sylvia J. Kerr

S. S. Tevethia

A. J. MacGillivray

G. Threlfall

Melnick

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WEINHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania

Consulting Editor

ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital London, England

Volume 75

(29

ACADEMIC PRESS

New York and London

1972

COPYRIGHT 8 1972, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRl"EN PERMISSION FROM THE PUBLISHERS.

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New qork, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

24/28 Oval Road, London NW17DD

Lmmy

OF

CONORESS CATALOO CARDNUMBER:52-13360

PRINTED IN THE UNlTED STATES OF AMERICA

CONTENTS CONTRIBUTORS TO VOLUME15 . CONTENTSOF PREVIOUS VOLUMES

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ix xi

Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of The Viral Genome

J . S. BUTEL.S. S . TEVETHIA. AND J . L . MELNICK I. Introduction . . . . . . . . . . . . 11. Oncogenic Potential of SV40 in Vivo . . . . .

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93 101 124 146 150

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163 168

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I11. Transformation of Mammalian Cells in Vitro by SV40 IV . Phenotypic (Antigenic) Changes in Tumor and Transformed Cells V . Genotypic Changes in Tumor and Transformed Cells . . . VI . Conclusions and Summary . . . . . . . . . . References . . . . . . . . . . . . . .

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Nasopharyngeal Carcinoma (NPC)

J . H . C . Ho I . Introduction I1. Histogenesis I11. Etiology . IV . Conclusion References

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Transcriptional Regulation in Eukaryotic Cells

A . J . MACGILLIVRAY. J . PAUL. AND G . THRELFALL

I. Introduction . . . . . . . . . . . . I1. Eukaryotic Chromosomes . . . . . . . . I11. Control of Transcription in Eukaryotic Cells . . . . IV . Theories of Transcriptional Regulation in Eukaryotic Cells References . . . . . . . . . . . .

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Atypical Transfer RNA’s and Their Origin in Neoplastic Cells

ERNESTBOREKAND SYLVIAJ . KERR

I . Introduction . . . . . . . I1. The tRNA Methylases of Tumor Tissues V

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CONTENTS

I11. Transfer RNA of Tumor Tissues . . . . . . . IV . Regulation of tRNA Methylase Activity . . . . . V . Elevated Excretion of Modified Purines and Pyrimidines in Tumor-Bearing Animals and Humans . . . . . VI . The tRNA Methylases in Reverted Oncogenic Systems . . . . . . . . VII . An Attempt a t Interpretation References . . . . . . . . . . . .

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172 178

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180 184 185 187

Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females

PHILIP J . FIALKOW I . Introduction . . . . . . . . . . . . . I1. Interpretation of G-6-PD Phenotypes in Tumors . . . . I11. Hematopoietic Neoplasms . . . . . . . . . IV . Carcinomas . . . . . . . . . . . . . V . Benign Tumors VI . Study of Genetic Markers in Established Tieaue Culture Lines VII . Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . .

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191 195 199 209 213 219 221 223

Electron Spin Resonance Studies of Carcinogenesis

HAROLD M . SWARTZ I . Introduction . . . . I1. The ESR Technique . . 111. Experimental Results . IV . Summary and Conclusions References . . . .

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227 228 233 248 248

Some Biochemical Aspects of the Relationship between the Tumor and the Host

V . S. SHAPOT I . Introduction . . . . . . . . . . . . . . I1. Respiration of Tumors in Vivo . . . . . . . . . I11. Glucose Levels in Ascites Cancer Cells and Their Medium in Vivo IV . Glycolysis and Destructive Tumor Growth . . . . . . V . Relative Glucose Deficiency of the Tumors Growing in the Body VI . Tumor as a Glucose Trap in the Body . . . . . . . VII . The Problem of Cancer Cachexia . . . . . . . . VIII . Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

253 254 257 262

264 268 279 281 282

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CONTENTS

Nuclear Proteins and the Cell Cycle

GARYSTEINAND RENATO BASERGA

I . Introduction . . . . . . I1. The Biochemistry of the Cell Cycle I11. The Control of Cell Proliferation . References . . . . . . AUTHOR INDEX. SUBJECTINDEX

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CONTRIBUTORS TO VOLUME 15 Numben in parentheses refer to the pages on which the authors’ contributions begin.

RDNATO BASERGA, Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania (287)

ERNEST BOREK, Departments of Microbiology and Surgery, University of Colorado Medical Center, Denver, Colorado (163)

J. S. BUTEL,Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas (1) PHILIPJ. FIALKOW, Departments of Medicine and Genetics, University of Washington, Seattle, Washington (191)

J. H. C . Ho, Medical and Health Department Institute of Radiology, Queen Elizabeth Hospital, Kowloon, Hong Kong (57)

SYLVIA J. KERR,Departments of Microbiology and Surgery, University of Colorado Medical Center, Denver, Colorado (163) A. J. MACGILLIVRAY, Beatson Institute for Cancer Research, Glasgow, Scotland (93) J. L. MELNICK,Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas (1) J. PAUL, Beatson Institute for Cancer Research, Glasgow, Scotland (93) V. S. SHAPOT, Institute of Experimental Clinical Oncology, Academy of Medical Sciences, MOSCOW, USSR (253)

GARYSTEIN,Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania (287)

HAROLD M. SWARTZ, Departments of Radiology and Biochemistry, The Medical College of Wisconsin, Milwaukee, Wisconsin (227) S. S. TEVETHIA, Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas (1)

G. THRELFALL, Beatson Institute for Cancer Research, Glasgow, Scotland (93) ix

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CONTENTS OF PREVIOUS VOLUMES Volume 1

Carcinogenesis and Tumor Pathogenesis I. Berenblum Ionizing Radiations and Cancer Austin M . Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D. Fenninger and G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards Calvin T. Klopp and Jeanne C. Bateman Genetic Studies in Experimental Cancer

Electronic Configuration and Carchogenesis C. A . Coulson Epidermal Carcinogenesis E. V . Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Aspects of Experimental Tumorigenesis T . U. Gardner Properties of the Agent of b u s No. 1 Sarcoma R. J . C . Harris L. W . Law Applications of Radioisotopes to Studies of Carcinogenesis and Tumor The Role of Viruses in the Production Metabolism of Cancer Charles Heidelberger C . Oberling and M . Guerin The Carcinogenic Aminoazo Dyes Experimental Cancer Chemotherapy James A . Miller and Elizabeth C. C. Chester Stock Miller AUTHOR INDEX-SUBJECT INDEX The Chemistry of Cytotoxic Alkylating Agents M. C. J . Ross Volume 3 Nutrition in Relation to Cancer Etiology of Lung Cancer Albert Tannenbaum and Herbert Richard Doll SilVeTStO?W The Experimental Development and Plasma Proteins in Cancer Metabolism of Thyroid Gland Richard J . Winzler Tumors AUTHOR INDEX-SUBJECT INDEX Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: Volume 2 New Developments A. Pullman and B . Pullman The Reactions of Carcinogens with Macromolecules Some Aspects of Carcinogenesis Peter Alexander P. Rondoni Chemical Constitution and Carcinogenic Pulmonary Tumors in Experimental Activity Animals Michael B. Shimkin G. M . Badger

xi

xii

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sidney Weinhowe AUTHOR INDEX-SUBJECT

Volume 4

INDEX

The Newer Concept of Cancer Toxin Waro Nakahara and Fumiko Fukuoka Chemically Induced Tumors of Fowls P. R. Peacock Anemia in Cancer Vincent E . Price and Robert E. Greenfield Specific Tumor Antigens L. A. Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K. Weisburger and John H . Weis burger

Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A. G. Galton AUTHOR INDEX-SUBJECT INDEX The Employment of Methods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian OrganVolume 6 ism Abraham Goldin Blood Enzymes in Cancer and Other Diseases Some Recent Work on Tumor Immunity P. A. Gorer Oscar Bodansky Inductive Tissue Interaction in Develop- The Plant Tumor Problem ment Armin C. Braun and Henry N. Wood Clifford Grobstein Cancer Chemotherapy by Perfusion Lipids in Cancer Oscar Creech, Jr., and Edward T . Frances L. Haven and W . R. Bloor Krementz The Relation between Carcinogenic Viral Etiology of Mouse Leukemia Activity and the Physical and Ludwik Gross Chemical Properties of Angular Radiation Chimeras Benzacridines P. C. Koller, A. J . S. Dauies, and A. Lacassagne, N . P. Buu-Hoi, R . Sheila M . A . Doak Daudel, and F . Zajdela Etiology and Pathogenesis of Mouse The Hormonal Genesis of Mammary Leukemia Cancer J . F . A. P. Miller 0. Miihlbock Antagonists of Purine and Pyrimidine AUTHOR INDEX-SUBJECT INDEX Metabolites and of Folic Acid G. M . Timmis Behavior of Liver Enzymes in HepatoVolume 5 carcinogenesis George Weber Tumor-Host Relations AUTHOR INDEX-SUBJECT INDEX R. W . Begg Primary Carcinoma of the Liver Charles Berman Volume 7 Protein Synthesis with Special Reference to Growth Processes both Normal Avian Virus Growths and Their Etiologic Agents and Abnormal J . W . Beard P . N . Campbell

CONTENTS OF PREVIOUS VOLUMES

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Xlll

Mechanisms of Resistance to Anticancer The Relation of the Immune Reaction to Cancer Agents Louis Caso R. W . Brockman Cross Resistance and Collateral Sensi- Amino Acid Transport in Tumor Cells R. M. Johnstone and P. G. Scholefield tivity Studies in Cancer Chemotherapy Studies on the Development, BiochemDorris J . Hutchison istry, and Biology of Experimental Hepatomas Cytogenic Studies in Chronic Myeloid Harold P. Morris Leukemia W. M . Court Brown and Ishbel M . Biochemistry of Normal and Leukemic Tough Leucocytes, Thrombocytes, and Bone Marrow Cells Ethionine Carcinogenesk I . F. Seitz Emmanuel Farber Atmospheric Factors in Pathogenesis of AUTHOR INDEX-SUBJECT INDEX Lung Cancer Paul Kotin and Hans L. Falk Progress with Some Tumor Viruses of Volume 10 Chickens and Mammals: The Prob- Carcinogens, Enzyme Induction, and lem of Passenger Viruses Gene Action G. Negroni H . V. Gelboin AUTHOR INDEX-SUBJECT INDEX I n Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin Volume 8 The Enzymatic Pattern of Neoplastic The Structure of Tumor Viruses and Its Tissue Bearing on Their Relation to Viruses W . Eugene Knox in General Carcinogenic Nitroso Compounds A. F. Howatson P. N . Magee and J . M . Barnes Nuclear Proteins of Neoplastic Cells The Sulfhydryl Group and CarcinoHarris Busch and William J . Steele genesis Nucleolar Chromosomes : Structures, J . S. Harington Interactions, and Perspectives The Treatment of Plasma Cell Myeloma M . J. Kopac and Gladys M . Mateyko Daniel E. Bergsagel, K . M . Grifith, Carcinogenesis Related to Foods ConA. Haut, and W . J. Stuckey, Jr. taminated by Processing and Fungal AUTHOR INDEX-SUBJECT INDEX Metabolites H . F. Kraybill and M. B. Shimkin Experimental Tobacco Carcinogenesis Volume 1 1 Ernest L. Wynder and Dietrich Hoffmann The Carcinogenic Action and Metabolism of Urethan and N-Hydroxyurethan AUTHOR INDEX4UBJECT INDEX Sidney S. Mirvish Runting Syndromes, Autoimmunity, and Volume 9 Neoplasia D. Keast Urinary Enzymes and Their Diagnostic Viral-Induced Enzymes and the Problem Value in Human Cancer of Viral Oncogenesis Richard Stambaugh and Sidney WeinSaul Kit house

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xiv

CONTENTS O F PREVIOUS VOLUMES

The

Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology William Regelson Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C.Arcos and Mary F . Argus

AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE INDEX

Volume 12

Antigens Induced by the Mouse Leukemia Viruses G. Pasternak Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G. I . Deichman Replication of Oncogenic Viruses in Virus-Induced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hannfusa Cellular Immunity against Tumor An tigens Karl Erik Hellstrom and Ingegerd Hellstrom Perspectives in the Epidemiology of Leukemia Irving I . Kessler and Abraham M . Lilienfeld AUTHOR INDEX-SUBJECT

INDEX

Volume 13

The Role of Immunoblasts in Host Resistance and Immunotherapy of Primary Sarcomata P. Alexander and J . G. Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarrett

The Function of the Dclayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processcs and Their Relevance to the Study of Neoplasia Gnjanan V . Sherbet The Characteristics of Animal Cells Transformed in Vitro Inn Macpherson Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and I . Hloz'cinek Cancer of the Urinary Tract D. B . Clayson and E . H . Cooper Aspects of the EB Virus M . A. Epstein AUTHOR INDEX-SUBJECT

INDEX

Volume 14

Active Immunotherapy Georges Math6 The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Ernest Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events Georges M e y e r Passive Immunotherapy of Leukemia and Other Cancer Roland M o tta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalj Complement and Tumor Immunology Kusuya Nishwka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . I. Abeler Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT

INDEX

ONCOGENICITY AND CELL TRANSFORMATION BY PAPOVAVIRUS SV40: THE ROLE OF THE VIRAL GENOME1 J. S. Butel, S. S. Tevethia,' and J. L. Melnick Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas

I. Introduction . . . . . . 11. Oncogenic Potential of SV40 in Vivo

. . . . . . . . . . . . . . . A. Role of the Host Animal . . . . . . . . B. Role of Defective Viral Genomes . . . . . . . C. Factors Affecting Viral Oncogenicity . . . . . . . 111. Transformation of Mammalian Cells in Vitro by SV40 . . . . A. Transformation of Permissive and Nonpermissive Cells by SV40 B. Transfocmation by Defective SV40 . . . . . . . C. Double Transformation of Cclls . . . . . . . . D. Reversion of Transformed Cells . . . . . . . . IV. Phenotypic (Antigenic) Changes in Tumor and Transformed Cells . . . . . . . . . . . . . A. Tumor Antigen B. Tumor-Specific Transplantation Antigen (TSTA) . . . . C. Surface Antigen . . . . . . . . . . . . D. Relationship of S-Antigen to TSTA . . . . . . . V. Genotypic Changes in Tumor and Transformed Cells . . . . A. Rescue of Infectious Virus from Transformed Cells . . . B. State of the Viral Genome in Transformed Cells . . . . C. Transcription of the Viral Genome in Transformed Cells . . . D. Basis for Lack of Virus Production by Transformed Cells . . . . .

E. Consideration of Whether the Viral Genome Is Essential for Maintenance of Transformation . . . . . VI. Conclusions and Summary References . . . . . . . .

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I. Introduction

Simian virus 40 (SV40) has undergone intensive study by many investigators in the decade following its discovery (Sweet and Hilleman, 1960). It is one of the most, if not the most, well-understood model tumor virus containing deoxyribonucleic acid (DNA). 'Supported in part by research grant CA 04600 from the National Cancer Institute and research contract PH 43-68-678 within the Special Virus-Cancer Program of the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. * Recipient of Research Career Development Award 5-K3-CA 38,614 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 1

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A number of attributes have encouraged the selection of SV40 as the viral agent for so many studies: it can be readily propagated and accurately assayed in tissue culture; it can transform cells in witro as well as induce tumors in wivo; i t has a limited content of genetic information; and its nucleic acid can be isolated in an infectious form. It is a member of the papovavirus group (Melnick, 1962) and the family Papotraviridae (International Committee on Nomenclature of Viruses, 1971). Several recent reviews have covered different aspects of SV40 tumorigenesis (Rapp and Melnick, 1966; Black, 1968; Deichman, 1969; Rapp, 1967, 1969), so this chapter will not attempt to recapitulate all the recorded facts about the virus. Rather, we have posed questions pertinent to the phenomenon of oncogenesis and surveyed the literature for possible answers. The subjects considered, among others, are: ( a ) the role of complete and defective viral genomes in oncogenesis and transformation; ( b ) the antigenic changes which accompany transformation and their relationship to each other; (c) the virogenic and genotypic changes in transformed cells; ( d ) the failure to recover virus from tumor cells; ( e ) the requirement for a persisting viral genome to ensure the maintenance of the transformed state, and (f) the factors which affect malignancy of transformed cells. Obviously, all the answers to the above questions are not yet known, but some intriguing clues have been obtained. We have attempted to evaluate critically the available knowledge of SV40 oncogenesis and to assess the implications for carcinogenesis by DNA-tumor viruses as a whole. I I . Oncogenic Potential of SV40 in Vivo

HOSTANIMAL The oncogenic potential of SV40 has been demonstrated only in hamsters (Eddy et al., 1962; Girardi et al., 1962; Ashkenazi and Melnick, 1963; Black and Rowe, 1964). Efforts to induce tumors by SV40 in mice, guinea pigs, rats, rabbits, and monkeys have been unsuccessful (Eddy, 1964). The latent period of tumor induction by SV40 in hamsters ranges from 3 months to more than a year, depending upon the concentration of virus employed and the age of the animal a t the time of inoculation. The incidence of tumors in animals inoculated as newborns is usually over go%, but it is variable in older animals. Girardi et al. (1963) reported that 23% of hamsters inoculated a t 1 month of age ultimately developed tumors with an average latent period of 360 days. In other studies, A. ROLE OF

THE

PAPOVAVIRUS

SV40

3

tumors failed to appear in hamsters inoculated at 3 weeks of age, although some hamsters inoculated a t 4 months of age developed tumors when held for long periods (Allison et al., 1967). Newborn hamsters are susceptible to the oncogenic effects of SV40 whether inoculated by the subcutaneous, intracerebral, intraperitoneal, or intrathoracic route. However, no tumors were observed when newborn hamsters were inoculated by the oral and intranasal routes (Eddy, 1964). Of the preceding methods of inoculation, the subcutaneous route is the one of choice since neoplasms are visible and can be scored soon after appearance. There seems to be some correlation between the concentration of the virus inoculum and the latent period for tumor development when animals are injected by the subcutaneous route (Eddy et al., 1962). DNA isolated from SV40-infected monkey cells is also oncogenic in these animals (Boiron et al., 1965). Histologically, SV40-induced tumors in subcutaneous tissues, lungs, and kidneys have been designated as undifferentiated sarcomas, although some fibrosarcomas have been reported (Eddy, 1964). Intracerebral inoculation of the virus into newborn hamsters resulted in the development of ependymomas (Kirschstein and Gerber, 1962). Induction of ependymomas by SV40 in mastomys has also been reported (Rabson et al., 1962) although no evidence was presented that the observed neoplasms were related to SV40. X-Irradiation of adult animals prior to virus inoculation enhances their susceptibility to tumor induction (Allison et al., 1967). Also, adult animals inoculated in the cheek pouch with SV40 were found to develop tumors after a latent period of only 97 days, as compared with a latent period of 490 days when animals of the same age were inoculated by the subcutaneous route. All tumors which developed were SV40 tumors based on the presence of SV40 transplantation antigen (Allison et al., 1967). The study indicated that potentially malignant cells can remain dormant in the animal for a long period of timc and that depression of the immune response of the host will result in the growth of such “dormant” cells. Tumor induction in mice by SV40 has not been demonstrated, even by the use of immunosuppressed animals (Tevethia, 1971). The virus can infect mouse cells rather easily, as shown by ( a ) transformation experiments in vitro using 3T3 mouse cells (Black and Rowe, 1963b; Todaro and Green, 1964) ; ( b ) the elevated enzyme levels and increased DNA synthesis following infection of mouse kidney cells (Kit et al., 1966b), and (c) the ability of the virus to induce SV40 specific transplantation immunity in mice (Kit e t al., 1969; Wesslh, 1970). It appears

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J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

that a mouse cell may possess many of the characteristics of transformed cells after infection with SV40 and yet not be able to grow as a tumor cell.

B. ROLEOF DEFECTIVE VIRALGENOMES Viral defectiveness may be divided into two broad categories-conditional and nonconditional. Conditionally defective viruses are unable to complete the cycle of replication under certain conditions, such as in a nonpermissive host cell, in the absence of a helper virus, or at an elevated temperature [ temperature-sensitive (ts) mutants]. I n contrast, nonconditionally defective viruses are not able under any known conditions to complete a replicative cycle. In this section, the significance in oncogenesis is considered of both conditionally defective helper-dependent viruses as well as other viral preparations which may, or may not, be nonconditionally defective. The defective viruses may be conveniently subdivided into those which are produced by some exogenous treatment of the virus stock, such as irradiation, and those which occur naturally (Table I). TABLE I TYPES OF DEFECTIVE SV40 GENOMES SHOWN TO BE ONCOGENIC A. Produced by exogenous treatment 1. Irradiated virus 2. Hydroxylamine-inactivated virus B. Naturally occurring 1. T-antigen-inducing defective particles 2. PARA(defective SV40)-adenovirus hybrid population

Defendi and Jensen (1967) demonstrated that, after inactivation by ultraviolet or by gamma radiation, SV40 not only retained its oncogenicity for newborn hamsters, but actually exhibited an enhanced tumorproducing capability when compared to that of the untreated virus. Most of the virus in the irradiated samples had been inactivated as determined by infectivity assays. Similar results were reported by Altstein et al. (1967a) using hydroxylamine-inactivated virus. The authors postulated that their results could be explained if the oncogenic potential of SV40 is actually due to the presence of defective particles in virus stocks. After inactivation of the virus preparation, the concentration of these hypothetical defective particles would increase, and these, in turn, would be responsible for the observed enhancement in oncogenicity. However, defective virions may not be the only explanation for oncogenicity because of some other unexplained observations: ( a ) complete virus genomes can be recovered from some transformed cells (Section V,A), ( b ) nononcogenic variants of defective SV40

PAPOVAVIRUS

SV40

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(PARA) have been described (see below) and (c) inactivated virus preparations that exhibit increased transforming capacity in vivo do not do so in vitro (Section 111,B). One other possible explanation offered for enhanced levels of oncogenicity is that tumors produced by defective viruses may lack tumor-specific transplantation antigens (TSTA) , the absence of which allows the tumors to grow without being influenced by the hosts’ immune mechanisms. However, no experimental evidence has been provided to support this claim. Naturally occurring SV40 particles which are defective for the synthesis of late viral proteins and infectious virus, but which are able to induce the synthesis of SV40 tumor (T) antigen, have been described (Sauer e t al., 1967; Altstein e t al., 196713; Uchida e t al., 1968). The concentration of defective particles in preparations of SV40 has been shown to increase after serial passage using undiluted inocula (Uchida et al., 1966) . The naturally occurring defective particles were oncogenic in newborn hamsters (Uchida and Watanabe, 1969). The tumors induced by the defective viruses contained SV40 T-antigen. A defective SV40 genome (PARA) carried by an unusual strain of human adenovirus type 7 has been described (Huebner et al., 1964; Rowe and Baum, 1964; Rapp e t al., 1 9 6 4 ~ )For . a current review of the properties of PARA-adenovirus populations, see Rapp (1971). The PARA genome is defective in that i t cannot code for SV40 coat protein; it is encased in adenovirus capsids supplied by helper adenovirions present in the mixed population (BoBye e t al., 1966; Butel and Rapp, 1966a; Rowe and Baum, 1965). PARA-adenovirus 7 produces SV40 type tumors in newborn hamsters (Huebner e t al., 1964; Rapp et al., 1966a), induces the synthesis of SV40 T-antigen in vitro (Rowe and Baum, 1964; Rapp e t at., 1 9 6 4 ~ )and ~ induces SV40 TSTA in weanling hamsters (Rapp et al., 1966b, 1967a). The discovery and subsequent characterization of PARA provided convincing evidence that late functions associated with the synthesis of capsid proteins and maturation of progeny virions are not required for the induction of SV40 T-antigen, TSTA, or oncogenicity. The tumors induced by the original PARA-adenovirus 7 population had latent periods similar to those induced by parental SV40, but the oncogenic potential vaned following “transcapsidation” (Rapp e t al., 196523) to a series of different human adenoviruses (Rapp et uZ., 1968). PARA particles which had been transcapsidated to adenovirus types 14, 16, and 21 were nononcogenic, whereas transcapsidant PARA-adenovirus population types 1, 2, 3, 5, and 6 were oncogenic in newborn hamsters. However, both the nononcogenic and oncogenic PARA-adenovirus populations were capable of inducing SV40-specific transplantation immunity in weanling animals. When PARA was transcapsidated from

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J . S. BUTEL, 6 . S. TEVETHIA, AND J . L. MELNICK

the nononcogenic adenovirus 21 population to adenovirus 6, it remained nononcogenic, whereas PARA from the oncogenic adenovirus 6 population retained its oncogenic potential upon transcapsidation to adenovirus type 21 (Rapp et al., 1970). Thcse results suggested that variants of PARA which differed in oncogenicity were present in the original population of PARA-adenovirus 7. Such variants were then isolated from the parental PARA-adenovirus 7 population by two successive plaque purifications in monkey kidney cells; 20 of the 112 cloned lines of virus tested were shown to be nononcogenic (Rapp et al., 1969). The nononcogenic variants of PARA were capable of transforming hamster cells in vitro (Butel et al., 1971c; Rapp and Duff, 1971) ; however, the transformed cell lines which were established varied in transplantability (Butel et al., 1 9 7 1 ~ ) . When PARA was transcapsidated from adenovirus type 7 to the highly oncogenic Huie strain of adenovirus type 12, it produced SV40 tumors in newborn hamsters with a short latent period of only 5-7 weeks (Butel et al., 1971a). All the early-appearing tumors induced by PARAadenovirus 12 contained SV40 T-antigen. Parental SV40 and PARAadenovirus 7 did not induce any tumors within the same time period. Artificial mixtures of adenovirus 12 and SV40 or adenovirus 12 and PARA-adenovirus 7 did not induce any tumors containing SV40 Tantigen within 5-7 weeks. Early SV40 tumors induced by PARAadenovirus 12 contained SV40 TSTA, ruling out the absence of antigen as the cause of the early appearance of the SV40 tumors. One possible explanation for the results with PARA-adenovirus 12 was that recombination had occurred between a portion of the adenovirus 7 region of the PARA genome and a portion of the adenovirus 12 genome. Such recombinant particles might contain the adenovirus 12 marker (s) responsible for the rapid appearance of tumors, and, in the process of tumor development, the linked SV40 information would be expressed. There is no direct proof yet available, however, that the postulated recombinational event actually occurred. Defective viruses can be very useful tools for determining viral functions necessary for oncogenicity. They can be replicated with the aid of a helper virus which, if it is nononcogenic, does not interfere with subsequent tests in vivo. With a conditionally defective virus which may have lost part of the parental genome, the system is not complicated by any possibility of leakiness or reversion on the part of the mutant. I n addition, strict conditions do not have to be imposed to ensure defectiveness as is the case, for example, with temperature-sensitive mutants. With the ts mutants, unfortunately, the body temperature of the intact animal required for oncogenicity studies is frequently that of the permissive temperature.

PAPOVAVIRUS

SV40

7

C. FACTORS AFFECTING VIRALONCOGENICITY As described above, if SV40 is inoculated into hamsters within 24 hours of birth, tumors will develop in a majority of the animals with latent periods ranging from 3 to 6 months. Without exception, all SV40induced tumors are antigenic and contain TSTA a t the cell surface (see Section IV,B). Polyoma virus can induce the development of tumors in certain strains of mice if inoculated into newborns. However, C57B1, strain A, and C3H/Lw mice are resistant t o polyoma virus oncogenesis (Law, 1969). If the resistant strains were made immunologically deficient by thymectomy a t birth, they became susceptible to polyoma virus tumor induction. Similar results were obtained when the resistant strains were treated with antilymphocytic serum. The immunologic capacity of the thymectomized mice and the resistance to polyoma virus oncogenesis could be restored by intravenous injection of syngeneic spleen or lymph node cells from intact animals, The role of immunocompetence in resistance to SV40 oncogenesis in hamsters was demonstrated by Allison et al. (1967))when they showed that tumor development in hamsters was dependent upon the age of the animal at the time of virus inoculation. SV40 produced tumors in adult animals only after X-irradiation of the host. Tumors also developed when virus was inoculated into the cheek pouch of animals (an immunologically privileged site). These studies suggested the following sequence of events upon inoculation of the virus into an adult animal: ( 1 ) cells are transformed, ( 2 ) TSTA a t the transformed cell surface sensitizes the host, and (3) potential tumor cells are eliminated. This sequence of events was first postulated by Habel (1962) and is supported . thymocyte by data in the report by Tevethia et al. ( 1 9 6 8 ~ )Antihamster serum, a potent suppressor of cellular immunity, prevented the sensitization of animals to SV40 TSTA when administered to hamsters during the period of virus immunization. The failure to become sensitized to TSTA was demonstrated by the fact that treated animals were unable to reject a challenge of SV40-transformed cells. In addition, spontaneous regression of primary tumors induced by SV40 in hamsters has been observed (Tevethia et al., 1968a; Deichman, 1969). The appearance of primary tumors in hamsters inoculated as newborns requires discussion in view of the fact that SV40 tumors have long latent periods and contain TSTA. The development of immunologic tolerance to SV40 TSTA can be ruled out, since SV40 oncogenesis can be blocked by immunization of the animals during the latent period with either virus or virus-transformed cells (Goldner et al., 1964; Girardi, 1965; Tevethia et al., 1968b). Such immunization, however, is ineffective

8

J . S. BUTEL, S. 8 . TEVETHIA, AND J . L. MELNICK

in thymectomized animals (Girardi and Roosa, 1967). Hamsters undergoing viral oncogenesis do not become sensitized to SV40 TSTA before the appearance of palpable tumors (Deichman and Kluchareva, 1964). However, animals bearing tumors become sensitized and can reject a challenge of SV40-transformed cells at a site distant from the primary tumor (Lausch and Rapp, 1971), which may explain the absence of widespread metastases in tumor-bearing animals. There are several possible mechanisms to explain the growth of primary SV40 tumors: 1. A transformed focus in a newborn animal may be established before development of immunocompetence. If the transformed focus reaches a certain critical size, it may sensitize the host without being rejected. However, this mechanism is unlikely since SV40 tumors have long latent periods (100-200 days) and the animals become immunocompetent long before the appearance of palpable tumors (Friedman and Goldner, 1970a). Also, infection of animals with SV40 does not lead to generalized immunosuppression (Friedman and Goldner, 1970b). 2. The amount of TSTA present in the developing focus of transformed cells might not be enough to sensitize the host. By the time the concentration of TSTA becomes sufficiently high to sensitize the host, the tumor might already have grown to a size such that it cannot be rejected by the immune lymphocytes. I n support of this, Deichman and Kluchareva (1964) demonstrated that hamsters inoculated with SV40 as newborns were as susceptible to challenge with SV40-transformed cells as control animals. The fact that SV40 oncogenesis can be prevented by immunization of hamsters during the latent period with either SV40 or transf,ormed cells indicates a block a t the efferent level of the immunological arc. Insufficient TSTA to sensitize the animals undergoing viral oncogenesis can be ruled out since only a single inoculation of virus during the latent period is enough to prevent tumor appearance. Deichman (1969) advanced a hypothesis to explain the difference between newborn and adult animals with respect to the development of transformed foci. She proposed that the development of TSTA in virusinfected cells and the transformation of the cells are separate functions. She postulated that TSTA is synthesized in cells of both newborn and adult animals infected with SV40. In newborn animals, however, TSTA would disappear from most of the cells perhaps because of the fast rate of cell division; the number of cells which ultimately transform would not be large enough to sensitize the host. In contrast, in adult animals TSTA would persist long enough to sensitize the host. I n support of this hypothesis, Kluchareva et al. (1967) showed that hamster cells infected

PAPOVAVIRUS

SV40

9

with SV40 in vitro developed SV40 TSTA which could immunize a host. 3. Blocking antibody (K. E. Hellstrom and Hellstrom, 1970) may be responsible for the growth of primary SV40 tumors. Such antibody molecules bind to TSTA and protect the antigenic sites from the action of immune lymphocytes. The possibility that blocking antibodies are responsible for the growth of primary tumors before they become palpable is unlikely, for the following reasons: ( a ) Blocking antibodies have been demonstrated only in tumor-bearing animals and cannot be detected before the development of the tumor or after the tumor is surgically removed. ( b ) Animals undergoing SV40 oncogenesis behave as relatively nonsensitized animals before the appearance of palpable tumors. Blocking antibodies may play an important role, however, in guarding the tumor cells from the action of immune lymphocytes once the animal has been sensitized. 4. The failure to demonstrate immunity to SV40 TSTA in hamsters undergoing SV40 oncogenesis may be explained by the immobilization of the immunoblasts in the lymph nodes draining the tumor site (Alexander et al., 1969). The release of the immunoblasts was achieved either by surgical removal of the tumor or by immunization of the host with tumor cells. These results may explain why SV40 oncogenesis can be prevented by immunization of the host during the latent period. Although it is well established that SV40 can induce oncogenesis in v i m , the role of the viral genome in the acquisition of malignant potential by cells transformed in vitro is not known. Transformed hamster cells can usually be readily transplanted in adult animals. I n contrast, mouse cells transformed by SV40 can be transplanted in syngeneic animals only with great difficulty, frequently requiring depression of the immune response of the host (Takemoto et al., 1968a). Prolonged cultivation in vitro of the transformed cells is also often necessary before a successful transplant in vivo can be achieved (Kit et al., 1969; Wesslkn, 1970). Enders and Diamandopoulos (1969) observed that hamster heart cells transformed by SV40 became highly transplantable after passage in vivo. Sixteen clones were isolated from the original population of transformed cells and a wide variation was found in the transplantability of the clonal lines. Some clones were highly transplantable whereas others failed to produce tumors unless more than a million cells were inoculated per animal. When the lines of apparent low transplantability were passaged in vivo, the oncogenic potential of the cells increased markedly. These studies indicated that cells of high oncogenicity may be selected in viva and that cellular mutations may be responsible for the increase in oncogenic potential. The authors concluded that the viral genome did

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J . S. BUTEL, S. S . TEVETHIA, AND J . L. MELNICK

not appear to be responsible for the increase in transplantability of the transformed cells. Variation in the transplantability of hamster lung and kidney cells transformed by various PARA-adenovirus hybrid populations has been observed by Butel et al. ( 1 9 7 1 ~ ) Virus . clones which appeared to be nononcogenic in newborn hamsters were able to transform hamster cells in vitro, but the transplantability in weanling hamsters of early passages of the transformed cells varied greatly. Some of the cell lines were readily transplantable whereas other lines transformed by the same clone of virus were either not transplantable or could be transplanted only with difficulty. The mechanism responsible for the observed differences in transplantability of transformed cells is not known. However, immunological factors which may operate a t either the level of the host or that of the tumor cell may be important. All cells transformed by SV40 contain TSTA which mediates the development of specific resistance against transplantation of the transformed cells. Most lines of SV40-transformed mouse cells are not transplantable in the syngeneic host, but can be transplanted in immunosuppressed animals (Takemoto et al., 1968a; Tevethia, 1971) indicating the role of immunological surveillance on the part of the host. This hypothesis is supported by the observation that an animal bearing a progressively growing SV40-transformed cell transplant is immune to a second transplant of the same cells a t a distant site. This concomitant immunity is viral-specific (Lausch and Rapp, 1971). However, SV40-transformed cells can become immunoresistant and are then capable of growing in immune animals. Deichman and Kluchareva (1966) reported the loss of SV40 TSTA from cells taken from metastatic tumors. SV40-immunized animals were unable to reject tumor cells derived from lung metastases. However, these cells were not tested for their capacity to immunize hamsters against a challenge of an immunosensitive cell line to definitely prove they lacked SV40 TSTA. There is the possibility that the cells from the lung metastases were simply immunoresistant in view of the fact that Tevethia et al. (1971) have isolated immunoresistant variants from a population of SV40-transformed cells. These variants are not rejected by SV4O-immunized animals but can immunize hamsters against a challenge of immunosensitive tumor cells, showing that the immunoresistant cells do contain TSTA. Major factors that may contribute to immunorcsistance include (a) a faster rate of cell growth in v i m , ( b ) lower concentrations of TSTA, ( c ) masking of TSTA by mucopolysaccharides, and ( d ) interference by blocking antibodies. Beyond these reports, which suggest that immunological factors may

PAPOVAVIRUS

SV40

11

play a role, very little is kn0w.n about the event(s) which determine the malignancy of a transformed cell. One factor which may affect the transplantability of transformed cells may be surface changes other than SV40 TSTA. These changes may be reflected in either increased or decreased transplantability of tumor or transformed cells. Deichman and Kluchareva (1966) reported that SV4O-transformed cells, when grown in vitro in the presence of sera from SV40 tumor-bearing animals, became immunoresistant. This change was not observed when the cells were grown in the presence of sera from hamsters which had rejected SV40 tumors or from animals bearing polyoma tumors. These findings strongly suggest the presence of antigens which can act as TSTA but are capable of “modulation.” Conclusive evidence favoring this hypothesis is still lacking. Ill. Transformation of Mammalian Cells in Vifro by SV40

Viral transformation has been defined as a n induced inheritable change in the properties of a cell, accompanied by the loss of regulatory controls of cell growth. The criteria for transformation of cells generally include the following (Enders, 1965) : (a) loss of contact inhibition, ( b ) altered morphology, (c) increased growth rate, ( d ) increased capacity to persist in serial subcultures, ( e ) chromosomal abnormalities, ( f ) increased resistance to reinfection by the transforming virus, (9) emergence of new antigens, and ( h ) capacity to form neoplasms. Black (1968) recently summarized the details of various SV40-host cell transformation systems. Consequently, only certain features of transformation by SV40 will be emphasized a t this time to provide a background for subsequent sections of the chapter.

A. TRANSFORMATION OF PERMISSIVE AND NONPERMISSIVE CELLSBY SV40 As discussed above, SV40 is maximally oncogenic in vivo in newborn hamsters. The narrow host range of the virus has been widely extended by in vitro transformation experiments. In addition to transformation of hamster cells in culture (Black and Rowe, 1963a; Rabson and Kirschstein, 1962; Ashkenazi and Melnick, 1963; Shein e t al., 1963), it has also been reported that SV40 can transform cells of human (Koprowski et al., 1962; Ashkenazi and Melnick, 1963; Shein and Enders, 1962), mouse (Black and Rowe, 1963b; Todaro and Green, 1964; Kit et al., 1966b), rabbit (Black and Rowe, 1963b), rat (Diderholm et al., 1966), bovine (Diderholm et al., 1965), guinea pig (Diderholm et al., 1966), and monkey (Fernandes and Moorhead, 1965; Koprowski et al., 1967; Wallace, 1967; Margalith et al., 1969; Shiroki and Shimojo, 1970) origin.

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J. S. BUTEL, S . 6 . TEVETHIA, A N D J. L . MELNICK

I n the hamster species, which is the most thoroughly studied transformation system for SV40, cells from a variety of organs and tissues have been shown to be susceptible to virus-induced transformation. Types of cells which have reportedly been transformed by SV40 include the following: ( a ) kidney (Black and Rowe, 1963a; Rabson and Kirschstein, 1962; Shein et al., 1963), ( b ) lung (Diamandopoulos and Enders, 1965; Nachtigal and Butel, 1970), (c) liver (Diamandopoulos and Enders, 1965), ( d ) heart (Enders and Diamandopoulos, 1969), ( e ) skin and subcutaneous tissue (Diamandopoulos et al., 1969), ( f ) lens epithelium (Albert e t al., 1969), (9) astrocytes (Shein, 1968), ( h ) prostate (Paulson e t al., 1968), and (i) pineal (Wells et al., 1966). The latter two types of transformed cells retained a certain level of enzymatic function (tartrate-inhibited acid phosphatase and hydroxyindole-0methyltransferase, respectively). Obviously, SV40 exhibits little, if any, particular tissue tropism in the in vitro studies described above since cells derived from nine different types of differentiated tissue were successfully transformed. Cells from patients with diseases associated with chromosomal abnormalities are highly susceptible to transformation by SV40. Human diseases which have yielded transformation-susceptible cells include Fanconi’s anemia (Todaro e t al., 1966), Down’s syndrome (Todaro and Martin, 1967; Potter et al., 1970) , and Klinefelter’s syndrome (Mukerjee et al., 1970). Using high concentrations of virus ( 1 0 9 . O TCID50/ml.), Todaro and co-workers observed that about 0.02-0.03% of normal human fibroblasts would transform. I n contrast, the cells from patients with Down’s syndrome were about three times as susceptible to transformation while those derived from people suffering with Fanconi’s anemia were at least ten times more susceptible. However, the mere presence of extra chromosomes per se was shown not to be sufficient to increase the susceptibility of cells to transformation (Payne and Schmickel, 1971). A triploid human fibroblast strain was no more susceptible to SV40 than normal human cells, based on the frequency of induction of T-antigen. It was speculated that the heightened susceptibility to transformation of the trisomic cells might be due to cellular replication errors which facilitated integration of the viral genome since the cells have chrornosome abnormalities. A recent report, however, suggests an alternative explanation for the observed differences in susceptibility of human cells to transformation by SV40 (Aaronson, 1970a). All the differences between various cell strains were eliminated when isolated SV40 DNA was employed. These results suggest that most human cells are resistant to transformation by SV40 because of a block a t an early step in infection, such as adsorption, penetration, or uncoating.

PAPOVAVIRUS

SV40

13

The preceding catalog of species of cells which have been transformed by SV40 contains cells which fall into three categories: (a) permissive, ( b ) semipermissive, and (c) nonpermissive. Permissive cells are those which are highly susceptible to SV40 and in which the virus undergoes a typical replicative cycle. Monkey kidney cells are permissive for SV40 (Hsiung and Gaylord, 1961; Mayor et al., 1962; Carp and Gilden, 1966; Kit et al., 1966a; Sauer et al., 1967). Human cells represent the semipermissive system. Small amounts of infectious virus are produced by the infected cells. Yet, even when free viral nucleic acid is used to infect the human cells, the yields of virus do not attain the levels produced by monkey cells (Swetly et al., 1969; Kit et al., 1970). All the other species of cells fall into the third category, nonpermissive. These cells are not susceptible to SV40 and do not produce detectable amounts of progeny virus. It has been shown with both hamster and mouse cells (Swetly et al., 1969) that even after exposure to SV40 DNA no infectious progeny virus can be detected in the cultures. The above distinction between permissive and nonpermissive types of cells is made in anticipation of the discussion below, in Section V,D. One cannot say that a transformed cell is resistant to superinfection by the transforming virus if the normal cells derived from that species are naturally resistant to the virus. It is much easier to transform nonpermissive cells by SV40 than those derived from permissive species. The mouse 3T3 cell line is sensitive to transformation by SV40 and constitutes the most widely employed quantitative transformation system for the virus (Black, 1966a ; Todaro and Green, 1966a). I n the 3T3 system, one transforming unit of virus corresponds to about lo3 infectious units and about lo5 physical particles. When high concentrations of virus are used, a t least 10% of the 3T3 population will transform. I n contrast, transformation of semipermissive human diploid cells is less efficient. An equivalent input of virus will transform only about 0.03% of the human cells. In general, laboratory manipulation of some type is used in order to achieve transformation of permissive cells. Tricks on the part of the researcher are necessary because the inoculum virus tends to replicate and destroy all the cells in the culture. Examples of such manipulation employed in successful studies include heavy ultraviolet irradiation of the inoculum virus (Shiroki and Shimojo, 1970), high multiplicities of infection (Margalith et al., 1969), and very low multiplicities of infection (Koprowski et al., 1967; Wallace, 1967; Fernandes and Moorhead, 1965). Transformation of permissive cells is a very rare event, far too rare to quantitate. Therefore, it has not been possible to differentiate between

14

J. S.

BUTEL,

S. S. TEVETHIA, AND J.

L.

MELNICK

the two alternative possibilities: ( a ) that a unique mutant cell in the population has been transformed or ( b ) that the virus has mediated a rare molecular event which aborted the normal replicative cycle and resulted, instead, in transformation of the permissive cell. BY DEFECTIVE SV40 B. TRANSFORMATION

Defective particles of SV40 can induce oncogenesis in vivo (see Section I1,B). Similarly, defective SV40 can mediate transformation in vitro as demonstrated by the following examples of defective virions: ( 1 ) PARA (defective SV40) -adenovirus hybrid population (Black and White, 1967; Black and Todaro, 1965; Diamond, 1967; Duff and Rapp, 1970a; Nachtigal and Butel, 1970; Butel et al., 1971c; Duff et al., 1970), ( 2 ) hydroxylamine-inactivated virus (Altstein et al., 1967a), and ( 3 ) Tantigen-forming defective virions (T-particles) of SV40 (Uchida and Watanabe, 1969). All stocks of SV40 naturally contain defective particles which arc capable of inducing the synthesis of T-antigen, but not of V-antigen (Sauer et al., 1967; Altstein et al., 1967b; Uchida et al., 1968). It remains to be determined whether the defective particles are genetically stable and perpetuated during passage or whether they are produced randomly during each replicative cycle of the virus. In either event, defective particles are prevalent. Since transformation is a relatively rare event (requiring a minimum of lo3 to > lo* PFU of virus per transformation event in the most susceptible cell systems (Black, 1966a; Todaro and Green, 1966a; Aaronson and Todaro, 19681, defective particles could conceivably be the agents which mediate the transformation event (s) . Irradiation experiments also indicate that the entire viral genome is not required for transformation. Aaronson (1970b) used human cells as the assay system with SV40 so that three viral functions (T-antigen, V-antigen, and transformation) could be determined under identical conditions. Infectivity was about 4 times more sensitive and the synthesis of V-antigen about 3 times more sensitive to inactivation by ultraviolet (UV) light than the property of transformation. The relative UV-sensitivity of the synthesis of T-antigen was more difficult to assess because the survival curve changed with time (time after infection before cells were stained for T-antigen). It was concluded, however, that UVsensitivity of T-antigen was intermediate between those of V-antigen and transformation. Previous reports had also indicated that T-antigen synthesis and thymidine kinase-inducing activity appeared to be more UV-resistant than plaque-forming ability by SV40 (Butel and Rapp, 1966b; Carp and Gilden, 1965; Carp et al., 1966). However, Yamamoto and Shimojo (1971) have reported that, owing to apparent multiplicity

PAPOVAVIRUS

SV40

15

reactivation, it is not possible to accurately estimate the size of the Tantigen gene on the basis of UV-irradiation data. Aaronson’s (1970b) above estimate of the proportion of SV40 genome involved in transformation compares well with that reported for polyoma virus by Latarjet e t al. (1967). Other authors had previously reported larger target sizes for the property of transforming ability by polyoma virus (Benjamin, 1965; Basilico and di Mayorca, 1965). Since only a portion of the genome is needed to mediate transformation, defective particles present in virus populations may be responsible for the oncogenicity of the virus. The concept that defective particles may be the transforming entities is considered further in Section V,D. C. DOUBLE TRANSFORMATION OF CELLS Transformation of a given cell by one member of the papovavirus group does not preclude its further transformation by a second member of the same virus group. Todaro and Green (1965) presented evidence based on colonial morphology that SV40 could further transform 3T3 mouse cells initially transformed by polyoma virus. Several isolated clones of the doubly transformed 3T3 cells were shown to contain specific T-antigens of both viruses (Todaro et al., 1965). Takemoto and Habel (1966) showed that SV40-induced hamster tumor cells became doubly transformed after superinfection with polyoma virus. This latter study demonstrated the presence of the specific transplantation (TSTA) and tumor (T) antigens induced by both viruses in the doubly transformed cells. Cells can be doubly transformed by SV40 and a human adenovirus, as well. Cells transformed either in vivo or in vitro by the PARA (defective SV40) -adenovirus hybrid populations sometimes contain both SV40 and adenovirus T-antigens and generally induce antibody against both SV40 and adenovirus T-antigens in tumor-bearing hamsters (Rapp et al., 1966a, 1969; Butel et al., 1971c; Richardson and Butel, 1971; Duff and Rapp, 1970a; Black and White, 1967). The morphology of the tumors and transformed cells will sometimes be characteristic of either adenovirus or SV40-transformed cells or, on occasion, histologically intermediate (Rapp et al., 196713; Igel and Black, 1967; Black et al., 1969). The most convincing evidence, however, that both SV40 and adenovirus information can be present in the cultures of PARA-transformed cells is that both adenovirus 7 and SV40 specific RNA’s were detected in such cultures after cloning (Levin e t al., 1969a). Therefore, the same cell can contain both SV40 and adenovirus genetic information. It should be recalled, though, that the PARA system is unique because the defective SV40 and the defective adenovirus 7

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J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

DNA’s appear to be linked (Baum et al., 1966; Rowe and Pugh, 1966; Levin et al., 1969a; Kelly and Rose, 1971). Therefore, integration of the SV40 portion of the PARA genome would carry into the chromosome the adenovirus 7 region also. However, cells transformed by a transcapsidant PARA-adenovirus 12 population sometimes contained both adenovirus 7- and adenovirus 12-specific RNA in addition to the SV40specific RNA (Levin et al., 1969a). This indicates that both PARA and adenovirus 12 genomes can be integrated into the same host cell. It has recently been reported (Rhim et al., 1971) that rat embryo cells infected with Rauscher leukemia virus show enhanced susceptibility to transformation by SV40. The underlying mechanisms responsible for the phenomenon remain to be determined. One important facet of multiple transformation by viruses has not yet been studied. That area concerns the number of different SV40 genomes which can be present in the same transformed cell. Such information would indicate how many different integration sites there are for SV40 in a single cell. Such a study could be approached using genetically defined temperature-sensitive mutants of SV40. The antigenic, genotypic, and virogenic properties of cells transformed in vitro by SV40 are analyzed in the following sections of this chapter.

D. REVERSION OF TRANSFORMED CELLS The transformation of a normal cell into a cancer cell has generally been held to be an irreversible change. Recently, however, Braun (1970) drew together examples from experimental systems ranging from plants to frogs, newts, mice, hamsters, and humans, and showed that the reversal of the neoplastic state can and does occur. For the purposes of this chapter, only those tumors induced by viruses will be considered with respect to the property of reversion. Nowcver, because of a dearth of information concerning the SV40 system, other viruses will also be considered in this section. Treatment of transformed cells with FUdR a t different cell densities resulted in the selection of certain cell variants (Pollack et al., 1968). These variant derivatives exhibited a flattened morphology, attained lower saturation densities, and, with the two variants tested, reduced tumor-producing capacity in vivo. This study included 3T3 mouse cells transformed by either SV40 or polyoma and a hamster tumor cell line originating from a polyoma virus-induced tumor. The variant clones still contained the virus-specific T-antigens and, with a t least one derivative cell line, still yielded infectious SV40 following fusion with monkey kidney cells. It would be informative to determine the transforming capabilities of the rescued virus. Such experiments would indicate

PAPOVAVIRUS

SV40

17

whether the observed reversion was due to a mutation of one or more essential genes of the transforming virus or whether reversion was due, instead, to an alteration in the expression of the essential virus genes in the transformed cells. Hamster embryo fibroblasts transformed by polyoma virus have reportedly undergone a reversion of properties characteristic of transformation (Rabinowitz and Sachs, 1968, 1969a,b, 1970a,b; Inbar et al., 1969). These variants showed a decreased cloning efficiency in soft agar, a decreased saturation density, and a loss of the ability to multiply a t 41°C. The variants also acquired the ability to form colonies on glutaraldehyde-fixed normal cells, manifested contact inhibition toward one another, and inhibited the parental transformed cells. The variant cells had an altered cell surface membrane in that they were no longer agglutinated by the carbohydrate-binding protein concanavalin A. The variants produced in vitro were less tumorigenic than the parental transformed cells, while those produced in vivo were more tumorigenic. All the variant cells continued to synthesize polyoma T-antigen. Eight different variant cell lines were shown to have suffered a partial or complete loss of detectable levels of polyoma-specific transplantation antigen. Further, it was observed that the reversion was initially unstable but that it could be stabilized by inhibiting cell multiplication to prevent a crowded condition. Sachs (1965) has proposed that the presence of the transplantation antigen on the cell surface results in a change in the control mechanism for cell replication. The revertant cells characterized by Sachs and his colleagues support his hypothesis because the variant cells behaved like normal cells in that they exhibited both contact inhibition and an absence of virus-specific transplantation antigen (Rabinowitz and Sachs, 1970a). However, there are other known examples which do not support this hypothesis. Hare (1967) reported that the lp-D strain of polyoma virus does not induce virus-specific transplantation immunity in weanling hamsters, yet is oncogenic. Furthermore, cells derived from lp-D-induced tumors do not contain polyoma transplantation antigen. The sp-D strain of polyoma virus, also lacking the marker for TSTA, was able to efficiently transform cells in vitro (Hare, 1967). An example with SV40 which fails to support the foregoing hypothesis is a fibroblastic line of H-50 cells (Tevethia, 1971). The cells are morphological revertants which are fibroblastic in appearance, attain similar saturation densities to that of the parental line, and have a reduced chromosome complement. Nevertheless, these revertant cells are highly transplantable and contain high levels of SV40 transplantation antigen. Eagle e t al. (1970) studied a variety of cell lines with respect to maximum

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J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

population density, inhibition by normal cells, serum requirement, and growth in soft agar and attempted to correlate such properties with xenogeneic transplantability. They concluded that none of the properties studied appeared to be a valid in vitro criterion for predicting tumorigenicity. However, it would be interesting to do such a study within a single species, e.g., SV4O-transformed hamster cells, the in vitro properties of which could then be correlated with transplantability in vivo in weanling hamsters. I n contrast to the cells studied by Rabinowitz and Sachs which still contained polyoma T-antigen, the revertant cells studied by Marin and Macpherson (1969) appeared to have lost the viral genes present in the parental cells. The revertant cells were less tumorigenic and both polyoma-specific T- and transplantation antigens were lost or reduced. The cells could be retransformed by polyoma virus. This latter fact ruled out the possibility that reversion was a consequence of the loss of cell genes required for the expression of the transformed phenotype. Some relevant experiments concerning reversion of transformed cells have been performed with the RNA-containing tumor virus Rous sarcoma virus (RSV). Macpherson (1966) reported the isolation of revertants from BHKPl cells transformed by the Schmidt-Ruppin strain of RSV. The revertant cells showed altered morphology, were not tumorigenic, did not contain detectable levels of COFAL antigen, and had decreased plating efficiencies in soft agar. The revertant cells appeared to be resistant to retransformation by RSV. Nevertheless, it appeared that, in this particular system, reversion was due to a loss of viral genes. A temperature-sensitive ( t s ) mutant (TI) isolated from the SchmidtRuppin strain of RSV (SR-RSV-A) has provided a good model for study (G. S. Martin, 1970). Cells transformed a t the permissive temperature by the mutant virus and then shifted to the elevated, nonpermissive temperature rapidly ( 2 4 hours) reverted back to a nontransformed phenotype. The t s mutant was able to replicate well a t the elevated temperature. It would appear from these experiments that the expression of some viral gene is required for the maintenance of the transformed state and the gene product is temperature-sensitive in the case of mutant TI. Furthermore, that gene product is evidently not required for viral replication. Toyoshima and Vogt (1969) also reported the detection of two ts mutants of RSV (strain B77) which could not transform cells a t the elevated temperature. As with Martin’s T1 mutant, cells transformed a t the permissive temperature and then shifted to the nonpermissive temperature reverted back to a normal phenotype. These mutants, however, could not replicate at the elevated temperature so they did not

PAPOVAVIRUS

SV40

19

allow a separation of viral functions required for replication and those essential for transformation. From these few scattered reports one can conclude that reversion of virus-transformed cells probably can and does occur. Some of the reports appear to have detected complete reversion, possibly due to a loss of the transforming viral genes. Some of the other observed cases of reversion would more accurately be denoted partial reversion in that some phenotypic properties were altered but virus-specific antigens were still present. In these latter examples, transplantability of the cells was sometimes lessened and sometimes increased. The available data are too few and too preliminary a t this time to prompt any generalizations about either the properties of cells which have reverted or the mechanism (s) of the observed reversion. IV. Phenotypic (Antigenic) Changes in Tumor and Transformed Cells

Transformation of mammalian cells by SV40 is accompanied by the synthesis of virus-specific proteins which are antigenic in the syngeneic host. These antigenic proteins are not a part of the mature virion but are specifically induced by the transforming virus. These virus-induced nonvirion antigens can be classified into two categories based upon their location within the transformed cell : (a) tumor (T) antigen-located in the nucleus of transformed cells. ( b ) surface antigen-located a t the surface of transformed cells. The surface antigens can be further subdivided: (i) tumor-specific transplantation antigen (TSTA)-detected in vivo by the transplantation rejection test, and (ii) S-antigen-detected by serological tests in vitro, using antibody from hamsters immunized with SV40 or SV40-transformed cells. In addition, the transformed cells may acquire some properties which are cryptic in normal cells. Of all thc changes that a cell undergoes during transformation by an oncogenic virus, acquisition of specific antigens a t the cell surface is one of the most important. In this section, we will attempt to analyze the nature of these antigens arid their relationship to each other and consider whether the changes are extrinsic (coded for by the virus) or intrinsic (due to derepression of the host cell genome). A. TUMOR ANTIGEN The T-antigen in SV40 tumor cells was first demonstrated by means of the complement fixation test using sera from hamsters bearing tumors induced by SV40 (Huebncr et al., 1963; Koch and Sabin, 1963). The nuclear localization of T-antigen was later established using the indirect immunofluorescence technique (Rapp et al., 1964a; Pope and Rowe,

20

J. S. BUTEL, S. S. TEVETHIA, AXD J . L. MELNICK

1964). A typical pattern of nuclear fluorescence by SV40 T-antigen is shown in Fig. 1. The T-antigen present in transformed cells was directly related to the transforming virus when it was shown to be synthesized in acutely infected monkey kidney cells (Rap]) e t al., 1964b; Sabin and Koch, 1964; Hoggan e t al., 1965). Kinctic studies showed that T-antigen appears 12-24 hours after infection with SV40, prior to the appearance of viral capsid antigen and progeny virions. The synthesis of T-antigen is not affected by inhibitors of DNA synthesis but is inhibited by actinomycin D, suggesting that i t is due to an early function of the viral genome (Rapp e t al., 1965a). T-antigens extracted from productively infected or transformed cells

FIG.1. Inimunofluorescence detection of SV40 T-antigen localized in nucleus of rell. X 400.

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SV40

21

originating from different species are antigenically similar. Conversely, T-antibodies obtained from various sources all give a typical intranuclear reaction when tested against cells either transformed or infected by SV40 (Rapp e t al., 1964a,b; Tevethia, 1970). These observations suggest that the synthesis of T-antigen is coded for by the viral genome. This hypothcsis is supported by the fact that isolated SV40 DNA is also able to induce the synthesis of T-antigen (Black and Rowe, 1965). Furthermore, three mutants of defective SV40 (PARA) have been described which induce the synthesis of an antigenically identical T-antigen in the cytoplasm of infected cells rather than in the nucleus (Butel e t al., 1969). The cytoplasmic T reaction is shown in Fig. 2. Subsequent studies by Duff et al. (1970) and Richardson and Butel (1971) showed that the

FIG.2. Immunofluorescence detection of SV40 T-antigen localized in cytoplasm cell. X 400.

22

J. S. BUTEL, S. 8 . TEVETHIA, AND J . L. MELNICK

cytoplasmic SV40 T-antigen was present in cells transformed by the variant viruses, as well as in cells from tumors induced by these variant viruses in newborn hamsters. The cytoplasmic localization of T-antigen in transformed or tumor cells was stable upon multiple passages in vitro. No apparent differences were detected when the properties of cytoplasmic SV40 T-positive cells were compared t o those of nuclear T-positive cells (Richardson and Butel, 1971). A summary of the results of that comparative study is given in Table 11. Although all the available evidence supports the concept that information for the synthesis of SV40 T-antigen resides in the viral genome, the host cell nevertheless does appear to exert some influence upon the expression of the T-antigen. Nachtigal et al. (1970) demonstrated that lung cells derived from hybrid animals originating from a cross of the Syrian and Rumanian species of hamsters did develop Tantigen when transformed by SV40. In contrast, after exposure to SV40, lung cells from Syrian hamsters frequently acquire the property of unlimited growth in vitro but do not develop detectable levels of T-antigen (Diamandopoulos and Enders, 1965; van der Noordaa and Enders, 1966; Nachtigal and Butel, 1970). SV40 T-antigen is heat labile and resistant to DNase and RNase, but is susceptible to trypsin (Gilden et al., 1965). The molecular weight of T-antigen has been estimated to be approximately 200,00(1-250,000 by Sephadex G-200 gel chromatography (Gilden e t al., 1965) and 70,000TABLE I1 COMPARISON OF PROPERTIES OF HAMSTER TUMORCELLLINESCONTAINING SV40 TUMORANTIQEN IN EITHER THE NUCLEUS OR T m CYTOPLASM OF CELLS

Propertya Contain nuclear SV40 T-antigen by IF Contain cytoplasmic SV40 T-antigen by IF Contain SV40 T-antigen by CF Contain SV40 S-antigen by IF Stable nuclear localization of T-antigen Stable cytoplasmic localization of T-antigen Indefinite life-span in vitro Readily transplantable in vivo Elicit SV40 T-antibody in vivo Induce SV40 transplantation immunity (immunogenic) Rejected by SV40 or PARA-immune animals (immunosensitive ) 0

T

=

Nuclear T-positive cell line

Cytoplasmic T-positive cell line

+ 0 + ++ 0 + + + + +

+ + + 0 + + + + + +

0

tumor; S = surface; IF = immunofluorescence; CF = complement fixation.

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SV40

23

80,000 by use of an immunoabsorbent (Del Villano and Defendi, 1970). Its physical properties indicate that T-antigen is not identical to any of the enzymes involved in DNA biosynthesis (Kit et al., 1967). The biological function of T-antigen remains obscure. Recently, a presumably new SV4O-specific antigen, designated the U-antigen, has been described (Lewis et al., 1969). This antigen has been localized in the perinuclear region of cells infected with a strain of adenovirus type 2 carrying a defective SV40 genome (AdB'ND,). Sera from monkeys immunized with cells infected with Ad2+ND, react with an intranuclear antigen present in both SV40-transformed and SV40infected cells (Lewis and Rowe, 1971). Antibody to U-antigen is also detected in sera from SV40 tumor-bearing hamsters. I n contrast to Tantigen, U-antigen is heat-stable. The existence of U-antigen and the difficulty in obtaining specific antisera (Lewis and Rowe, 1971) reveal that future studies aimed at characterizing T-antigen will need to be carefully designed and evaluated.

B. TUMOR-SPECIFIC TRANSPLANTATION ANTIGEN(TSTA) The first evidence for the presence of TSTA in cells transformed by DNA viruses was provided when Sjogren et al. (1961) and Habel (1961) demonstrated that mice or hamsters inoculated with polyoma virus were capable of rejecting a challenge of polyoma tumor cells. TSTA was later demonstrated in SV40-induced tumor cells using the transplantation rejection technique (Khera et al., 1963; Habel and Eddy, 1963; Koch and Sabin, 1963; Defendi, 1963). The transplantation rejection test involves immunizing hamsters with one to three doses of live SV40 and later challenging immune and nonimmune hamsters with varying numbers of transplantable SV40 tumor cells. The results are generally expressed as the number of cells required to produce tumors in 50% of the animals (TPD,,). A 10-fold difference in the TPD,, of the immune animals when compared to that of the control, nonimmunized group is taken as evidence of the presence of TSTA in the tumor cells. TSTA is not a capsid antigen since antiviral antibodies have no effect on the growth of SV40 tumor cells (Khera et al., 1963). Also, SV40 tumor cells carrying TSTA were shown to be free from both infectious virus and virion antigens (Melnick e t al., 1964). Live virus is essential in inducing immunity against the tumor cells. Habel (1962) postulated that virus, upon inoculation into adult hamsters, transforms certain cells in the animals which then develop TSTA identical to those present on tumor cells subsequently used for challenge. The virus thus codes for the synthesis of new antigens unrelated to capsid antigen which sensitize the host against challenge of SV40 tumor cells.

24

J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

The TSTA present in cells transformed by SV40 in vitro have been shown to be identical to those present on virus-induced tumor cells (Khera et al., 1963). The evidence that SV40 TSTA is present at the surface of transformed cells is 2-fold: ( a ) the rejection of tumor cells by immune animals required contact between sensitized lymphoid cells and TSTA (Tevethia et al., 1970b; Coggin et al., 1967), and ( b ) cell membranes isolated from SV40 tumor cells induced a state of immunological tolerance to SV40 TSTA in newborn hamsters (Tevethia and Rapp, 1966). The tolerance was demonstrated by the failure of adult hamsters, which had received cell membranes as newborns, to be immunized by SV40 against a challenge of SV40 tumor cells. The membranes from SV40 tumor cells were later shown to immunize adult hamsters against SV40 tumor cells (Coggin et al., 1969). The TSTA in SV4O-transformed cells can also be demonstrated by immunogenicity tests. Transformed cells are tested either for their ability to immunize adult hamsters against a challenge of transplantable SV40 tumor cells or for their ability to block SV40 oncogenesis in hamsters inoculated as newborns with the virus (Girardi, 1965; Lausch et al., 1968). The oncogenicity of SV40 can also be prevented by inoculating hamsters in the latent period before tumor appearance with nontransplantable syngeneic or xenogeneic SV40-transformed cells. Using this method, Girardi (1965) demonstrated the cross-reactivity between the TSTA of SV40-transformed human cells and that of SV40-transformcd hamster cells. F. Jensen and Defendi (1968) used the same technique to demonstrate the absence of SV40 TSTA in simian cells transformed by ultraviolet light-irradiated defective SV40 (PARA), However, it should be pointed out that these latter cells were not tested for their ability to immunize hamsters against challenge with an immunosensitive SV40 tumor cell line. This is an important point, since Lausch et al. (1968) showed that hamster cells transformed by defective SV40 (PARA) were not effective at preventing SV40 tumorigenesis, yet were rejected by SV40-immunized hamsters. Recently, Girardi and Defendi (1970) showed that SV40 TSTA was synthesized during the cytolytic cycle of the virus in monkey cells. The synthesis of TSTA could be blocked by inhibitors of protein synthesis and by actinomycin D, but not by inhibitors of DNA synthesis.

c. SURFACE ANTIaEN Since in vivo tests for the measurement of immunosensitivity and immunogenicity of SV40-transformed cells are time-consuming, many efforts have been made to develop in vitro tests for the detection of

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SV40

25

SV40 TSTA. The attempts to devise in vitro tests fall into two general categories: ( a ) cytotoxic tests which use specific antibodies or immune lymphocytes, and ( b ) serological tests based on the detection of antigenantibody interaction. Immunofluorescence and mixed agglutination tests belong to this latter category. A tumor virus may bring about changes in transformed cells other than the development of TSTA, and some of these changes may represent new antigenicities. Therefore, it cannot be said that different in vitro tests all measure TSTA. For this reason, the antigen or antigens detected by in vitro tests will be designated as surface or S-antigen(s) . I n this section, we will analyze the S-antigen(s) with respect to their detection, specificity, and nature. Tevethia et al. (1965) first demonstrated S-antigen in SV40-transformed cells by the indirect immunofluorescence test using sera from SV40-vaccinated hamsters that had rejected a transplant of virus-free SV40 tumor cells. Live cells were used in the immunofluorescence test, and the reaction was localized at the cell surface in the form of a ring around the cell (Fig. 3 ) . The reaction was specific for SV4O-transformed cells since antibody against S-antigen did not react with either normal hamster cells or cells transformed with unrelated viruses. The S-antigen was present whether the hamster cells had been transformed by SV40 in vivo or in vitro. Antibody against S-antigen was present in hamsters which had rejected a challenge of transformed cells, but was absent from tumor-bearing animals. Tevethia et al. (1968a) later demonstrated that hamsters synthesized S-antibody when immunized with SV40 alone, thereby ruling out the participation of isoantigens in the membrane reaction. Even though the hamsters that were used in the preceding

FIG.3. Immunofluorescence detection of SV40 S-antigen localized a t surface of cell. X 400.

26

J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

studies were not inbred, this probably was not a serious detriment because it has not been possible to demonstrate isoantibody in hamsters (Billingham and Silvers, 1964). Kluchareva et al. (1967) confirmed the presence of S-antigen in SV40-transformed cells, and further established the specificity of the reaction by using sera which were prepared in hamsters inoculated with live SV40 during the latent period of viral oncogenesis. This latter approach avoided any possible isoantigen incompatibilities in the test system since the antibody was from animals which had never been exposed to foreign cells. A similar membrane reaction was later demonstrated in polyoma virus-transformed cells by the immunofluorescence test (Malmgren et al., 1968; Irlin, 1967). The S-antigen of polyoms virus-transformed cells was specific for that virus. Hayry and Defendi (1968) and Metzgar and Oleinick (1968) independently demonstrated the presence of S-antigen in SV40-transformed cells by the mixed hemadsorption technique. The S-antigen detected with this method was virus-specific and the antibody against the antigen did not react with cells transformed by unrelated viruses. The authors also showed that the S-antigen in SV40-transformed human cells would cross-react with the S-antigen of SV40-transformed hamster cells, further establishing the specificity of the antigen. Tevethia et al. (1970a) confirmed the cross-reaction between the S-antigens of SV40-transformed human and hamster cells by means of the immunofluorescence test using sera prepared according to the method of Hayry and Defendi (1968). Tevethia e t al. (1970a) used the colony inhibition test originally described by I. Hellstrom and Sjogren (1965) to demonstrate specific S-antigen in SV40-transformed cells. The sera prepared in hamsters either against SV40-transformed human or marmoset cells or against purified SV40 was active only against SV40-transformed cells. The observed inhibition of colony formation was dependent upon the presence of active guinea pig complement. There was a positive correlation between results obtained using the colony inhibition and the immunofluorescence tests, suggesting that the two tests may be detecting the same or similar antigen. These results were confirmed by Smith et al. (1970) , who demonstrated S-antigen by the in vitro cytotoxic test using sera from mice hyperimmunized with syngeneic nontransplantable SV40transformed mouse cells. The sera were cytotoxic only against SV40transformed cells. Recently, Wright and Law (1971) used the cytotoxic test and humoral antibody prepared in mice by the repeated inoculation of syngeneic SV40-transformed cells to demonstrate the presence of tumorspecific antigens a t the cell surface. SV40-transformed cells labeled with chromium-51 were selectively killed with the antisera in the presence of rabbit complement. The fact that only the cells which possessed

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SV40

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SV40 TSTA were killed by the cytotoxic antibody suggests that the cytotoxic action of humoral antibody may be directed against TSTA. Since TSTA and S-antigen(s) are both present a t the cell surface, there has been a temptation to assume that the in vitro tests are actually measuring TSTA. Tevethia et al. (1968b) were the first to point out the lack of relationship between TSTA and S-antigen in certain SV40exposed hamster cell lines which became oncogenic. The cell lines in question were derived by Diamandopoulos et al. (1968) by exposing cloned hamster embryo fibroblasts to SV40. Four of the seven virusexposed cell lines became oncogenic and developed SV40 S-antigen. However, only two of the cell lines also contained intranuclear T-antigen. Neither T- nor S-antigen was detected in lines established from cultures which had not been exposed to SV40. When the various lines were tested for the presence of SV40 TSTA, only S'T' cell lines were rejected by SV40-immunized hamsters ; S'T- cells were not rejected by the immune animals (Tevethia et al., 1968b). This finding, plus the observation that S'T- cells were unable to block SV40 oncogenesis in hamsters when inoculated during the latent period (Tevethia et al., 1968b) , suggested that the S'T- cells lacked detectable amounts of SV40 TSTA. Wright and Law (1971) using hyperimmune mouse sera in the cytotoxicity test also failed to demonstrate SV40 TSTA in S'T- cell lines. The S'T- cells have subsequently been shown to lack detectable amounts of both SV40 messenger RNA (Levin et al., 196913) and SV40 DNA (Levine et al., 1970). The absence of detectable quantities of SV40 genetic information in the S'T- cells suggests that the S-antigen in these cells may not be coded for by a persistent viral genome. One possibility, proposed by Tevethia et al. (1968133, is that the Santigen in the S'T- cells may be the result of derepression of the host cell genome by the virus. Duff and Rapp (1970b) recently demonstrated that sera from pregnant hamsters reacted specifically with SV40-transformed hamster cells, indicating the presence of some derepressed embryonic antigens on SV40-transformed cells. It was somewhat puzzling that the sera from pregnant hamsters did not react with cells transformed either by adenoviruses or by chemical carcinogens. Even if each tumor virus depresses only a specific region of the host cell chromosome, pregnant hamsters should be exposed to all these antigens during embryogenesis, The definite proof that the S reaction is due, altogether or in part, to an embryonic antigen is still lacking, since no attempts have been made to determine whether anti-S sera will react with embryonic cells. Baranska e t al. (1970) demonstrated cross-reactions between SV40transformed mouse cells and mouse eggs using antimouse egg sera prepared in guinea pigs. The sera also reacted with a tumor line induced by a chemical carcinogen, but not with normal cells. The nature of the antigen detected remains obscure.

28

J. S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

It is also possible that, upon infection of hamster cells, SV40 either directly or indirectly activates an unrelated virus previously latent in the hamster cells (Huebner and Todaro, 1969), which, in turn, is responsible for the membrane reaction. Another possibility is that cells infected with SV40 may undergo abortive transformation, but in the process conditions are altered such that S-antigen synthesis is initiated. A recent report (Vasconcelos-Costa, 1970) indicated that cells transformed by adenovirus 12 may lose T-antigen but retain S-antigen. It would be of great interest to ascertain whether those cells also lose adenovirus genetic information and TSTA. Recent studies by Hayry and Defendi (1970) using the mixed hemadsorption test have suggested that S-antigen is a normal cell antigen which is specifically unmasked by SV40 during transformation. This conclusion was based on the observation that, after brief treatment with trypsin, spontaneously oncogenic or polyoma virus-transformed cells reacted with SV40 S-antibody. This antigen on the non-SV40-transformed cells could also be exposed by treatment of the cells with chemotrypsin, but not with ficin, papain, or neuraminidase. Similar reactivity was detected using the immunofluorescence test. On the basis of adsorption studies, it was concluded that normal cell antigens cross-reacted with the SV40 antigen. However, complete absorption of anti-S activity from the sera using either SV40-transformed cells or trypsin-treated nontransformed cells was not achieved. Burger (1969) has described presumably “tumor-specific” sites a t the surface of SV40-transformed cells. The sites were demonstrated by an agglutination test, using agglutinin isolated from wheat germ lipase. The agglutinin binding sites which were present on transformed cells were cryptic in normal cells, but could be unmasked by treatment with trypsin. The agglutination reaction could be inhibited with N-acetylglucosamine. Agglutination of transformed cells by Concanavalin A has been reported by Inbar and Sachs (1969). Normal cells were agglutinated only after mild trypsinization, again suggesting the cryptic nature of the agglutination sites on normal cells. The authors claimed that the sites were specifically exposed during viral transformation. SV40-infected 3T3 cells wcre also agglutinated by Concanavalin A, but only if they had undergone a t least one cell generation (Ben-Bassat et al., 1970). Of interest is thc report (Benjamin and Burger, 1970) that the change in tlic cell membrane which renders a cell agglutinable by Concanavalin A was not detected after infection with mutants of polyoma virus previously denionstrated to be defective in their ability to transform cells. The agglutination sites were acquired by cells after lytic infection with the wild-type polyoma virus.

SV40

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29

D. RELATIONSHIP OF S-ANTIGENTO TSTA I n view of the many surface changes which have been described with SV40-transformed cells, their relationship to each other and to TSTA is considered below and summarized in Table 111. TABLE 111: RELATIONSHIP BETWEEN SURFACE CHANGES OBSERVED AFTER TRANSFORMATION BY SV40 ~~

Surface change

Properties Specific for transforming virus Appear after viral transformation Present on normal cells in cryptic form Forssman antigen Antigenic in syngeneic hosts

Normal Agglutination cell sites TSTA S-antigen antigen 0

+ + ? 0

+ +

+ +

0

0

+0

+

0

Embryonic antigen

0

0

+ +

+

0 ?

? ?

?

The agglutination sites described by Burger (1969) and Inbar and Sachs (1969) may not be TSTA, since all tumor cells bind wheat germ agglutinin and Concanavalin A regardless of whether they are transfoimed by SV40 or by other tumor viruses. I n contrast, TSTA is specific for the transforming virus. Also, normal cells possess the agglutination sites in a cryptic form, and TSTA has not been demonstrated in normal cells. The possibility that the agglutination sites are similar to the Santigen detected in vitro is also unlikely because cells transformed by polyoma and other viruses do not react with SV40 anti-S sera but do bind the agglutinins. I n addition, SV40-transformed cells which have been pretreated with anti-S serum and then washed to remove the excess serum can still be agglutinated by Concanavalin A a t a rate similar to that of untreated cells or of cells pretreated with normal hamster serum, indicating that there are agglutination sites on the tumor cells which are not the S-antigen (Tevethia, 1971). The agglutination sites may also differ from the sites containing the normal cell antigen described by Hayry and Defendi (1970). The normal cell antigen is exposed a t the surface of SV40-transformed cells but is cryptic in polyoma and spontaneously transformed cells, whereas agglutinin sites are exposed in all the transformed cells. It is not yet

30

J. S. BUTEL, S.

S. TEVETHIA, AND J .

L. MELNICK

known whether the agglutination sites are antigenic in syngeneic hosts. The relationship between the normal cell antigen described by Hiiyry and Defendi (1970) and SV40 TSTA is not clear a t this time, but the available cvidence suggests that the two are different. Experiments carried out in the authors’ laboratory, as well as by others, indicate that spontaneously oncogenic cells or normal cells that are treated with typsin do not immunize hamsters against a challenge of SV40 tumor cells. Furthermore, if the normal cell antigen were to act as TSTA, then animals should be tolerant to it unless the antigen is masked during both embryonic and adult life (which is unlikely), and SV40 would not be able to immunize hamsters against a challenge of SV40 tumor cells. The transplant rejection test used to detect TSTA is dependent upon the fact that SV40 can immunize adult animals. It is not known whether sera prepared in hamsters against trypsinized normal, polyoma-transformed, or spontaneously oncogenic cells will react with nontrypsinized SV40-transformed cells. Such tests would help clarify a possible identity between normal cell antigens and SV40 S-antigen. The L‘normal” cell antigen is not a Forssman antigen such as that described by Robertson and Black (1969), because extensive treatment of SV40 S-antisera with sheep red blood cells failed to adsorb antibody against the normal cell antigen (Hayry and Defendi, 1970). Duff and Rapp (1970b) discovered that sera from pregnant hamsters reacted with the membrane of SV40-transformed cells in the immunofluorescence test, indicating that embryonic antigens are present a t the surface of the transformed cells. Since the pregnant animals developed antibody against these antigens, the synthesis of the antigens in the developing embryo must be shut off before the development of the lymphoid system. Although there is no direct evidence that embryonic antigens may act as SV40 TSTA, Coggin e t al. (1970) reported that hamsters immunized with 10-day-old hamster or mouse embryo cells temporarily stopped the replication of SV40-transformed cells. These experiments were carried out by placing Millipore chambers containing the transformed cells in the peritoneal cavities of immune animals. In a previous study Coggin and Ambrose (1969) demonstrated that antibody diffuses into the chambers and produces a cytostatic effect on the tumor cells. The effect is only temporary and, after 5 days, the multiplication of the tumor cells in immunized animals is equal to that in the controls. The evidence that the immunofluorescence and the cytotoxic tests using humoral antibody may be measuring SV40 TSTA is circumstantial. Tevethia et al. (1970a) showed that antibody prepared in hamsters against SV40-transformed cells or against purified SV40 reacted with the S-antigen of SV40-transformed cells by the immunofluorescence test and

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SV40

31

also reacted in the complement-dependent colony inhibition test. TSTA is apparently detected in the colony inhibition test in which the inhibition of tumor cell growth in vitro is measured. This opinion is based on the reasoning that the colony inhibition test in vitro and the reaction of the immune host in vivo against the target tumor cells are similar in that both result in the death (inhibition) of the neoplastic cells (I. Hellstrom and Sjogren, 1966). Further, the common antigens shared by SV40transformed human and hamster cells demonstrated by Tevethia et al. (1970a) using the in vitro immunofluorescence and colony inhibition tests are compatible with the common antigens previously demonstrated by the use of in vivo techniques (Girardi, 1965; F. Jensen and Defendi, 1968). Common antigens between polyoma virus-transformed mouse and hamster cells have also been demonstrated by in vivo and in vitro tests (I. Hellstrom and Sjogren, 1966). Further evidence that some in vitro tests may be detecting TSTA was provided by Girardi (1967) when he demonstrated that sera from SV4O-immunized animals gave a positive reaction by immunofluorescence and also enhanced the growth of SV40 tumor cells in immune animals. This immunological enhancement is thought to proceed through immunoglobulin molecules which cover TSTA sites and thus protect the tumor cells from the action of immune lymphocytes (K. E. Hellstrom and Hellstrom, 1970). More direct evidence that the cytotoxic test with immune serum may be detecting SV40 TSTA was provided by Smith et a2. (1970). They isolated a solubilized SV40 TSTA which could immunize animals and which could also absorb the cytotoxic activity of immune serum. Recently, Klietmann and Seemayer (1971), using the mixed hemadsorption test, showed marked differences in the concentration of specific surface antigen(s) on hamster cells transformed by normal SV40 and those transformed by UV-irradiated SV40. UV-irradiated virus also had a reduced capacity to induce SV40 transplantation immunity in vivo. The cells which had lower concentrations of surface antigen(s) as measured by the mixed hemadsorption test showed equal rates of growth in intraperitoneally implanted diffusion chambers in either immune or normal hamsters. These cells, however, were not checked for the presence of SV40 TSTA by determining their capacity to immunize animals against a challenge of immunosensitive tumor cells. Transformation of normal cells by oncogenic viruses may produce cellular changes other than the development of TSTA. These changes could result in an alteration of surface structure, which in turn would create new antigenic sites either specified by the viral genome or by the derepressed host cell genome. Since TSTA is present a t the surface of cells transformed by oncogenic DNA-containing viruses, it appears to be a t least one of the antigens detected by the various in vitro tests.

32

J . S. BUTEL, S. S. TEVETHIA, AND J .

L.

MELNICK

Isolation and characterization of the various cell membrane antigens in pure form will help elucidate the relationships between the various surface changes that have been observed after transformation by SV40. V. Genotypic Changes in Tumor and Transformed Cells

A. RESCUEOF INFECTIOUS VIRUSFROM TRANSFORMED CELLS Cells transformed either in vivo or in vitro by SV40 are generally virus-free, although occasional tumors and cell lines have been reported to spontaneously release small amounts of infectious virus (Sabin and Koch, 1963a,b; Gerber and Kirschstein, 1962; Black and Rowe, 1963a; Ashkenazi and Melnick, 1963). SV40 tumors which had been allowed to grow in an animal for long periods of time more frequently contained detectable amounts of infectious virus than did tumors taken soon after transplantation (Sabin and Koch, 1963b). The minute amounts of virus found in extracts of such tumors amounted at most to 109.3TCID,,/gm. of tumor tissue. This small accumulation of virus, supposedly from those rare cells which, out of the millions in the tumor, spontaneously released a small amount of virus, probably had not had time to reach detectable levels in tumors excised less than 4 weeks after transplantation. Procedures known to induce lysogenic bacteria to produce infectious bacteriophages, such as exposure to mitomycin C, proflavin, hydrogen peroxide, and X-irradiation, were only minimally effective when applied to SV40 tumor cells (Sabin and Koch, 196313; Gerber, 1964; Melnick et al., 1964; Burns and Black, 1968, 1969; Rothschild and Black, 1970). Combinations of inducing agents were not more effective a t induction of infcctious SV40 than were the individual agents alone. I n addition, there was no dose-response relationship between the concentration of inducer employed and the yield of virus obtained (Rothschild and Black, 1970). Yields of SV40 following induction by mitomycin C were in the range of 10-1000 PFU/lO' cells. Thus, only the very rare tumor cell had been induced to release infectious virus in these experiments. Gerber (1966) discovered that recovery of infectious SV40 was more efficient if the tumor cells were placed in direct contact with susceptible indicator cells, such as primary African green monkey kidney cells. The sensitivity of the indicator cell system was increased by the use of inactivated Sendai virus to form heterokaryons (Harris and Watkins, 1965) of tumor and indicator cells (Gerber, 1966; Watkins and Dulbecco, 1967; Koprowski et al., 1967). The fusion technique increased the yield of virus about 1000-fold over simple cocultivation, but the vast majority of the heterokaryons were nonproductive (Watkins and Dulbecco, 1967; Koprowski et a,!., 1967). Therefore, while this is the most sensitive means

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SV40

33

of virus rescue currently available, it also succeeds in inducing only a rare tumor cell to produce virus. The frequency of successful virus recoveries and the amount of virus rescued in the fusion experiments ( a ) varied widely between clonal lines of transformed cells derived from the same parental cell line (Watkins and Dulbecco, 1967)) ( b ) varied a t different passage levels of the same clone (Watkins and Dulbecco, 1967), (c) did not correlate with the number of viral DNA equivalents estimated to be present in each transformed cell (Westphal and Dulbecco, 1968), and ( d ) was not dependent upon the multiplicity of infection at the time transformation occurred (Kit and Brown, 1969). Burns and Black (1968) observed considerable heterogeneity with respect to inducibility by mitomycin among a series of clonal lines derived from an SV40-transformed hamster kidney cell line. The fluctuation in rescue obtained at different passage levels of a given cell line suggests a possible change in the status of the viral genome in the cell (see Section V,B). However, some SV40-transformed cell lines have consistently resisted repeated attempts to rescue infectious virus (Black, 1966b; Koprowski et al., 1967; Melnick et al., 1964; Westphal and Dulbecco, 1968; Dubbs and Kit, 1968; Kit and Brown, 1969). It has been suggested that these lines might have been derived from cells transformed by defective viruses and that the entire genome is not present. An alternative explanation for the nonyielding lines is that there has been a mutation which precludes viral replication, e.g., the absence or inability to function of an enzyme required to release the viral genome from integration. I n line with this reasoning, it would be interesting to know whether SV40 can be rescued, and how readily, from transformed xeroderma pigmentosum cells which appear to be deficient in the enzyme(s) involved in host cell reactivation (Aaronson and Lytle, 1970) since these might be the enzymes involved in excision of the integrated viral genome. Single cell clones have been isolated from many SV40-transformed cell lines. The vast majority of such clonally derived lines were found to yield small amounts of infectious SV40 (Gerber, 1966; Black, 1966b; Dubbs et al., 1967). These studies revealed that nearly every SV40transformed cell contained the complete viral genome. The fact that many of the single cell clones were established in the presence of SV40 antiserum ruled out any essential role of extracellular virus in the maintenance of transformation. The cloning experiments do not rule out the possibility, however, that some exceptional cell lines might contain only defective viral genetic complements. I n support of this hypothesis, Knowles et al. (1968) reported that virus could be rescued when two different virus-free transformed cell

34

J. S . BUTEL, S . S. TEVETHIA, AND J. L. MELNICK

lines were fused in the presence of monkey kidney cells. It was postulated that this rescue occurred because the transforming genomes were defective a t different loci. However, the rescued progeny were not characterized to reveal whether this rescue was a consequence of complementation or of recombination of the defective genomes. If complementation, one would expect the yield of rescued progeny to be a mixture of defective viruses unable to replicate independently. If due to recombination of defective genomes, one would expect the rescued virus to consist of complete infectious virions. T o adequately study this phenomenon, it would be necessary to utilize cells transformed by genetically marked parental viruses differing in recognizable properties. This would enable one to prove that recombination of transforming genomes had occurred if the two genetic markers appeared linked in the rescued virus. “Partial” induction of SV40-transformed cells has been achieved by heat shock (45°C. for 30 minutes), which induced the synthesis of SV40 viral antigen, but not infectious virus, in transformed BSCl cells (Margalith e t al., 1970b). Oddly enough, the synthesis of the viral antigen after heat treatment was not inhibited by actinomycin D, although SV40 capsid antigen synthesis is inhibited by the antibiotic in productively infected cells (Margalith et al., 1970a). The mechanism of this heat induction phenomenon is not clear, but a plausible explanation is that only a portion of the transforming viral genome has been derepressed and possibly only one of the viral coat protein polypeptides synthesized. It is not yet known whether the heat induction procedure results in the detachment of an integrated viral genome (see Section V,B) prior to capsid antigen synthesis. In general, virus which has been recovered from tumor cells has resembled the parental transforming virus (Tournier et al., 1967; Dubbs et al., 1967; Takemoto et al., 1968b). However, distinctive genetic markers were not available for these studies. In one interesting exception (Todaro and Takemoto, 1969), rescued virus was more efficient at transformation than the parental type. The authors postulated that the enhanced efficiency of transformation of the rescued virus could be due either to a selection of more efficient transforming viruses from the original stock or to some host-induced modification of the virus. There is no direct evidence available that the infectious virus recovered in any of the rescue experiments is the virus responsible for the transformation event, the genome of which has been excised from integration and replicated. Extrachromosomal copies of the virus may exist, and these may be detected in the virus recovery attempts. Rescue experiments to date have not succeeded in eliciting infectious virus from more than a minor proportion of a transformed cell population. One type of

PAPOVAVIRUS

SV40

35

evidence which would be supportive would be the isolation of specialized transducing particles containing a portion of the viral genome linked to a region of the host cell DNA. Such particles would be analogous to Xdg (Campbell, 1957) and would arise as a result of excision a t the LLwrong’’ sites on the chromosome. It has been shown that SV40, under the proper growth conditions, will encapsidate host cell DNA to form pseudovirions (Trilling and Axelrod, 1970; Levine and Teresky, 1970), so it is reasonable to assume that linked viral and cell DNA could also be encapsidated if it were the correct size. In addition, it has been demonstrated that polyoma pseudovirions are adsorbed to, and uncoated by, host cells (Osterman et al., 1970) and that the monkey cell DNA in SV40 pseudovirions is able to enter the nuclei after infection of mouse embryo cells without loss of physical integrity (Grady e t al., 1970). Therefore, transduction should be possible. The SV40 pseudovirions associated with virus grown in monkey cells appear to be analogous to generalized transducing bacteriophages in that they appear to contain only host DNA and do not show a preference for the region of host DNA incorporated (Grady et al., 1970). Experiments designed to detect transduction by either rescued virus or pseudovirions cannot be carried out until a wide variety of genetic markers have been identified for the host cell. One interesting virus which should be considered with respect to the possibility that host cell DNA might be carried in virus stocks and express certain functions in infected cells is the MAC-adenovirus hybrid population (Butel et al., 1966). MAC (the monkey cell-adapting component) is defective and can replicate in simian cells only in the presence of a helper adenovirus because MAC is dependent upon the adenovirus for the provision of a capsid. In turn, MAC complements the human adenovirus so that it can replicate in simian cells. MAC can be transcapsidated to heterologous types of adenoviruses (Butel and Rapp, 1967). Although similar to PARA in many respects, MAC does not carry the genetic information for any known SV40 markers, including T-antigen (Butel and Rapp, 1967) and transplantation antigen (Rapp et al., 1967a). In addition, no SV40 DNA could be detected in cells infected with MAC-ftdcnovirus 7 (Butel, 1967). So, all available evidence indicates that MAC did not originate from SV40. Furthermore, sera which reacted with three different groups of simian adenovirus tumor antigens failed to react with GMK cells infected with MAC (Butel, 1967). Therefore, the origin of MAC remains obscure. However, it is conceivable that the defective genetic material of MAC is actually of host cell origin which, in some way, became associated with adenovirus type 7 under conditions of mutual dependence such that its persistence was assured.

36

B.

J. S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

STATE O F THE VIRAL

G ~ N O MI NE TRANSFORMED CELLS

Since all, or nearly all, transformed cells can produce infectious virus, but do so only rarely, what is the state of the viral genome in such cells? Assays of nucleic acid isolated from SV40-transformed cells have failed to reveal the presence of any infectious viral DNA (Sabin and Koch, 1963b; Kit et al., 1968). Reich et al. (1966) first reported that SV40-transformed hamster cells contain DNA with homology t o SV40 mRNA prepared in vitro. This complementary RNA bound to some extent to normal hamster cell DNA, but the extent of binding was greater in the transformed cells. Westphal and Dulbecco (1968) confirmed these observations using RNA prepared from highly purified component I of virus DNA as template and were able to estimate the number of viral DNA equivalents per transformed cell. They found a wide spectrum ranging from 7 equivalents in one SV40-transformed mouse cell line (SV3T3-56) to as many as 58 in a hamster tumor cell line (H-50). The estimated number of equivalents was stable for a given cell line even when grown under variable physiological conditions. A more recent report (Levine e t al., 1970), using a further refined hybridization system, lowered the estimate of SV40 viral DNA equivalents/cell in the H-50 cell line to 9. Other SV40-transformed cell lines contained 2-5 DNA equivalentsjcell. Although the estimates vary, these reports, plus an additional one (Tai and O’Brien, 1969), all indicated that there were apparently multiple copies of the SV40 genome per transformed cell. I n contrast, analysis of renaturation kinetics of DNA ( C o t values) resulted in an estimation of only 1-3 SV40 DNA equivalents per transformed cell (Gelb et ul., 1971). Such estimates, of course, do not indicate whether or not some of the “equivalents” might be defective (incomplete) viral genomes, especially since virus has never been rescued from the H-50 tumor cell line (Melnick e t al., 1964) in spite of trials in many laboratories. What, then, is the state of these multiple copies of viral nucleic acid within the transformed cells? Using the DNA-RNA hybridization technique and an SV40-transformed line of 3T3 mouse cells, Dulbecco and his colleagues (Westphal and Dulbecco, 1968; Sambrook et nl., 1968) established the following points about the physical state of the viral DNA: ( a ) It is in the nucleus; no hybridizable DNA was detected in cytoplasmic extracts. ( b ) It is associated with the cellular chruinosomes. Chromosomes were isolated from metaphase cells, and the DNA isolated from the chromosomes was shown to hybridize to the same extent as DNA from interphase cells. (c) I t is not in the form of circular molecules. After centrifugation to equilibrium in cesium chloride

PAPOVAVIRUS

SV40

37

in the presence of ethidium bromide, the hybridizable material was found in the cellular DNA band in the gradient. ( d ) It is not present in the form of free molecules the size of a single SV40 genome. The Hirt (1967) salt precipitation method, which separates large and small molecules of DNA, showed that the hybridizable regions were found in the precipitate of cellular DNA. ( e ) The viral and cellular DNA’s are not separated by alkaline denaturation and centrifugation in a sucrose gradient. The main conclusion to be drawn from these studies is that the SV40 DNA is apparently covalently bound to the chromosomal DNA of the transformed cell. The site or sites of insertion of the multiple copies of the genome are still unknown. The fluctuation in the rescue of infectious SV40 a t different passage levels of the same cell line suggests that the state of the viral genome might possibly vary in the transformed cells. If the rare cells which release infectious virus do so because the viral DNA is detached from the host chromosome, the quantity of viral nucleic acid present in a free state would be too small to be detected by the biochemical tests employed in the studies just described. I n addition to the above evidence which points strongly to the fact that the transforming viral DNA is predominantly, if not always, integrated into the host cell genome, a totally different type of evidence supports the same concept. Weiss et al. (1968) prepared somatic hybrids between mouse cells and SV40-transformed human cells. On passage of cells in culture, the chromosomes of human origin were gradually lost from the hybrid cells, and this gradual loss was correlated with a reduction of SV40 tumor antigen in the hybrid cells. The T-antigen was not compIetely lost until all or nearly all the human chromosomes had been lost. Hybrid cells which had lost T-antigen were capable of synthesizing the antigen after reinfection by SV40, thereby showing that the loss of T-antigen had not been due to the loss of a cellular gene required for the expression of the viral genome (Weiss, 1970). Karyological analyses of the hybrid cells failed to implicate a single specific integration site in the human chromosomes. Rather, the results suggested that the multiple viral genomes were scattered among various human chromosomes. Wherever the integration sites may be, there must be different ones for each virus, since both hamster and mouse cells can be doubly transformed by SV40 and polyoma virus (see Section II1,C). The use of interferon has also suggested the integration of the viral genome into that of the host cell. The induction of T-antigen by SV40 in 3T3 cells is sensitive to inhibition by interferon. However, serial passage of SV40-transformed 3T3 cells in the presence of interferon had

J . S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

38

no effect on the synthesis of T-antigen (Oxman et al., 1967). Previous investigations had established that fixation of the transformed state in the infected cell required only one cell generation (Todaro and Green, 1966b) and that treatment of the cells a t the time of infection with interferon would prevent transformation (Todaro and Baron, 1965). The use of synchronized cell cultures revealed that if the cells were not synthesizing DNA, transformation remained interferon-sensitive, but if the cells were rapidly synthesizing cellular DNA (S phase), transformation readily became interferon-resistant (Todaro and Green, 1967). Since there is evidence that interferon blocks translation of viral mRNA (Marcus and Salb, 1966; Joklik and Merigan, 1966), these observations can be explained by the assumption that the viral DNA becomes integrated into the cellular chromosomes during the S phase of cell growth. Once integrated, the viral mRNA is masked by attached regions of cell rnRNA so that interferon no longer recognizes i t as being viral in origin. C. TRANSCRIPTION OF THE VIRALGENOME IN

TRANSFORMED CELLS

The viral DNA shown by the above experiments to be present in SV40-transformed cells was obviously being transcribed to some degree. This was evident in that virus-specific antigens were associated with such transformed cells (Section IV). It was important to determine the degree to which transcription did occur. Benjamin (1966) first reported that a small fraction of pulse-labeled RNA from SV40-transformed cells was able to hybridize with SV40 DNA. Aloni et al. (1968) modified the hybridization technique and compared the virus-specific RNA in transformed mouse cells with the mcssenger RNA (mRNA) synthesized in BSC-1 cells productively infected with SV40. They found that mRNA formed during lytic infection prior to viral DNA synthcsis (“early” RNA) was different from the mRNA present after the onset of viral DNA replication (“late” RNA). Approximately one-third of the SV40 genome was represented in the early RNA whereas a t least 75% of the genome was represented in the late RNA. Competition experiments between virus-specific RNA from transformed cells and late RNA from infected cells suggested that only about onethird of the genome was transcribed in the transformed cells. Unfortunately, competition experiments between early RNA and transformed cell RNA were not technically feasible a t that time. However, they did show that the base composition of the virus-specific RNA in transformed cells was not identical to the basc composition of either the early or the late mRNA in productively infected cells. Oda and Dulbecco (1968) and Sauer and Kidwai (1968) both confirmed the existence of early and late mRNA after infection with SV40.

PAPOVAVIRUS

SV40

39

Although approximately the same proportion of viral genome was represented in early RNA and virus-specific transformed mouse cell RNA, the two types of RNA were not identical. A late gene seemed to be transcribed in the transformed cells. Similar results were obtained with transformed mouse cells, but approximately 80% of the SV40 genome appeared to be transcribed in a transformed green monkey kidney cell line. I n all cases, it appeared that the lack of expression of certain viral genes in transformed cells was at the level of transcription. A more recent study of the regulation of SV40 gene activity in transformed cells utilized randomly labeled lytic mRNA (M. A. Martin and Axelrod, 1969). A series of SV40-transformed mouse cell lines were analyzed. The extent of transcription in the individual lines varied, ranging from 30 to 100% of that seen during lytic infection. In the latter cell line, there must be some block beyond the point of transcription which prevents the synthesis of infectious virus. This study also emphasized that the extent of transcription of the SV40 genome is variable from one transformed cell line to the next, even within a single species of host cell. Of interest is a recent report (Sauer, 1971) which demonstrated that in an SV40-transformed green monkey kidney cell line, production of late viral mRNA sequences was not prevented when DNA synthesis was inhibited. This was in contrast to a productive infection by SV40 in which the inhibitors of DNA synthesis prevented the appearance of late mRNA. The mechanism(s) responsible for the apparent differences in transcriptional control is not known a t this time. High molecular weight heterogeneous RNA that contains virusspecific RNA occurs in the nucleus of transformed mouse cells (Lindberg and Darnell, 1970). Polysomal mRNA of lower molecular weight also contained virus-specific RNA. The authors suggested that the large nuclear molecules may be precursors of the cytoplasmic mRNA. Of particular interest is the fact that the largest nuclear molecules containing SV40 sequences were longer than one SV40 genome; therefore, either cellular RNA must be covalently linked to virus-specific RNA, or several SV40 DNA molecules must be integrated in tandem. The latter possibility was not supported by the chromosome data described above (Weiss et al., 1968; Weiss, 1970), but different transformed cell lines were employed in the two studies. If cellular mRNA regions are shown to be adjacent to the virus-specific sequences, this would be very formidable evidence in support of the concept of integration of the viral genome into that of the host cell. It would also strengthen the postulated rnechanism described above of the interferon resistance ( a ) of T-antigen synthesis in transformed cells and ( b ) of transformation after S phase growth of SV40-infected 3T3 cells. Evidence for such polycistronic

40

J. S. BUTEL, 6 . S. TEVETHIA, AND J . L. MELNICK

“viral-cell” RNA molecules has, in fact, been obtained with RNA from adenovirus 2-transformed rat embryo cells (Green et al., 1970).

D. BASISFOR LACKOF VIRUS PRODUCTION BY TRANSFORMED CELLS The preceding sections have established that the majority of SV40transformed cells, if not all, contain one or more copies of the viral genome, probably in an integrated state. Most cell lines can be induced to release small amounts of infectious virus, but only a small fraction of the individual cells in a given cell line appear to synthesize virus in such induction experiments. By far, the general rule is that the transformed cells do not produce infectious virus. What, then, is the mechanism by which transformed cells prevent the completion of the virus replicative cycle? This important question is central to the understanding of the mechanism of viral carcinogenesis and can be viewed from three different perspectives. ( a ) A virus-specific “repressor” is synthesized and the presence of this material in some way blocks the replicative cycle. ( b ) The transformed cells may lack some essential factor required by the virus for replication. ( c ) Defective viral genomes which are inherently unable to replicate because of a lack of certain genetic information may be the transforming agents (see Section II1,B) , It will be recalled that in Section II1,A a distinction was made between cells which were permissive and cells which were nonpermissive for SV40. Normal cells derived from nonpermissive species are not susceptible even to the naked viral nucleic acid (Swetly et al., 1969). Certain species of cells seem to lack essential factors required for SV40 replication and are, therefore, nonpermissive. Alternatively, a cellular “repressor” could be present, making the system nonpermissive. I n either case, it is meaningless at this point to say that transformed, nonpermissive cells are resistant to superinfection when the normal cells from that species cannot be productively infected. There are no data available to indicate whether the replication of a superinfecting SV40 genome in transformed cells is stopped p i o r to the block imposed by the “natural resistance” of the nonperrnissive cells. In contrast, permissive cells generally support viral replication. However, when such permissive cells are transformed (see Section II1,A) and cell lines are established, the lines tend to be virus free. These cell lines would, therefore, appear to be the most logical ones to study in an attempt to delineate which events determine whether a cell becomes transformed or productively infected following exposure to SV40. Based on the information currently available about transformed permissive

PAPOVAVIRUS

SV40

41

cells, it is difficult to distinguish between the alternate hypotheses proposed above, One can assume that since the parental cells were susceptible to SV40, proving that all essential replication factors were present, there must be some repressorlike substance present. However, since transformation of permissive cells is a rare event, such transformation may occur in those rare deficient cell mutants which lack the essential replication factors, and thus they are analogous to nonpermissive cells. Many of the SV40-transformed monkey kidney cell lines will not yield virus under any conditions which have been employed (Margalith et al., 1969; Rapp and Trulock, 1970; Fernandes and Moorhead, 1965; Shiroki and Shimojo, 1970; Koprowski et al., 1967; Butel et al., 1971b). These cell lines may well have been transformed by defective SV40 particles. However, some SV40-transformed monkey kidney cell lines, as well aa many SV40-transformed human cell lines, will yield infectious virus (Koprowski et al., 1967; Kit et al., 1970; Ashkenazi and Melnick, 1963). Therefore, one cannot conclude that permissive cells can be transformed only by defective particles. Cassingena and colleagues (1968, 1969) described the presence of a specific “repressor,” protein in nature, in cells of different species transformed by SV40, as well as in cells productively or abortively infected by SV40. This repressor was extracted from transformed cells by disrupting the cells by sonic oscillation, followed by removal of cell debris by centrifugation. The repressor in the supernatant fluid was found to inhibit plaque formation by SV40 in monkey kidney cells. The inhibitory activity could be detected only if polylysine or polyornithine was employed to increase the permeability of the host cell membrane to the extract. The repressor activity was specific for SV40; it had no effect on plaque formation by vesicular stomatitis virus, poliovirus, vaccinia, or herpes virus. The correct dilution of repressor was critical. The concentrated extracts did not show inhibitory activity. Maximum plaque reduction achieved in these tests a t the most potent dilution of extract was in the range of 50%. Conversely, other investigators, using a variety of approaches, have failed to obtain results compatible with the existence of a cytoplasmic virus-specific repressor. F. C. Jensen and Koprowski (1969) were able to rescue infectious SV40 when virus-free transformed hamster or mouse cells were fused with virus-free transformed monkey or human cells. If a repressor substance were present in the transformed cells, such completion of viral replication should not have occurred. It was found (Swetly et al., 1969) that transformed monkey and human cells were susceptible to infection by SV40 DNA although they were resistant to complete SV40 and that when uptake of virus was enhanced, a productive

42

J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

cycle of infection proceeded (Barbanti-Brodano et al., 1970). Using a line of stable monkey kidney cells (BSC-1) transformed by SV40, Rapp and Trulock (1970) found it was resistant to superinfection by complete SV40 but supported the replication of a defective SV40 genome (PARA) enclosed in an adenovirus capsid. Experiments with transformed permissive cells in the authors’ laboratory (Butel et al., 1971b) have confirmed and extended those described above. Four monkey cell lines transformed by complete or defective SV40 were analyzed with respect to susceptibility to superinfection. A spectrum of susceptibility was observed. Two cell lines transformed by defective SV40 (defective T-antigen-inducing particles of SV40 and irradiated PARA-adenovirus 7, respectively) were sensitive to superinfection by complete SV40. The remaining two cell lines, transformed by complete SV40, were resistant to challenge by SV40. However, one did support the replication of SV40 DNA, confirming the observations of Barbanti-Brodano et al. (1970) that some transformed cell lines are resistant to SV40 apparently because of surface changes. However, the fourth cell line was markedly resistant to SV40 DNA, suggesting that alterations a t the cell surface are not the only explanation for resistance to superinfection in SV40-transformed cells. As Rapp and Trulock (1970) had observed with one cell line, all the transformed cell lines in this study were susceptible to superinfection by PARA. Experiments in the authors’ laboratory (Butel and Guentzel, 1971) have also confirmed the observation that extracts of SV40-transformed cells reduce plaque formation by SV40 in the presence of polyornithine. However, the same extracts, when added to SV4O-infected cultures every 6 hours in the presence of polyornithine had no effect on the kinetics of the viral replicative cycle. There was no change in the duration of the latent period, the rate of increase of infectious virus, the time a t which peak virus titers were obtained, or the final yield of virus from the cultures. Of course, i t is possible that an improper concentration of extract or improper multiplicity of infection (-1 PFUJcell) was employed in the growth studies which resulted in a negation of the repressor activity. The rescue of virus following fusion of transformed and susceptible cells is not the consequence of a phenomenon akin to “zygotic induction” in lysogenic bacteria. Wever et al. (1970) showed that when virus replication was activated by fusion of transformed cells with permissive monkey kidney cells, the initial site of synthesis was in the nucleus of the transformed cell. Only secondarily were the permissive cell nuclei in the heterokaryons infected with virus. The speculated series of events

PAPOVAVIRUS

SV40

43

which may lead to the rescue of infectious virus from an SV40-transformed cell is presented diagrammatically in Fig. 4. The technical problems involved in obtaining transformation of a permissive cell line by SV40 should be considered. If live virus is used initially, transformed cells which may arise but are not resistant to superinfection will eventually be destroyed by the virus in the culture. If inactivated (e.g., heavily UV-irradiated) virus is used to infect the cells, transformation would very likely be mediated by defective genomes. Consequently, technical problems are such that the transformed permissive cells currently available may not be representative of all the different types of transformation theoretically possible. Experimental conditions would tend to select those cells resistant to superinfection or those transformed by defective viruses. NORMAL SUSCEPTIBLE S V 4 0 - TRANSFORMED CELL UYYYYY HOST CELL CHROMOSOME

-m VIRAL DNA x

ESSENTIAL FACTOR OR ANTIREPRESSOR VIRUS PARTICLE

@@ ( 1 ) Cell fusion mediated by inactivated Sendai Virus

( 7 ) Release of rescued infectious virus

Secondary cycle of sv40 reDlication in normal cell nucleus

( 2 ) Transfer of ( a ) essential factors , or ( b ) antirepressor from normol cell to transformed cell

t

(3)Release of viral oenome J trom integration-

G (J& i-ji

( 5 ) Transfer of ( a ) virus or ( b ) viral DNA from Ironsformed cell nucleus to normal \ cell nucleus

/(4)

Replication of S V 4 0 in tronsformed cell nucleus

F I ~4.. Speculated series of events which lead to rescue of infectious virus from SV4O-transformed cells.

44

J . 8. BUTEL, 8. 8. TEVETHIA, AND J . L. MELNICK

One can visualize that an entire spectrum of transformed permissive cells should be obtainable, the variation noted by Butel et al. (1971b) being only part of that spectrum. If particles defective a t different points in the genome were to mediate transformation, the resulting transformed cell lines should exhibit variations in properties, such as differences in susceptibility to superinfection by virus or viral nucleic acid or differences in the extent of surface changes. The realization of these many variables makes it difficult to generalize since very few transformed permissive cell lines have been studied in detail. At this time, the diverse available evidence can best be synthesized in the following way: The nonpermissive cells lack some essential factor required for virus replication. These factors or, alternatively, an antirepressor for the nonpermissive cell-eoded repressor, may be supplied by permissive cells following fusion and heterokaryon formation, so that virus rescue can occur. Transformed permissive cells may possess a block which prevents the replication of the resident genome. This may well be some type of repressor substance, but it does not behave in tests like a lambda bacteriophage repressor molecule. Defective viral genomes may well be responsible for some observed cases of transformation. Obviously, this area, which is critical to our understanding of transformed cells, warrants further attention. A series of temperature-sensitive mutants defective in different viral functions would be one logical approach to the problem.

E. CONSIDEXUTIONOF WHETHER THE VIRALGENOME Is ESSENTIAL FOR MAINTENANCE OF TRANSFORMATION The strongest evidence in support of an affirmative answer to the above question is G. S. Martin’s t s mutant of RSV described in Section II1,D. His results (1970) strongly suggest that a viral gene not involved in the replication of the virus is required for the maintenance of the transformed state. This separation of replication and transformation by a tumor virus is supported by other evidence. Also working with RSV, both Gold6 (1970), using SR-RSV-D, and Toyoshima et al. (1970), using B77, irradiated the parental viruses and succeeded in isolating virus variants unable to transform cells but able to replicate well. I n addition, they also detected defective variants of virus able to transform cells but unable to replicate. While neither group was able to positively rule out the possibility that the nontransforming virus was actually a contaminating avian leukosis virus preexistent in the parental RSV stock, each felt that was an unlikely explanation and favored the interpretation that the virus-producing and cell-transforming capabilities of RSV had been separated.

PAPOVAVIBUS SV40

45

I n Section III,D, examples of complete reversion of polyoma and RSV-transformed cells were described which appeared to be due to a loss of viral genes. Those reports would, therefore, suggest that the presence of a t least part of the viral genome is required for the maintenance of the transformed state. The vast majority of SV40-transformed cells synthesize virus-specific antigens (Section IV) and virus-specific mRNA(Section V,C) and frequently yield virus under proper conditions (Section V,A) , leaving little doubt that viral information is, indeed, present. There are a few perplexing exceptions, however, which preclude any simple conclusions that the persistence of viral genetic material is obligatory to maintain transformation. Diamandopoulos and Enders (1965) reported that lung and liver cells from Syrian hamsters underwent transformation following exposure to SV40, but no T-antigen or infectious virus could be detected. These results were subsequently confirmed by others (van der Noordaa and Enders, 1966; Sahnazarov et al., 1970; Nachtigal and Butel, 1970). Hamster embryo fibroblasts were used in analogous experiments and four of seven virus-exposed cultures developed SV40 S-antigen while only two synthesized SV40 T-antigen (Diamandopoulos et al., 1968). The S+Tcells were found to lack detectable levels of SV40 transplantation antigen (Tevethia et al., 1968b), complementary RNA (Levin et al., 1969b), and viral DNA (Levine et al., 1970). The specificity of the S-antigen reaction was discussed in Section IV,C. These studies are pertinent in that we are faced with cells which transformed following exposure to SV40, but which lack demonstrable amounts of viral DNA or complementary RNA. Until such time as it is definitely ruled out, the possibility exists that exposure to SV40 activated a latent hamster tumor virus (Huebner and Todaro, 1969). Another possible mechanism is that the virus mediated a somatic mutation of the cell genome. However, Braun (1970) considered this possibility in depth and, after showing the diversity of tumors able to revert to normal phenotype, concluded that mutation was an unlikely explanation for neoplasia. Two experimental approaches are readily apparent which could be pursued to answer this question. One would be the use of temperaturesensitive mutants of SV40 to delineate one or more specific viral genes required for the maintenance of transformation. Cells transformed by such mutants should revert to normal under restrictive conditions where the temperature-sensitive gene product would be inactive. A second approach would be to study one of the cell types, such as Syrian hamster lung cells which exhibit the unique response to SV40.

46

J. S. BUTEL, S. S. TEVETHIA, AND J . L. MELNICK

The key question is whether the observed cases of transformation were actually mediated by SV40 in spite of the absence of T-antigen in the transformed cells. After transformation by PARA (defective S V N ) , hamster lung cells are capable of synthesizing SV40 T-antigen (Nachtigal and Butel, 1970). These results demonstrate that there is no intrinsic host cell block which prevents the expression of this virus-specific marker. It is possible that several different types of virus-host cell interactions can occur after exposure to SV40. Once the spectrum of virushost interactions is established, the next step would be to determine the relative significance of each type of interaction. The type of interaction most common in the SV40-hamster system might be least common in the human system, and vice versa. It is, of course, critical for both the diagnosis and treatment of human tumors to know whether the persistence of the viral genome is required for maintenance of transformation. VI. Conclusions and Summary

This chapter has attempted to evaluate the available knowledge pertaining to SV40 oncogenesis. A summary of the different tests which can be employed to detect virus-induced changes in SV40-transformed cells is given in Fig. 5. Inoculation of such cells into susceptible hosts usually results in the production of tumors, the ultimate criterion necessary to establish that malignant transformation has indeed occurred. No in vitro test is known that will accurately predict whether a cell with all the properties of a transformed one will actually be transplantable

in vivo. Fusion or cocultivation of the transformed cell with normal susceptible cells may sometimes succeed in the rescue of infectious virus. The optimum conditions required to elicit production of virus from a majority of the transformed cells have not yet been established. Transformed cells are usually immune to superinfection by the transforming virus. The mechanism underlying this resistance to SV40 requires further delineation. Nucleic acid hybridization experiments indicate that multiple copies of the SV40 genome are present, probably integrated into the host cell chromosome. The integration sites are not known. Virus-specific mRNA is present in the transformed cells. The extent of transcription of the viral genome seems to vary from one transformed cell line to the next. No relationships have been established yet between the extent of transcription in a given transformed cell line and the number and/or magnitude of virus-specific changes of that cell line. Another in vivo test, the transplant rejection test, serves to detect

PAPOVAVIRUS

A. Intracellular changes 1. T-antigen a. Immunofluoreacence b. Complement fixation 2. Viral DNA

a. Hybridization

3. Viral-specific mRNA a. Hybridization

4. Infectious viral genome a. Rescue by fusion 5. Immunity to SV40

a. Superinfection

SV40

47

B. Cell surface changes 1. Santigen a. Immunofluoreacence b. Mixed hemadsorption c. Colony inhibition d. Cytotoxicity 2. TSTA a. Immunogenicity b. ImmunoseIlsitivity c. Neutralization by immune 1YmPhOCY~ d. Colony inhibition? 3. Normal cell antigens a. Immunofluorescence b. Mixed hemadsorption 4. Agglutination sites a. Concanavalin A b. Wheat-germ agglutinin 5. Embryonic antigens a. Immunofluoreacence 6. Immunity to SV40 a. Superinfection

FIG.6. Methods of detection of virus-induced changes in SV-40-transformed cells.

the presence of the tumor-specific transplantation antigen in the transformed cell. When this antigen is present, the cell is incapable of growing in animals immunized by the transforming virus. A series of in vitro tests detect a variety of surface changes on the transformed cells. These tests include immunofluorescence, agglutination, cytotoxicity, colony inhibition, and mixed hemadsorption. An important goal is to develop an in vitro test which can detect TSTA, and the colony inhibition test seems to be closest of those available. The relationships between the surface changes detected on SV40-transformed cells by the various tests are discussed in Section IV,D and summarized in Table 111. Results obtained using the tests outlined above enable us to visualize a transformed cell, as shown in Fig. 6. The transforming genome is integrated into the cellular chromosome in the nucleus. SV40-specific mRNA is found in both the nucleus and cytoplasm. T-antigen is synthesized and localized in the nucleus. A virus-specific repressor may or may not be present. If such repressor molecules do exist, they could conceivably be in either the nucleus or the cytoplasm or in both places. A multitude of changes occur a t the cell surface, including the appearance of TSTA, S-antigen, agglutination sites, embryonic antigens, and

48

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J. S. BUTEL, S. S. TEVETHIA, AND J. L. MELNICK

HOST CELL CHROMOSOME

0

Hm VIRAL DNA

0

it

8

\ .

VIRAL mRNA 1 - A NTI G EN REPRESSOR SWO VIRUS PARTICLE

UNCOVERED NORMAL CELL ANTIGENS

sANTIGEN CI

MALIGNANT

AGGLUTINATION SITES

Fia. 8. Diagrammatic comparison of normal and SV40-transformed cells.

normal cell antigens. The cell is immune to superinfection by SV40. Finally, it may be malignant. By comparison, all these changes are absent or masked in a normal cell, which in addition, is susceptible to infection by SV40. In spite of many efforts, the virus-mediated change which is the key to the neoplastic conversion of a normal cell into a cancer cell remains unknown at this writing. It may be one or a combination of the changes detected in the tests described in the preceding sections of this chapter, or it may be an event which has escaped detection so far. The last few years have seen a large increase in the storehouse of. knowledge pertaining to properties of papovavirus-transformed cells. Preliminary results obtained with mutant viruses and variant transformed cells, coupled with the advent of new tests to detect virusinduced changes, suggest that key events related to virus-induced carcinogenesis are on the verge of becoming unraveled.

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Rhim, J. S., Greenawalt, C., Takemoto, K. K., and Huebner, R. J. (1971). Nature New Biol. 230, 81-83. Richardson, L., and Butel, J. S. (1971). Int. J. Cancer 7, 75-85. Robertson, H. T., and Black, P. H. (1969). Proc. SOC.Exp. Biol. Med. 130, 363-370. Rothschild, H., and Black, P. H. (1970). Virology 42, 251-256. Rowe, W. P., and Baum, 8. G. (1964). Proc. Nat. Acad. Sci. U . S. 52, 1340-1347. Rowe, W. P., and Baum, S. G. (1965). J. Exp. Med. 122,955-968. Rowe, W. P., and Pugh, W. E. (1966). Proc. Nat. Acad. Sci. U . S. 55, 1126-1132. Sabin, A. B., and Koch, M. A. (1963a). Proc. Nat. Acad. Sci. U. S. 49, 304-311. Sabin, A. B., and Koch, M. A. (196313). Proc. Nat. Acad. Sci. U. S. 50, 407417. Sabin, A. B., and Koch, M. A. (1964). Proc. Nat. Acad. Sci. U. S. 52, 1131-1138. Sachs, L. (1966). Nature (London) 107, 12721274. Sahnazarov, N., GraEe, L. H., Nachtigal, M., and Ionescu-Homoriceanu, S. (1970). R e v . Roum. Injramicrobwl. 7, 79-85. Sambrook, J., Westphal, H., Srinivasan, P. R., and Dulbecco, R. (1968). Proc. Nat. Acad. Sci. U.S. 80, 1288-1295. Sauer, G. (1971). Nature New Biol. 231, 135-138. Sauer, G., and Kidwai, J. R. (1968). Proc. Nat. Acad. Sci. U. S. 61, 125f3-1263. Sauer, G., Koprowski, H., and Defendi, V. (1967). Proc. N a t . Acad. Sci. U. S. 58, 594806. Shein, H. M. (1968). Science 159, 1476-1477. Shein, H. M., and Enders, J. F. (1962). Proc. Nat. Acad. Sci. U. S. 48, 1164-1172. Shein, H. M., Enders, J. F., Levinthal, J. D., and Burket, A. E. (1963). Proc. Nal Acad. Sci. U.S. 49, 28-34. Shiroki, K., and Shimojo, H. (1970). Personal communication. Sjogren, H. O., Hellstrom, J., and Klein, G. (1961). Exp. Cell Res. 23, 204-208. Smith, R. W., Morganroth, J., and Maor, P. T. (1970). Nature (London) 227, 141-146. Sweet, B. H., and Hilleman, M. R. (1960). Proc. SOC.E z p . B i d . Med. 105, 420-427. Swetly, P., Barbanti-Brodano, G., Knowles, B., and Koprowski, H. (1989). J. Virol. 4, 348-356. Tai, H. T., and O’Brien, R. L. (1969). Virology 38, 698-701. Takemoto, K. K., and Habel, K. (1966). Virology 30, 20-28. Takemoto, K. K., Ting, R. C., Ozer, H. L., and Fabisch, P. (1968a). J. Nat. Cancer I m t . 41, 1401-1409. Takemoto, K. K., Todaro, G. J., and Habel, K. (1968b). Virology 31, 1-8. Tevethia, S. 5. (1970). J. Immunol. 104, 7278. Tevethia, S. S. (1971). Unpublished observations. Tevethia, S. S., and Rapp, F. (19as). Proc. SOC.Exp. B i d . Med. 123, 612-615. Tevethia, S. S., Katz, M., and Rapp, F. (1965). Proc. SOC.Ezp. B i d . Med. 119, 896-901. Tevethia, S. S., Couvillion, L. A,, and Rapp, F. (1968a). J. Immunol. 100, 3!%362. Tevethia, 6. S., Diamandopoulos, G. T., Rapp, F., and Enders, J. F. (198813). J. Immunol. 101, 11924198. Tevethia, S. S., Dreesman, G. R., Lausch, R. N., and Rapp, F. (196%). J. Immunol. 101, 1105-1110. Tevethia, S . S., Crouch, N. A., Melnick, J. L., and Rapp, F. (1970a). Int. J . Cancer 5, 176-184. Tevethia, S. S., Lausch, R. N., and Rapp, F. (1970b). In “Immunity and Tolerance in Oncogenesis,” pp. 183-199. 4th Quadrennial Int. Conf. Cancer, Perugia, Italy.

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NASOPHARYNGEAL CARCINOMA (NPC) J. H. C. Ho Medical and Health Department Institute of Radiology, Queen Elizabeth Hospital, Kowloon, Hong Kong

I. Introduction . . . . 11. Histogenesis . . . . 111. Etiology . . . . . A. Early Cases . . . B. Genetic Factors . . C. Environmental Factors IV. Conclusion . . . . References

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78 88 89

I. Introduction

Nasopharyngeal carcinoma (NPC) has recently attracted worldwide interest because a high titer of antibodies to Epstein-Barr virus (EBV), which is suspected to have oncogenic properties, has been found with unusual frequency among patients with this neoplasm compared with controls from the general population and also with patients with head and neck tumors other than NPC (de Schryver et al., 1969; W. Henle et al., 1970). It is of interest also that immune responses by the host against tumor-associated antigens have been shown in a number of neoplasms including NPC (Klein, 1970). This neoplasm is rare among most groups of people, but Chinese, particularly those originating from the southern province of Kwangtung, have a very high risk of developing the disease. Lilly (1966) has shown that in inbred strains of mice the incidence of lymphomas and leukemias is linked to a genetically determined susceptibility to tumor induction by oncogenic viruses. The discovery of an association between a virus infection and NPC which has a predilection for an ethnic group has, therefore, stimulated much interest in the nature of the association, which may shed some light on the tantalizing question whether viruses, which are now known to give rise to a variety of tumors in certain mammals and birds, also cause cancer in man. It is the purpose of this chapter to review the etiology of NPC. It. Histogenesis

Many types of nasopharyngeal cancers have been found, but carcinoma is by far the predominant type in people of all races. I n China 57

58

J. H. C. H O

and Southeast Asia, where the incidence is exceptionally high, a t leaat 99% of the malignant neoplasms have been reported to be carcinomas (Liang, 1964; Yeh, 1967; Shanmugaratnam and Muir, 1967). In Australia, Scott and Atkinson (1967) detected essentially no difference in the histopathological distribution of nasopharyngeal cancers among Caucasian and Chinese patients. In their series of 213 histologically proven cases of malignant neoplasms, 202, or approximately 95% of them, are carcinomas. I n America, where the incidence of nasopharyngeal cancers is low, the ratio of carcinoma to sarcoma is between 8 : l and 9 : l (Fletcher and Million, 1965; Vaeth, 1960). I n England, also a lowincidence area, the ratio of carcinoma (epithelioma including lymphoepithelioma) to sarcoma is 3.5:l in a series of 208 cases with known histology reported by Lederman (1961). Also in Kenya, Africa, carcinoma is the predominant neoplasm (Clifford, 1965). Table I shows the histological types of neoplasms arising primarily in the nasopharynx of 1606 cases diagnosed a t Queen Mary Hospital, Hong Kong, during the 5-year period 1959-1963. More than one case of reticulum cell sarcoma was diagnosed initially, but later development showed all of them, except one, to be undifferentiated squamous carcinoma. It is anticipated that, if electron microscopy is used routinely, there will be a reduction in the frequency of nasopharyngeal lymphoreticular neoplasms being diagnosed, a t least in Chinese patients. Svoboda et al. (1967) have shown by electron microscopy that the undifferentiated carcinomas, including the so-called lymphoepithelioma, often show evidences indicative of their squamous origin. Although similar evidences-desmosomes and cytoplasmic filamentous inclusionsare occasionally encountered in malignant lymphoma and chordoma (Friedman, 1967), the mere fact that most nasopharyngeal carcinomas

TABLE I HISTOLOQICAL TYPESOF NASOPHARYNQEAL CANCERS DIAQNOSED AT QUEENMARYHOSPITAL (1959-1963) No. of

% Carcinomas (squamous, undifferentiated and anaplastic) Malignant melanoma Reticulum cell sarcoma Not histologidly confirmed

From Ho (1972), by permission from the editor.

1571 2 1 32

98 2

-

-

1606

100

NABOPHARYNGEAL CARCINOMA (NPC)

59

contain more than one histological type of malignant cells in the same tumor and that typical squamous features are not uncommonly found in sections among predominantly undifferentiated carcinoma cells has led to the belief, which is now generally held, that the undifferentiated carcinoma including lymphoepithelioma are histogenetically variants of squamous carcinoma, the predominant neoplasm in the nasopharynx. It is in patients with this neoplasm, and not others, arising in the nasopharynx that an association with EB virus infection has been discovered, and also it is this cancer that has a predilection for people of Chinese descent. Ali (1967) in his study based on the histological examination of nasopharyngeal mucous membranes obtained at 100 medicolegal autopsies from apparently healthy individuals between ages of 10 and 80, found that under normal conditions only 60% of the total nasopharyngeal epitheIia1 surface was lined by stratified squamous epithelium and that the proportion of squamous epithelium in the nasopharynx appeared to be constant after the first decade of life. Liang et al. (1962) proposed, “Squamous metaplasia would be a prerequisite for the formation of different types of nasopharyngeal carcinoma.” This hypothesis is based on their finding that among 54 cases of malignancy (including precancerous change, carcinoma in situ, and early invasive carcinoma) arising from the nasopharyngeal mucosa, 27, or 50%, arose from the squamous epithelium of the mucosa, and that at the frequent site of carcinomatous origin, i.e., the superior two-thirds of the nasopharynx, the mucosa was normally lined by cylindrical cell epithelium. Further, in their histological examination of 300 complete nasopharyngeal mucosa specimens of cadavers above the age of 15 years, Liang et al. found that 129 specimens showed multiple small foci of squamous metaplasia. They, therefore, held squamous metaplasia to be of great significance in the histogenesis of NPC. On the other hand, they stated in the same paper that, with the exception of cylindrical cell carcinoma which arose from the surface cylindrical cell epithelium of the nasopharyngeal mucosa (including the lining of the crypts), all types of nasopharyngeal carcinoma (including squamous cell carcinoma) could arise either from the cylindrical cell epithelium or from the squamous cell epithelium. This finding was confirmed by Ch’en (1964a) from a study of 90 biopsies of the nasopharyngeal mucosa which showed malignant change. In another study Ch’en (1964b) found that anaplasia could start from a single cell or from a group of cells in the undifferentiated basal layer of the mucosal epithelium, or in the middle layer already beginning to differentiate, or in the well-differentiated superficial layer. Shanmugaratnam and Muir (1967) also found that all forms of nasopharyngeal carcinoma, both

60

J. H. C. H O

classical squamous cell carcinomas and undifferentiated carcinomas, might arise from squamous, transitional, or respiratory epithelium. Experience in Hong Kong is in agreement (Ho, 1972). It would appear, therefore, that carcinoma could start de novo without the cells first undergoing squamous metaplasia. Multicentricity of foci of carcinomatous change is not uncommonly seen in Hong Kong cases. Occasionally a new carcinoma may even appear on the opposite side of the nasopharynx from one to a few years after a course of radiotherapy which appeared to have caused the original tumor to disappear. These are unlikely to be recurrences of tumors too small to be noticed during the initial examination, because the smaller the tumor the greater is the chance of its being eradicated or controlled, and in the radiation treatment of NPC the whole nasopharynx is as a rule given the same high dose. Such new carcinomas raise a very important question: If radiotherapy had no prophylactic effect on the process of carcinomatous transformation in these cases, did it play any part in causing or accelerating the transformation, since radiation is known to be carcinogenic? The short interval between irradiation and the development of an apparently new carcinoma does not exclude such a possibility since it is not unlikely that other parts of the nasopharyngeal mucosa might already be primed by the time the first carcinoma was treated or already in the late stage of carcinomatous transformationprecancerous stage, in which case the interval can be very short. A similar example is to be found in the radiation treatment of carcinoma of the buccal mucosa. A new carcinoma may develop in the originally normal looking buccal mucosa on the opposite side, which already had received a good dose of radiation when the first tumor was treated. That a radical course of radiation treatment has little or no prophylactic effect on cells at a certain phase of carcinomatous transformation, a t least in some cases, is beyond doubt. It is, however, of importance to ascertain whether it has any promoting effect on the malignant change. Animal experiments designed to study the effect of radiation on cells at different stages of malignant transformation might help in producing an answer. 111. Etiology

A. EARLY CAB= It is not known whether NPC is largely a product of our environment within the last one and a half centuries or has afflicted man since antiquity, as has been claimed by Clifford (19701, who thinks that the oldest pathological specimens of NPC at present known were derived from

NASOPHARYNGEAL CARCINOMA (NPC)

61

inhabitants of Northeast Africa and the Middle East from the period 3500-3000 B.c., on the basis largely of the works of Wells (1963, 19641, Krogman (1940), Smith and Dawson (1924), and Derry (1909). The most important evidence is supposed to be found in the works of the first three, but close scrutiny of these works reveals considerable doubt that they are indicative of NPC, and not of other diseases. Wells (1964) stated that only 3 or 4 cases of indubitable carcinoma have been recognized among the tens of thousands of ancient Egyptian mummies and skeletons that have been examined, and that hardly a score of such cancers have been identified from all the cemeteries of the pre-Renaissance world. He thought that several of this very small number appeared to be of nasopharyngeal origin. Through the courtesy of Dr. J. C. Trevor, Director of the Duckworth Laboratory, and the kind cooperation of Professor J. Mitchell and Mr. J. A. Fairfax Fozzard of the University of Cambridge, the author had the opportunity to examine radiographs and photographs of probably the most important specimen, a skull (No. 236) kept a t the Duckworth Laboratory. This specimen shows only destruction of the posterior part of the left maxillary alveolus forming the floor of the left maxillary sinus, the adjacent part of the hard palate and pterygoid laminae, with antemortem loss of the 2nd and 3rd molars. These findings are shown in Figs. 1 and 2. The destruction is indicative rather of carcinoma or myeloma of the maxillary alveolus or of the floor of the maxillary sinus than of NPC, as the destroyed parts are situated in front of the nasopharynx, whereas the bones immediately overlying the nasopharyngeal cavity, which are the ones most commonly involved, were spared. Furthermore, the multiple circular holes in the tables of the cranial vault unaccompanied by any evidence of sclerotic bony reaction around them are far more typical of myelomatosis than NPC metastases. A “strongly probable one” according to Wells (1964) was from Tepe Hissar, Iran, ca. 3000 B.C. (Krogman, 1940). The specimen referred to is 33-23-36, the skull of a male of Mediterranean type from Hissar 11. It shows extensive destruction of the left facial bones-maxilla, palatine, and zygoma. There is an extension of the destruction to the floor of the left orbit. The left maxillary sinus is completely obliterated; the floor of the left nasal fossa is penetrated, and the penetration extends to the right of the midline. All upper teeth on the left side and the right central incisor are missing. The mandible is, however, intact, a fact which, according to Krogman, testifies against injury being the cause. He stated: “It may be conjectured whether the condition is primarily due to sinus infection, brought about by dental disease.” There was no description of destruction of bone in the immediate vicinity of the naso-

62

J. H. C. H O

Fro. 1. Photograph of skull specimen No. 236. Dotted line indicates the nasopharyngeal boundaries on the right side (R). Destruction of bone is limited to the floor of the left maxillary sinus and adjacent hard palate and pterygoid laminae.

NASOPHARYNGEAL CARCINOMA ( N P C )

63

FIG.2. Superoinferior X-ray projection of skull specimen No. 236. Dotted line indicates the nasopharyngeal boundaries on the right side (R).Site of lesion is snowed.

pharynx. There are, therefore, no grounds to interpret from these findings that the primary condition could be nasopharyngeal carcinoma. Wells (1964) thought that one of the Romano-Egyptian cases described by Smith and Dawson (1924) was another example of carcinoma of nasopharyngeal origin. The case referred to was reported to be a male

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J. H. C. HO

pre-Christian Nubian (ca. fourth to sixth century, A.D.) with extensive destruction of the base of skull from the cribiform plate to the basiocciput almost reaching the foramen magnum. The site and appearance of the destruction are consistent with a diagnosis of either carcinoma of the sphenoid sinus or nasopharyngeal carcinoma. It is, however, difficult and sometimes impossible to differentiate between the two even in examining patients. Judging from the evidence presently available, we are still far from sure whether nasopharyngeal carcinoma had afflicted human inhabitants of Northeast Africa and the Middle East some 5000 years ago. Since NPC is a prevalent disease in China, and in the province of Kwangtung has even earned the name of “Kwangtung tumor,” it would be pertinent to search old Chinese medical writings for reference to the disease. In a book of 50 volumes called, “Aetiology and Symptomatology of Various Diseases” written by CHOU Uen Fung or CHOU Yuan Fang ( # $ $), a royal physician of the Sui Dynasty (A.D. 589-617), there is a description of various types of superficial tumorous swellings. Only in “shu-lu,” or rat tumor, was there a description of the disease appearing in the neck with its root in the lungs. Jung and Yu (1963) found it impossible to determine whether all, some, or none of the cases of “lo li” (glandular enlargement of the neck) were carcinomatous metastases rather than tuberculosis or other diseases. In The Encyclopaedia of Chinese Medical Terms edited by Wu (1921) a disease, called “shih ying” or (‘shih jung” disease, meaning, respectively, loss of nutrition is described as belonging to one of the four fatal diseases and has the following clinical picture: Masses appear in front of and behind the ear and in the neck. They are stony hard, immobile, neither hot nor cold to the touch and painless at first. As they grow in sire pain gradually appears and fungation sets in. The discharge is sero-sanyineous but not pussy. Ultimately, a crater lined with necrotic tissue is formed. By this time pain becomes intense. Brisk bleeding from the ulcer soon occurs and the patient may die of haemorrhage after 1 or more bleeding episodes or of gradual loss of nutrition.

There is no doubt from this description that the masses are metastatic, not tuberculous, lymph nodes, and that they are most likely caused by a primary carcinoma in the nasopharynx. No mention is made in this encyclopedia as to when this disease was first reported in Chinese medical writings. Now that the name of the disease is known, the task of searching for such information is made easier. It is hoped that workers in mainland China and Taiwan, where one can get access to old Chinese medical writings, will do some work in this direction.

65

NASOPHARYNGEAL CARCINOMA ( N P C )

B. GENETICFACTORS 1. Sex Incidence In most countries the male to female incidence ratio is over 2:1, but in Sweden it is below 2:l. The crude and age-standardized annual incidence rates of nasopharyngeal carcinoma (with all other neoplasms of the nasopharynx excluded) for different parts of the world where such figures are available are given in Table I1 for comparison. TABLE I1 SEX INCIDENCE RATIOOF NABOPHARYNOEAL CARCINOMA Crude rates per 100,000 Population HongKong (Chinese) Singapore (Chinese) Sweden

Age-standardized rates per 100,OOO

Male

Female

Ratio M:F

Male

Female

Ratio M:F

20.68

9.42

2.19:l

24.66

10.36

2.38:l

1968

-

-

-

22.2

8.9

2.49:l

1959-65

0.76

0.49

0.30

1.86:l

Period 1965-9

1.55:l

0.56

2. Racial Susceptibility Nasopharyngeal carcinoma is rare in most parts of the world except in China and many parts of Southeast Asia inhabited largely by people of mongoloid stock who have had close relationship with Chinese for many centuries dating back to the early Ming dynasty in the 14th century. Although Japanese and Koreans are also mongoloid people and have had an even longer period of association with Chinese, incidence among the Japanese is rare (Miyaji, 1967), and probably this applies also to the Koreans (Clifford, 1970). Although the frequency of the disease is highest among people of Chinese descent both in and outside China, in China itself there is a drop in frequency from south to north according to the relative frequency expressed as percentages of all malignant tumors diagnosed by biopsies reported by major hospitals and medical schools in certain cities and provinces. This is illustrated in Table I11 and Fig. 3. The difference in frequency in different parts of China could be due to geographic reasons or to ethnic differences. It is, therefore, important to determine whether Chinese originating in different parts of China and living in the same locality have any significant differences in susceptibility. Hong Kong is not the ideal place for such a study because of the

J. H. C. H O

66

TABLE 111: RELATIVE FREQUENCY OF NABOPIIARYNGEAL CARCINOMA I N CHINESE MAINLAND AND TAIWAN ESTIMATED AS PERCENT OF ALL MALIQNANT TUMORS DIAGNOSED BY BIOPSY Percent of all cancers by biopsy

Male

Female

56.9 of 3010 3 1 . l o f 672

17.4 of 4O26 10.0of 748

16.2 of 1010 23.2 of 5436 7 . 3 of 8332 6 . 7 o f 879

4 . 7 of 1068 6 . 2 of 8636

5 . 1 of 2738 7 . 9 of 1562 4 . 0 of 5137

-

Place South Canton (Kwangtung province) Kwangei province Central Fukien province Taiwan Shanghai (Kiangeu province) Sian (Shensi province) North Tsinan (Shangtung province) Tientein (Hopei province) Peking

-

-

From Ho (1972), by permission from the editor.

constant influx of refugees. However, this influx became insignificant after 1962. The incidence rates by place of origin for Chinese males and females domiciled in Hong Kong in 1969 are given in Tables IV and V. Excluded in the analysis are patients who came from outside Hong

IOOOE

I 10°E

FIG.3. Map of China.

I2OOE

1309

67

NASOPHARYNQEAL CARCINOMA (NPC)

INCIDENCE RATES OF &INESE

Place of origin

Hong Kong Canton, M a w , and adjacent places Sze Yap area Chiu Chau area Elsewhere in Kwangtung and Kwangsi Elsewhere in China outside Kwangtung or Kwangsi

TABLE IV NASOPHARYNQEAL CARCINOMA FOR HONQKONQ &LEB BY PLACE OF ORIQIN, 1969 Population 1966 byNo. of c e n s u ~ ~ CBBB~C

Crude rate per100,OOO

Agestandardized rateper100,000

120,620s 875,530

1od 182

8.29 20.78

15.05 25.69

355,650 214,410 115,440

68 26 28

19.11 12.12 24.25

25.27 17.66 28.54

137,140

11

8.02

10.99

__

,, Barnett (1966). b Include an unknown number of non-full-blood Chinese. c Include only cases seen at Medical and Health Department Institute of Radiology. All full-blooded Chin-.

Kong for medical consultation or treatment. The rates for people of Chiu Chau origin of both sexes are significantly lower than those for people who claim origin from other parts of Kwangtung with Hong Kong excluded ( P < 0.01). The people from Chiu Chau are ethnically more TABLE V INCIDENCE RATESOF NASOPHARYNOEAL CARCINOMA FOR HONQKONQ CHINESE FEMALES BY PLACE OF ORIGIN, 1969

Place of origin

Hong Kong Canton, M a w and adjacent p l a w Sze Yap area Chiu Chau area Elsewhere in Kwangtung and Kwangsi Elsewhere in China outside Kwangtung or Kwangsi

Population 1966 bycensus0

No. of cases0

Crude rate per 100,OOO

Agestandardized rate per 1 0 0 , ~

124,63@ 875,110

6d 78

4.81 8.91

9.56

347,210 184,230 103,240

23 3 9

6.62 1.62 8.71

7.40 2.17 10.29

143,300

4

2.79

3.92

6.54

Barnett (1966). Include an unknown number of non-full-blood and Chinese. 0 Include only cases seen at Medical and Health Department Institute of Radiology. d All full-blooded Chinese. 0

b

68

J. H. C. H O

closely related to the people from Fukien than those from other parts of Kwangtung. The rates for people originating from parts of China outside Kwangtung and Kwangsi are significantly lower than those for people from Kwangtung and Kwangsi with Chiu Chau and Hong Kong excluded ( P < 0.01 for males and P = 0.025 for females). Hong Kong was a part of Kwangtung until it was ceded to Great Britain in 1841. These findings are in general agreement with those obtained in an earlier study on the incidence of nasopharyngeal cancer in the Chinese population of Hong Kong in 1961 (Ho, 1967a) and also with those reported by Mekie and Lawley (1954) on the Chinese population in Singapore. They found that the Teochews (people from Chiu Chau) and the Hokkienese (people from Fukien province) had lower frequencies of nasopharyngeal cancer than the Cantonese, Kheks, and people from Hainan Island. The Kheks are people scattered in different parts of China, but the majority of them are from Kwangtung. Muir and Shanmugaratnam (1967) have shown that in multi-racial Singapore the age-adjusted minimum incidence rates for the 12-year period 1950-1961 were the highest for Chinese and lowest for Indians and Pakistanis, those for Malays being in between. This finding is confirmed by Shanmugaratnam (1970) in a later analysis of the incidence rates based on histologically diagnosed cases only for Chinese, Malays, and Indians in Singapore for the 5-year period 1960-1964. The crude rates per 100,000 per annum for the 3 groups are, respectively, 13.3, 3.2, and 0.4, and the corresponding age-standardized rates 20.2, 5.8, and 0.2 for males. For Chinese and Malay females the crude rates are 6.4 and 0.9, and the age-standardized rates 9.0 and 2.0, respectively, there being no cases among Indians. Singapore, unlike Hong Kong, has relatively few immigrants. Chinese living there have been found to have risks according to their places of origin in China similar to those in Hong Kong. Furthermore, Indians who are normally of low risk and living in Singapore among Chinese and Malays do not share their high risk of developing the disease. It would appear, therefore, that it is the ethnic factor, rather than the geographic locality of domicile, which determines the risk. 3. Eject of Distant Migration on People of High Risk

Chinese in Hong Kong or Singapore constitute the majority of the local population and have, therefore, retained largely the customs and ways of life of their ancestors. Chinese in Australia, Hawaii, and California are in the minority and because of the vast distance between these places and the Orient they are likely to retain less. This applies especially to those born in their countries of adoption.

NASOPHARY NGEAL CARCINOMA ( N P C )

69

I n Australia, Scott and Atkinson (1967) found little difference in the risk of suffering from the disease whether a person of Chinese descent is born in Australia or outside. On the other hand, Worth and Valentine (1967) reported incidence rates for the population over the age of 14 years of Chinese descent to be 35.1 per 100,000 for males born in China and Hong Kong and 10.2 for those born in Australia. For females they are, respectively, 29.1 and 11.1. The corresponding crude rates for those of all ages are, respectively, 31.6 and 7.1 for males and 19.2 and 7.8 for females. Their report was based on an analysis of a series of 15 Chinese cases during a 10-year period 1953-1963. However, they pointed out that of the 11 China-born male cases, it was possible only to ascertain that five had been in Australia more than 5 years when diagnosed, one had been in Australia only 1 month, and the duration of residence was unknown for the other five. If these six were not immigrants but patients who went to Australia from Southeast Asia for treatment or other purposes and excluded from the list then the rate for Chinese males over 14 would be only 16.0 which is only 57% higher than the corresponding rate for non-Australian-born Chinese males. In such a small series a difference of this magnitude is without significance. In a place like Sydney, where all the case material was obtained, it is not at all unusual for Chinese from Southeast Asia traveling there during the 1953-1963 period to seek treatment because of the inadequate radiotherapy facilities in their place of domicile at the time. I n Hawaii, Quisenberry and Reimann-Jasinski (1967) analyzed a series of 14 cases of Chinese descent reported in the Hawaii Tumor Registry during 1960-1962; they found rates for 9 subjects born in the United States including Hawaii to be 6 times higher than for subjects born elsewhere. An annual rate of nasopharyngeal cancer of 54.2 per 100,000 for all ages and both sexes combined for Chinese other than those born in the United States including Hawaii is about 3.5 times higher than the corresponding rate of 15.2 per 100,000 and over 3 times the age-standardized rate of 17.2 for the mean total population of Hong Kong during the 5-year period 1965-1969 with patients from elsewhere excluded. This makes one wonder whether this group might have included Chinese patients who went to Hawaii for medical purposes. An alternative explanation is that Chinese by migrating to Hawaii had become more susceptible to the disease whereas the local-born Hawaiians of Chinese descent were no more susceptible than the Chinese in Hong Kong-a most unlikely explanation. I n the State of California, Zippin et al. (1962) investigated the place of birth of 31 Chinese males reported to the California Tumor Registry during a 16-year period 1942-1957; they found the ratio of observed-to-

70

J. H. C. H O

expected ( O / E ) number of cases by age group to be more than 8 times higher in Chinese under the age of 55 born outside United States than in those born inside. No difference was found between the two groups over 55. They stated that a difference in coverage might exist between Chinese born in the United States and those born outside, but they did not mention whether cases in the latter group might have included patients who went to California from the Far East specifically for treatment. These patients are likely to be in the younger age group because the older patients are less fit as well as less willing to make the trip. If such patients were unknowingly included, the O/E ratio would be unnaturally high. In addition, two assumptions were made in the calculation of the O / E ratio. One is that the age-sex-specific rates of New York State applied equally well to each of the age-sex-race-nativity-specific groups in California. This is not valid because the incidence rate for Chinese males reaches a peak at the age period of 50-54, after which the rate declines sharply (Fig. 5) whereas for Caucasian males the peak is reached one to two decades later. The second assumption is that the. age-distribution of the United States-born and foreign-born Chinese populations in California according to the 1950 census constitute the mean age-distributions for the two groups during 1943-1957. This is not necessarily valid. Finally, as the authors themselves have rightly emphasized, caution must be exercised in the interpretation of results based on numbers so small as those in some categories of their series. Buell (1965) , on the other hand, studied the California mortality records of deaths from cancer of the nasopharynx in 67 men and 13 women of Chinese descent during the 14 years from 1949 through 1962. The number of cases analyzed is again small and spread over a long period. He found that the risk of nasopharyngeal cancer in the local-born Chinese is considerably higher than in the white population, but lower t,han that in the immigrant Chinese. The factor of increase is about 20fold for both men and women of Chinese descent born in the United States, and 30- to 40-fold for the men and women born in China. Buell further stated that the Chinese immigrants to California have carried with them as much, if not more, risk of cancer of the nasopharynx as the immigrants to Singapore. While a lower incidence among the United States-born Chinese would support an environmental hypothesis, both Zippin et al. (1962) and Buell were of the opinion that it did not rule out a genetic etiology. It could be the result of a genetic-environmental interaction or a selection against a genotype as a cause of a reduction in the filial generation. Buell feels that there is no doubt that the immigrant generation had a lower fertility than their fathers, for, even as late as 1950, the ratio of

NASOPHARYNGEAL CARCINOMA (NPC)

71

single adult men to single women was 2:l and several decades earlier the ratio was several times higher. He finds evidence that marriage was often postponed, as revealed by the disparity in the parental age on some birth certificates of Chinese, a paternal age of 40 or 50 and a maternal age of 20 or 30 being not infrequent. Also according to Buell, about 28% of the nasopharyngeal cancer cases were reported to have died unmarried, and some married men were separated from their wives for long intervals, partly due to the Extension Acts of 1882 and 1924 (Lee, 1960). I n conclusion, it could be said that we are still left in doubt whether distant migration has altered the risk of Chinese born in their country of adoption, but the risk of those born in their place of origin appears to be unaffected. Further studies are called for.

4. Risk in People of Part-Chinese Ancestry Ho (196713)reported a crude average annual incidence rate estimated to between 20.0 and 26.7 per 100,000 during 195S1963 among Hong Kong “Macaonese,” a term which had been used by the Macao Government until a few years ago to describe “local” Portuguese as distinct from “continental” Portuguese. Macao has been colonized by Portugal for over 4 centuries, and the “Macaonese” are largely products of intermarriage between the two races, but all of them are Catholics by religion. Many of them have migrated to Hong Kong. In fact, the great majority of the people of Portuguese nationality in Hong Kong are “Macaonese.” According to a communication dated August 15, 1966 from the Commissioner of Registration of the Hong Kong Government, the number of persons resident in Hong Kong of Portuguese nationality who had registered with his department for Hong Kong Identity Cards comprised of 833 males and 735 females over the age of 6, and of 117 males and 121 females between the ages of 6 and 17 years. Those below the age of 6 were not registered, and it is not possible to ascertain the number of those who have adopted British nationality, and have not registered as Portuguese. Those registered as Portuguese included, on the other hand, a small proportion of “continental” Portuguese. With the help of some of the members of their community, it has been estimated that the total Portuguese population in Hong Kong was between 3000 and 4000 a t the time of the study. Even taking the lower estimate of 20.0 per 100,000, the crude incidence rate is unusually high, being higher than the highest of the crude rates for the various ethnic groups of Chinese in Hong Kong, e.g., 13.2 for the people originating from the Sze Yap area (Ho, 1967a). There was probably an underestimation of the “Macaonese” population accounting for its unusually high incidence,

72

J. H. C. H O

but there could be no doubt that it was much higher than that for the other non-Chinese ethnic groups in Hong Kong, because among the 53,230 persons classified as non-Chinese in the 1961 census there were five cases of nasopharyngeal carcinoma diagnosed during the 5-year period 1959-1963-four of them “Macaonese,” and one a Malay. There was not a single case among the rest, which include Europeans in the British civil service and armed forces and the foreign business community, Gurkhas in the Gurkha Infantry Brigade, Indians, Pakistanis, and other minority racial groups. I n fact, the author in over 20 years’ practice as a radiotherapist in Hong Kong has seen only one case of NPC among European Caucasians and none among the Gurkhas, Indians, and Pakistanis, although their total number far exceeds that of Macaonese in the population. In Thailand, Garnjana-Goochorn and Chantarakul (1967) engaged in a prospective survey of 1000 consecutive cancer patients in the Tumour Clinic of the Siriraj Hospital in Dhonburi with the intention of finding the relative frequencies of nasopharyngeal cancer among the three racial groups-Chinese, Chinese of part-Thai ancestry, and Thais. Out of the 1000 cases, there were 170 Chinese, 195 Chinese of mixed descent, 628 Thais, and 7 of other nationalities. Nasopharyngeal cancer was found in 27 Chinese (15.9%),20 Chinese of mixed descent (10.3%), and 29 Thais (4.6%). From this study they estimated the ratio of relative frequencies in the 3 groups to be respectively 3.4:2.2:1.0, and believed that this estimation was as near the correct proportion as they could get in Thailand. They further reported that nasopharyngeal cancer constituted 3.5% of all malignant neoplasms diagnosed a t Siriraj Hospital during a 6-year (1957-1962) period. Thais are mongoloid people, mostly Buddhists, but Portuguese are Caucasian Catholics. Yet in both, the products of intermarriage with Chinese appear to inherit at least a part of the high risk of their Chinese ancestors. 5. Familial Aggregations

Reports on familial aggregations of cases of nasopharyngeal carcinoma in literature are scarce. Pang (1959) records finding two pairs of related cases, mother-son and sister-brother combinations, in a series of 34 consecutive cases of nasopharyngeal cancer, including 27 Chinese, in Hawaii. Buell (1965) found one pair, mother-son combination, in his study of 80 certificates of death due to nasopharyngeal cancer in California. Ho (1967b) found 15 instances of familial aggregations in two separate series of 1180 cases of nasopharyngeal carcinoma. I n eight instances two brothers in the family had the disease, in one instance

73

NASOPHARYNGEAL CARCINOMA (NPC)

three brothers, in two instances a pair of brother and sister, in one instance the son and his father, one nephew and aunt, one sister and her brother, and finally in one instance a female patient had a family aggregation of cases extending over three successive generations. A pedigree study, retrospective and prospective, of this family is shown in Fig. 4. The family was originally from Canton, but generation I1 had settled in Hong Kong. Nos. 3 and 4 of this generation, the two younger brothers of the paternal grandfather of the propositus, died of a disease with a clinical history highly suggestive of NPC, e.g., nasal bleeding, regurgitation of food through the nose, and loss of voice. These are the only symptoms recalled by I11 (No. 1) and related by members of generation IV. When a Chinese of Canton origin dies of a disease with these symptoms, it is most likely NPC with involvement of the last four cranial nerves. The father of the propositus was a proven case of NPC; he was treated at Queen Mary Hospital by the late Professor K. K. Digby, who advised him to go to Shanghai for radiotherapy as there was no such facility in Hong Kong at that time. He died at the age of 38, leaving a wife with seven children and a concubine with three children. There has been no contact between the wife and the concubine, who live separately, since his death. However, it is known from hearsay that all 3 children by the concubine are now over 40 and none suffer from NPC, whereas six of the seven children by the wife had developed the disease verified by biopsy. The wife is still alive a t 78 and well. 0-0

1

n

Ip

1. 2. 1 2 8 dx50

D D D dead

4. 5. 6O 7. eo go ion 4x28 dx42 dx35 dx34 %W" W W " "AW" D D A3R AW D AW a l i v e & w e l l A3R a l i v e without recurrence

3.

t age at death 7

no d a t a

''

dx age at diagnosis

hearsay

0

NPC

I'not v e r i f i e d

FIQ. 4. Pedigree study of a family with aggregation of NPC cases extending over three successive generations. From Ho (1972), by permission from the editor.

74

J . H. C. H O

Pedigree studies of Chinese patients in Hong Kong are handicapped by the fact that many of them have lost contact with some or even all of their kinfolk as a result of wars, rebellions, and revolutions which have ravaged China over the last several decades. Some patients do not know the causes of death of relatives because of inadequate diagnostic service. Others were adopted when very young and know nothing about their relatives. Under the assumption that these handicaps probably apply equally to family studies of NPC patients and of patients suffering from other cancers (OC) in a large series, a study has been carried out a t the Medical and Health Department Institute of Radiology, Hong Kong, in 1969 to determine whether close blood-linked relatives of NPC patients have a higher risk of developing NPC than those of patients suffering from other cancers. The results of this study are given in Tables VI and VII. The source of data in the study consists mainly of medical records; these are, as a rule, inadequate for genetic studies, as clinicians are usually more concerned with medical diagnosis and treatment than with obtaining a detailed family history of diseases. T o minimize this inadequacy the medical staff has been requested to inquire for family histories of NPC in both groups of patients. Positive questions were asked in order to counterbalance the memory bias of patients who tend to remember relatives suffering from a disease similar to their own better than those suffering from other diseases. In doubtful cases, the patients were recalled for further questioning by the author. A greater risk of getting NPC is found in relatives of NPC patients than in relatives of patients suffering from other cancers. It is interesting to note that 3 TABLE VI FREQUENCY OF FAMILY HISTORYOF NPC IN PATIENTS WITH NPC AND IN THOSE WITH OTHER CANCERS (OC) DIAGNOSED AT THE MEDICALAND HEALTHDEPARTMENT INSTITUTE OF RADIOLOGY, H O N Q HONG,1969 Cancer

NPC

oc

Families with history

Families with no history

12” 2

385 687

-

14 X’ = 14.77785 ( P < 0.001) t

-

Total

397‘ 689”

-

1072 1086 = 3.864234 (P= 0.00011)

From Ho (1972),by permission from the editor. Of the 12 families, 3 are “boat” people. b Seventy-two were excluded because a family history wm unobtainable, unreliable, or not obtained. In this group, 515 c a m were excluded, 230 for the above reasons and 285 because the cancers are sex-determined, e.g., gynecological, penile, etc.

75

NAsOPHARY NGEAL CARCINOMA (NPC)

TABLE VII FAMILIAL AQOREQATIONB OF NP CARCINOMA Families 1

2

Vertid

PATIENTS SEEN IN 1969

Horizontal

+

Daughter (II/2M 4F) and father Son (V/2M 3F) and mother.

Remarks

1 Female paternal 1st cousin

+

3 4

IN

2 Siters (I and II/6M

Son (IV/4M

+ 5F) and

+ 3F)

-

“Boat” people

mother 5

6

+

Son (V/4M

+ 3F) and

father

+

7 8

9 10

11

2 Paternal 1st cousins: Male (III/3M 2F) and M OM)

+

Daughter (IV/lM 5F) and fathero Son (only child) and father

-

Mother (II/lM daughter

+ 3F) and

12 Total = 7

Male (V/3M 2F) and brother (11) and brother (11

Male (only child) and his half-brother by same father; latter’s halfbrother by same mother was well

“Boat” people

-

+

Brother (?/4M 1F) and 1 brother Total = 7

“Boat” people

From Ho (1972), by permission from the editor. 0 D iagnosis was based on typical history only. (II/2M 4F) means propositus is the second child (11) of a family of two sons and four daughters.

+

of the 12 NPC families with aggregations of cases are “boat” people, a very small group of people who have a tendency to marry within their group and have been living in boats, junks, and sampans for centuries until recently when some of them have settled ashore and intermarried with land dwellers. The marine population of Hong Kong, according to the 1966 By-Census (Barnett, 1966), constitutes only 2.76% of the total population and consists largely of “boat” people. Table VII shows the directions of aggregation in the 12 families. If we accept the two unverified cases with only hearsay typical clinical histories (mother of propositus No. 2, and father of propositus No. 8) as positive cases, then the incidence of aggregations in this series is as

76

J. H. C. H O

great in the vertical as in the horizontal direction. From sheer numbers at risk, one would expect the aggregation in the horizontal direction to be greater than in the vertical. It is also more likely for a patient to know of or remember diseases suffered by relatives belonging to the same generation than by those in earlier generations. Diagnostic facilities were also poorer in the early days. Duration at risk is on the side of the older generations but not to a great extent, as the risk of getting NPC declines after the fifth decade in Chinese except in recent years, when a second but lower peak has become increasingly apparent in the seventh decade. It would appear in the present and past studies that if there were vertical transmissions in NPC they are not sex-linked. The random nature of the aggregations is indicative of a multifactorial etiology, and if genes are involved they are likely to be polygenic. 6. Nasopharyngeal Carcinoma in Twins No report of nasopharyngeal carcinoma occurring in twins has been found in the literature. Of some 6000 cases of nasopharyngeal carcinoma diagnosed at the Queen Mary and Queen Elizabeth Hospitals, where the Medical and Health Department Institute of Radiology is based, during a 20-year period 1951-1970 there was only one verified instance of NPC occurring in a pair of twins, probably dizygotic, of Sze Yap origin. One member, who died of NPC, was a patient of the Institute. The other, who migrated to Canada over 10 years ago, received radiotherapy in Canada for NPC 8 years after the death of his twin brother a t the age of 41, 1.5 years after the clinical onset of his disease. There are an elder and a younger brother and three younger sisters. None of them or their parents are known to have suffered from the same disease. The eldest brother is alive and well. Nothing more is known about the others. A twin birth occurs in about every 100 deliveries among Chinese in Hong Kong. At Tsan Yuk Maternity Hospital there were 673 twin deliveries during the 10-year period 1959-1968. Of these the nature of the twinning is recorded in the case of only 631. Three hundred and fifty (55.6%) of them are biovular and 281 (44.4%) are uniovular (Chun and Lee, 1970).

7 . ABO Blood Group Distribution If a genetic etiology is suspected, it is a call for more data, not for immediate speculations. So far only the ABO group distribution in NPC cases and normal controls have been investigated (Ho, 196713; Clifford, 1970). Clifford, comparing the ABO blood group distributions in 233 Kenyan patients with nasopharyngeal carcinoma and in controls, found a significance level of 3% in the comparison A/O and considers this to

77

NASOPHARYNGEAL CARCINOMA (NPC)

TABLE VIII ABO BLOODGROUPDISTRIBUTION IN PATIENTS WITH NASOPHAF~YNGEAL CARCINOMA AND CONTROL^ Population (all Chinese)

0

A

B

AB

NPC patients: males NPC patients: females Total Percentage Controls (Grimmo and Lee, 1961) Controls (Tong el al., 1963) Total Percentage

291 99 390 39% 258 5,747 6,005 41.6%

197 69 266 26.6% 181 3,650 3,831 26.5%

205 69 274 27.4% 180 3,515 3,695 25.6%

57 13 70 7% 51 856 907 6.3%

Total 750 250

1,OOO 100% 670 13,768 14,438

100%

From Ho (1972), by permission from the editor. For A/O comparison: t = 0.82093 ( P = 0.41); for B/O comparison: t = 1.62801

(P = 0.104).

be highly suggestive that, in Kenya, group-A persons are “protected” or a t less risk of nasopharyngeal carcinoma and that persons with other blood groups are consequently relatively a t greater risk. He feels that there seems to be an inherited susceptibility to, or protection against, nasopharyngeal carcinoma in some Kenyan Africans. I n Hong Kong a study of the ABO blood group distribution in loo0 consecutive Chinese patients with nasopharyngeal carcinoma failed to reveal any significant difference in the distribution in this group when compared with normal controls in the A/O or BJO comparison. The results are given in Table VIII. Shanmugaratnam (1970) also failed to find any significant difference between NPC cases and controls in Singapore. The distributions of ABO blood groups in Chinese in different parts of China, Singapore, Sumatra, New York City, and Hong Kong are given in Table IX for comparison. There is no great difference in the distributions between those reported by Grimmo and Lee (1961) and Tong et al. (1963) for Chinese in Hong Kong and those reported by Dormanns (1929) and Allen and Scott (1947) for Chinese in Canton and in Singapore, respectively. A very high incidence of the disease is found in all three places. I n the Yangtse River region, Hunan, Hupeh, and Kiangsu, where the relative frequency is lower (Hu and Yang, 1959), there is a definite excess of A group over B, but then in Peking, where the relative frequency is lowest, it is just the reverse. It would appear, therefore, that if certain genes are associated with a greater susceptibility to nasopharyngeal carcinoma in Chinese they are unlikely to be associated with the ones which determine the possession of blood

78

J . H. C. H O

TABLE IX ABO BLOODGROUPDISTRIBUTIONB AMONG CHINEBE OUTSIDEOF HONGKONQ" AND AMONG CHINESEIN HONGKONG Percentage Place China Yangtge River Hunan Hupeh Kiangsu Kwangtung Canton Canton Peking Peking Hong Kong Singapore Sumatra (East coast) New York City

Authors

Size

0

A

B

AB

29.5 1096 42.9 Yang (1925)" 93 43.01 31.18 Yang (1928)" 197 42.13 32.49 Yang (1928)" Yang (1928)" 228 52.63 21.05 196 40.82 32.65 Yang (1928)" 992 45.87 22.78 Dormanns (1929) 101 45.54 29.70 Alley and Boyd (1943)" 1000 30.70 25.10 Liu and Wang (1920)" 427 65.34 4.68 Hung and Steffan (1928)" 670 38.51 27.02 Grimmo and Lee (1961) Tong el al. (1963) 13788 41.74 26.51 Allen and Scott (1947) 624 43.11 24.04 592 40.20 25.00 Bias and Verhoef (1924)O

19.7 7.9 19.35 6.45 17.26 8.12 15.79 10.53 18.37 8.16 25.20 6.15 18.81 5.94 34.20 10.00 19.67 10.30 26.86 7.61 25.53 6.22 27.72 5.13 27.33 7.26

Levine and Wong (1943)"

25.33

150 30.00 34.00

10.67

From Ho (1972), by permission from the editor. Cited in Mourant and Domaniewska-Sobczak, 1958.

0

group A, B, or 0 substance. A study of the HLA pattern in Southern Chinese and non-Chinese living in Singapore will soon be conducted. 8. Physical Anthropology

There have been regrettably few attempts to survey the physical characteristics of patients with nasopharyngeal carcinoma for comparison with unaffected subjects matched for ethnic origin, sex, and age. Walsh (1967) concluded from his study that to date no single physical characteristic has been found that is common to the population of those countries with a high incidence of the disease and absent from others. He emphasized, however, that this does not mean that the possibility has been exhausted of finding a unique multifactorial set of characteristics in those populations with high risk, and that many more surveys must be undertaken and submitted to computer analysis.

C. ENVIRONMENTAL FACTORS 1. External Factors a. Inhalants. Dobson (1924) postulated that the high incidence of the disease in Chinese was related to the poorly ventilated houses in

NASOPHARYNGEAL CARCINOMA (NPC)

79

which they lived and inhaled much of the domestic smoke from burning grass, wood, tobacco, candles, incense (joss sticks), kerosene, and peanut oil lamps. Then Clifford and Beecher (1964) proposed that the inhalation of smoke from burning wood from exotic trees (eucalyptus and wattle) and indigenous acacias in ill-ventilated huts for several hours a day over a period of years may have some bearing on the distribution and incidence of the disease in Kenya, especially when significant quantities of carcinogenic substances, such as benzopyrene and benzanthracene, have been found in the soot taken from the roof of the huts of 46 patients with nasopharyngeal cancer on analysis by Hoffman and Wynder at the Sloane-Kettering Institute, New York. On the other hand, Booth et al. (1968) found similar living conditions among the one million and more people living in the Highlands of Australian New Guinea, but nasopharyngeal carcinoma is a rarity among them. It might be argued that the smoke in the two cases contains different substances, but then the crude annual incidence rates of nasopharyngeal carcinoma for most Kenyan tribes are below 0.5 per 1OO,OOO, and even among the Nandi tribe, which has the highest rate, the rate was only 0.94 (Clifford, 1967), which is only slightly higher than for the Swedes, who live in well-ventilated houses. Furthermore, Ho (1967a) found the incidence rates for the marine population in general and the “boat” people in particular, who live and spend most of their lives in sampans and junks and cook their food in open air, to be significantly higher than that for the land dwellers in Hong Kong. It would appear, therefore, unlikely that domestic smoke plays a significant role in the genesis of nasopharyngeal carcinoma, and certainly it cannot account for the high incidence in Hong Kong Chinese. Opium has also been suspected since it was first introduced into China in the early part of the nineteenth century through Canton, the capital of Kwangtung province. Digby (1951) observed that many Chinese who developed nasopharyngeal cancer were not opium smokers, and Polunin (1967) quotes a personal communication to him by Leong (1964) who estimates that he has seen about 5000 Chinese opiumsmoking men in Singapore, following them up for a period of a year each. Their average age was 48 years, and they had smoked opium for about 20 years, but he did not find a single case of nasopharyngeal cancer among them. Shanmugaratnam and Higginson (1967) found 87% of 63 Chinese male patients with nasopharyngeal carcinoma never smoked opium as against 84% of matched controls. In a retrospective survey of the records of 685 patients with nasopharyngeal carcinoma diagnosed during the 2-year period 1962 and 1963 a t the Medical and Health Department Institute of Radiology, Hong Kong, only four patients admitted to having smoked opium. There may have been some underrecording, but it is unlikely to be more than slight. I n a later study

80

J. H. C. H O

in 1969 there were 6 cases of opium addiction and three of heroin among 471 cases of nasopharyngeal carcinoma. None of the rest indicated that they had smoked opium at any time. In contrast there were four cases of opium addiction and 2 of heroin among 173 cases of bronchial carcinoma, and 11 cases of opium addiction among 47 cases of laryngeal carcinoma. There is, therefore, no evidence to suggest that opium plays a part in the causation of the high incidence of the disease in Chinese. Oriental incense or joss sticks are burnt not only in Chinese Buddhist and Taoist temples but also on a small scale in many Chinese homes in Hong Kong, China, Southeast Asia, and the United States. Those used in Hong Kong are made from sawdust from sandal-wood imported from Hainan Island and other parts of Kwangtung, Indonesia, India, and Australia in factories in Macao and Hong Kong. During the IO-year period 19551964, and average of 71.4 tons of joss sticks were exported or reexported from Hong Kong to the United States per year according to the Hong Kong Department of Commerce and Industry. Sturton e t al. (1966) found a difference (significant a t the 5% level) between incense burning and nonincense burning for male cases under 50 years of age and for male cases as a whole, but not for those over the age of 50, when a group of 29 male NPC cases was compared with a group of 38 male cases of other cancers. There was, however, no significant difference in the female patients of the two groups. Perhaps, the latter finding is more important than the former, because it is the females, not the males, who attend to the burning of joss sticks in Chinese homes. Furthermore, the incense smoke hypothesis is not in keeping with the finding by Ho (1967a,b) that NPC is rare among Buddhist monks, nuns, and Taoist temple attendants, who spend much of their time in incense-laden atmosphere. In fact, among over 3500 cases of nasopharyngeal carcinoma diagnosed a t the Medical and Health Department Institute of Radiology during the ll-year period 195&1966, there were only one Buddhist nun and one Taoist temple keeper. According to Sturton et a,?. (1966) there were 2000 temples and shrines in the city of Hangchow in central China and about 10,000 monks and a smaller number of Taoist monks or priests in a population of about 800,000 in that city before the institution of the present Chinese Government. Sturton (1965), in his work as a doctor and radiologist from 1921 to 1952 in Hangchow, did not observe any higher incidence of nasopharyngeal cancer than one would find in a large general hospital in England. The apparently high incidence of nasopharyngeal carcinoma in Macaonese who are traditionally Catholics and do not burn joss sticks in their homes or go to Buddhist or Taoist temples for worship, the relatively high incidence in Malays in Southeast Asia, who are traditionally Muslims and do not burn joss sticks, and the

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high incidence in the “boat” people of Hong Kong, who are less exposed to the smoke from burning joss sticks by virtue of their living in open boats than the land dwellers, are other contradicting evidences. It is, liowever, not possible to draw any conclusion at this stage that, because nasopharyngeal carcinoma appears to be rare in Buddhist monks, nuns, and Taoist temple attendants, joss sticks may have a protective action against the risk of getting the disease. These people belong to a small occupational group and the actual incidence of nasopharyngeal carcinoma in them has yet to be determined. Of the other inhalants, mention must be made of antimosquito coils which have been in common use in Chinese homes in China for many years and in Hong Kong until recent years, when urban areas have been rid of mosquitoes. The coils are still being used in some rural homes. Shanmugaratnam and Higginson (1967) did not find any significant difference between a group of 100 patients with nasopharyngeal carcinoma and a group of matched controls of the same number in their use of antimosquito coils in Singapore. Neither did they find any significant difference between the two groups in their use of cigarettes, pipe tobacco, snuff, and Chinese medicinal balms, oils, drops, and powders for inhalation or intranasal applications. The use of tobacco among Chinese is not as prevalent as among people of western nations, and the introduction of its use as a social habit in China occurred only within this century. Consequently, it could not have been responsible for the high frequency of the disease in China which most probably have existed for a longer period. b. Ingestants. We must not look for carcinogenic inhalants in the environment only of people a t high risk. It has been shown by Druckrey et al. (1964a) that compounds of the nitrosamine group can act systematically and are organ specific. They have further shown that a single dose of such a carcinogen can initiate a train of events which will culminate in cancer development after a long latent period without a further dose (Druckrey et al., 1964b). Bonser (1967) suggests that nitrosamine is not itself carcinogenic, but has to be converted enzymatically to an active carcinogenic metabolite. The enzymes capable of effecting such a conversion are different for different nitrosamines, so that the location of specific enzymes determines the site of the cancer. Nitrosamines are formed when nitrates are used as food additives or preservatives. Fish preserved in salt containing nitrate has been for many years a common and favorite item of food among most Chinese, rich or poor, in or outside China in Southeast Asia or the United States. This is also true for Macaonese in Hong Kong and Macao and to a less extent the Malays in Malaysia. Shanmugaratnam and Higginson (1967) noted

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no significant difference in food habits between a group of 100 male patients with nasopharyngeal cancer and a group of matched normal controls. The frequency of use of rice, and a variety of other foodstuffs, including salted and dried food, was approximately the same in the two groups. They pointed out, however, that their negative findings are not conclusive. Further epidemiological studies aa well as investigation into their carcinogenic properties are called for. It is more important to determine whether Chinese who never eat salted fish or food have the same risk of getting the disease as those who do than to compare the frequencies of eating such food between nasopharyngeal cases and matched controls. c. Nasopharyngeal Carcinoma Occurring in Both Marital Partners. During the 14-year period 1956-1969 only two verified instances of the disease occurring in both the husband and the wife of a family are found among 5070 cases diagnosed at the Institute of Radiology, Hong Kong. The husband in the first instance developed symptoms of the disease a t the age 39, just over 6 years after marriage, and the wife a t the age of 36 about 10% years after marriage. Both of them are Catholic, have no family history of nasopharyngeal cancer, and lived together in Hong Kong after their marriage. The husband in the second instance developed symptoms a t the age of 52, and the wife a t the age of 40. Neither of them had a family history of the disease. The exact time of their marriage is not known, but the number of years between marriage and symptomatic onset cannot have been less than 13 years in the case of the wife and 18 years in the case of the husband, judging from the age of their only surviving daughter. The length of stay in Hong Kong before onset was 14 years in the case of the wife and 20 years in the case of the husband. Both have since died, and their daughter is untraced. This incidence must be considered as minimal, because after the death of a spouse the surviving one-might return to China or migrate elsewhere or would not return to the Institute which had failed to cure his or her spouse of the disease to seek treatment. There could not be many such cases because there are few places available for treatment, and the Institute is the only place in Hong Kong where the poor can get free treatment as well as social welfare benefits. Among the 5070 cases, about 200 are patients who came from China or Southeast Asia specifically for treatment, after which they returned to their homes. Of the 121 who came from Southeast Asia during the period 1964-1969, many are, however, still in contact with the Institute. Nasopharyngeal carcinoma is a prevalent disease. Even allowing for some probable underrecording, one cannot but be impressed by the infrequency of such occurrences. It would appear, therefore, that those environmental factors,

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which affect husband and wife alike and are found in most homes, are of little etiological importance, at least during adult life. d. Occupation and Socioeconomic Level. In Singapore, Shanmugaratnam and Higginson (1967) found a wide range of occupations to be represented in both nasopharyngeal cancer and control groups in a retrospective survey, and Polunin (1967) found a lack of a clear-cut association between ways of living and nasopharyngeal cancer. I n Hong Kong, the carcinoma does not appear to occur more frequently in any particular occupation, as in the case of Singapore. In the highly competitive Hong Kong society, where much free enterprise exists, there is a definite possibility that someone who came originally from a poor family could become rich later in life, and vice versa. It is more important to study the early part of the life of a subject in a survey, than the later part. There are in Hong Kong, however, sufficient cases of nasopharyngeal carcinoma who are known to belong to the upper, middle, and lower socioeconomic levels throughout their preceding lives to justify the impression that a higher or lower risk is not associated with any socioeconomic level. Andrews and Michaels (1968a,b) reported three cases of nasopharyngeal carcinoma in Canadian bush pilots, a very small occupational group. One is of French extraction, one is Finnish, and one English, and they had been on their jobs for 10, 28, and 25 years, respectively. Such flying in unpressurieed aircraft subjects the pilots to very frequent and rapid air pressure changes which are likely to produce “otitic barotrauma,” which is thought to be a precursor of nasopharyngeal carcinoma. All three were cigarette smokers. It was thought that cigarette smoke and other potentially carcinogenic substances may be specifically directed to the nasopharyngeal area as a result of pressure changes. It was also pointed out that there was a greater speed of pressure changes experienced by these pilots than that experienced by divers. There was, however, no subsequent report of another case in these pilots or in other groups of airmen working under similar conditions of barometric change, e.g., crop-duster, air taxi, and helicopter pilots. There must be many such airmen all over the world, although the climatic conditions under which they work may not be similar to those for the Canadian bush pilots. For example, the Canadian air in winter is very cold and dry (Lancet Annotations, 1968). Although coincidence cannot be ruled out, the finding of three cases in so small an occupational group certainly deserves further investigation. 2. Internal Factors a. Malnutrition and Vitamin Deficiency. Contrary to the findings by Clifford (1965) in Kenyans, a good nutritional state without clinical

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evidence of vitamin A or B group deficiency is the rule rather than the exception in Hong Kong patients except in the terminal stage of the disease. Many of them also gave no history of malnutrition or chronic ill health during childhood. b. Hormonal Factors. Kenyans with nasopharyngeal carcinoma had significantly lower plasma levels of dehydroepiandrosterone sulfate (DS) as compared with the control group, but plasma levels of androsterone sulfate (AS) in both groups were similar (Wang et al., 1969; Clifford, 1970). In Singapore, similar low levels of DS and AS were noted in Chinese patients with nasopharyngeal carcinoma, but studies on the plasma levels of DS, AS, cortisol, and transcortin in apparently normal male subjects from racial groups with high (Chinese), intermediate (Malays), and low (Indians) incidence of the disease revealed no differences (Wang et al., 1969). Plasma estrogens were, however, not measured in the studies carried out in Kenya or Singapore. c. Chronic Infection of the Upper Respiratory Passage and Vasomotor Rhinitis. Although nasal sinus infection is a common complication of established nasopharyngeal carcinoma, only a minority of the patients in Hong Kong gave a past history of chronic upper respiratory infection or vasomotor rhinitis. Vasomotor rhinitis may be common in Chinese, but it is certainly very rare as an antecedent disease in nasopharyngeal carcinoma as seen in Hong Kong. d. Virus. If a virus is involved in the causation of nasopharyngeal carcinoma it has to be one which is ubiquitous to explain the regular high incidence of the disease in Chinese living in widely scattered parts of the world. Furthermore, to explain why Indians, who live in similar environments in Singapore as Chinese and Malays, have a much lower risk of the disease than the other two racial groups, the virus has to be one to which Chinese and, to a lesser extent, Malays are more susceptible than Indians, or, alternatively, one which functions together with other cocarcinogenic or carcinogenic factors, which are peculiar to or selectively affect Chinese and Malays. So far, only an immunological relationship between Epstein-Barr virus (EBV) , a member of the herpes group, and nasopharyngeal carcinoma has been found. Old et al. (1966) found in the sera of nasopharyngeal carcinoma (NPC) patients precipitating antibodies similar to those present in Burkitt’s lymphoma (BL) sera. De Schryver et a2. (1969) found in the sera from African and Chinese postnasal (same as nasopharyngeal) carcinoma an unusual frequency of high titer of membrane reactive antibodies (Anti-MA) when EBV-carrying permanent lymphoblastoid cell lines derived from Burkitt’s lymphoma and infectious mononucleosis (IM) were used as

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target cells. Much lower frequencies were found in control sera from African healthy individuals and patients with neoplastic diseases other than NPC and nonneoplastic diseases, and from Indian donors with buccal, oro- and hypopharyngeal carcinomas. W. Henle et al. (1970) found that 100% of the sera from East African and Chinese patients with NPC had antibodies against E B viral capsid antigens (VCA) and that 84% of them had high titer ( 2 1:160) with a geometric mean titer (GMT) of 1:340, whereas only 10% of the controls from the general population, unmatched for sex and age, and 14% of the patients with head and neck tumors other than N P C showed high titer, with a G M T of 1 : l O and 1:41, respectively. They further found that when the NPC patients from Hong Kong were grouped according to the stage of the disease, presumed to correlate with the tumor burden, the incidence of high titers increased successively from 45% in stage I to ultimately 100% in stage V. The GMT also rose correspondingly from 1:103 in stage I to 1:788 in the stage V. G. Henle and Henle (1972) detected antibodies to early EBV-induced antigens (EA) in infectious mononucleosis, Burkitt’s lymphoma, and nasopharyngeal carcinoma. Whereas in infectious mononucleosis the antibodies to EA disappear usually within 6 months, in Burkitt’s lymphoma and nasopharyngeal carcinoma they usually persist and are frequently found at high titers. EpsteinBarr virus is ubiquitous on a worldwide basis, but no tumor has been induced to date by EBV-carrying materials in laboratory primates. There are, however, some observed effects of EBV infection in vitro which are consistent with-but by no means proof of-an oncogenic potential for the virus. There is a possible analogy with the herpes virus producing malignant proliferation of lymphoid cells in Marek’s disease of fowls, but the virus has not yet been recovered from disrupted cells, although it can spread from lymphoma cells to kidney cells when they are ruptured. I n nasopharyngeal carcinoma, herpes-type viral particles, similar to the EBV particles observed in cultures from B L and IM, have been observed in some of the degenerating lymphoblastoid cells derived from cultures of biopsy specimens from Hong Kong (de-The et al., 1969, 1970). A new human virus, unassignable to any known morphological group, has been observed in cultures of a nasopharyngeal carcinoma from Kenya (Epstein, 1972). This virus was found only in suspension cultures of lymphoblastoid cells released from the original monolayer after 105 days in vitro. Since nasopharyngeal carcinoma is of epithelial, not of lymphoreticular origin, EBV or a similar virus may play no role in the etiology of the disease. If this is so, an explanation has to be found for its close immunological association with EBV, which

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is not found in carcinomas arising elsewhere in the head and neck including other parts of the Waldeyer’s ring and tumors other than carcinoma arising in the nasopharynx. If, on the other hand, nasopharyngeal carcinoma provides only a favorable medium for the multiplication of this virus, we should find among the NPC patients a proportion of HTV-negative persons, reflecting the HTV-negative portion of the general population. Furthermore, if the virus were merely a passenger, then why other tumors, such as leukemias, lymphomas, Hodgkin’s disease, reticulum cell sarcoma, and multiple myelomas should not offer similarly favorable habitats. Unfortunately, the increase in anti-EBV titers with the advancement of the disease does not differentiate between a passenger role and a causal relationship. T o determine whether EBV or a similar herpes-type virus plays a role in the genesis of NPC, it is essential to study first the natural history of the virus in man, its prevalence in populations of high and low risks for NPC in different as well as the same parts of the world, its mode of spread, etc. Such a study has already commenced in Southeast Asia, Japan, and France. Then a prospective seroepidemiological study may be called for in an attempt to establish the type of association, causal or noncausal, direct or indirect, which exists between the suspected virus and NPC. As separate exercises, it is of great importance to obtain long-term cultures of the epithelioid cells derived from NPC for investigating the possible presence of herpes-type viral particles or their indicators in such cells, and also look for them in peripheral lymphocytes. 3. Pattern of Age Distribution

Figure 5 gives the age distributions of nasopharyngeal carcinoma in Chinese men in Hong Kong and Singapore and the male population of Sweden for comparison. There is much similarity in the Hong Kong and Singapore patterns, both of which show a rapid, almost uninterrupted and fairly regular increase in incidence after 20-24 years of age, two decades earlier than in Sweden and also earlier than most other epithelial cancers. The incidence then declines after 50-54 in both places, again two decades earlier than that in Sweden. This would suggest that the disease in Hong Kong and Singapore Chinese was not due to continued exposure to an external carcinogen throughout life, as is postulated for most of the common epithelial cancers, or, alternatively, that the susceptibility to the carcinogen is influenced by an internal factor, possibly hormonal in nature, which is responsible for the rapid increase in incidence soon after adolescence and the decrease after 50-54 years of age. Doll (1970) in a personal communication to the author commented that the Hong Kong nasopharyngeal carcinoma pattern is very similar to that

:I

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80 r

20

/:-

FIG.5. Male age-specific incidence rates of nasopharynge41 carcinoma for Sweden and for Chinese in Hong Kong and Singapore. Only transitional cell, squamous, and undifferentiated carcinomas are included for Sweden. K e y : -per 100,OOO (Hong Kong), 2019 cases (1965-9) ; --- per 2 million (Sweden), 202 cases (195% 65); . . . . per 100,ooO (Singapore), 839 cases (1950-81). From H o (19721, by permission from the editor.

of cancer of the uterine body and suggested that the possibility of hormonal factors contributing to both should be borne in mind. Figure 6 shows the age incidence curves for nasopharyngeal carcinoma in the two sexes in Hong Kong, where 98.7% of the population are of Chinese descent (Barnett, 1966), and Fig. 7 shows the curves in Sweden. In both places the curves for the two sexes differ only in height but not greatly in shape; the Swedish curves, however, differ significantly both in height and shape from the corresponding ones for Hong Kong. I n Sweden the curves reveal a pattern similar to that for bronchial carcinoma in cigarette smokers and most of the common epithelial cancers.

”i 20

10

FIQ.6. Age-specific incidence rates of nasopharyngeal carcinoma by sex in Hong Kong, 1960-1964 and 1% ! 51-969. Patients from elsewhere are excluded from the calculation. K e y : 19604 , male, 1492 cases; 1965-9 ---, male, 2019 cases; I-.... , female, 672 cases; 1966-9 -. .-, female, 887 cases.

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40

t

10

F I ~ 7. . Age-specific incidence rates of nasopharyngeal carcinoma (transitional cell, squamous, and undifferentiated) in Sweden by sex, 1959-1965. K e y : - male, 202 caaes; female, 129 caaes. -.a-

IV. Conclusion

The predominant cancer arising in the nasopharynx of people of different races is a carcinoma of the squamous type showing, in the majority of cases, poor differentiation or absence of differentiation. It is this tumor which shows a predilection for people of Chinese descent, especially those from the southern provinces, and an immunological relationship with EBV ar a similar virus. Chinese migrants in different parts of the world appear to have as much risk of the disease as Chinese inside China including Hong Kong, but whether the risk for people of Chinese descent born and raised in their countries of adoption is altered is not clear. Results of previous studies are far from conclusive. People of part-Chinese ancestry tend to share partly the high risk of their Chinese ancestors. Close blood-linked relatives of nasopharyngeal carcinoma patients have been found to have a higher risk of the disease than those of patients with other cancers, and the aggregation of nasopharyngeal carcinoma appears to be a t least as frequent in the vertical direction as in the horizontal. These are all highly suggestive, but not necessarily, the result of gene action. They may be the result of certain social customs, dietary habits, family recipes for treating minor ailments, etc., passed down to subsequent generations and shared by members of the same generation. We should, therefore, look for environmental factors likely to affect all ethnic groups of high risk. That such factors are also common in people of low risk does not necessarily exclude the possibility of a causative role played by these factors, because they may act together with other factors, especially genetic, which are present only in people of high risk, but their rarity or absence would suggest that they

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may be of etiological importance. Of the environmental factors, fish and probably eggs preserved in salt, which may be a source of carcinogenic or cocarcinogenic nitrosamines acting specifically on the nasopharynx of people with a genetically determined susceptibility, are traditionally common and favorite items in the diet of Chinese, especially those from Kwangtung, whether in China, Hong Kong, Southeast Asia, Australia, or the United States. The Chinese salted fish is always steamed or fried. In Japan, a low incidence area, a different kind of salted fish is often eaten, but it is always baked over a hot iron grill instead. The Chinese salted fish and eggs and the herpes-type virus which has an immunological relationship with nasopharyngeal carcinoma well deserve a thorough investigation. Chinese incense or joss sticks by virtue of the fact that they are commonly used by people of high risk should also be investigated as a possible etiological factor, although evidence so far obtained is all negative. There are still many unknown factors and missing links. All we can say a t present is that the etiology of nasopharyngeal carcinoma is most likely to be multifactorial, and that if a genetic factor were involved it is certain to be polygenic, not sex-linked or related to the ABO blood-group, at least in Chinese. There is an old saying: “A pinch of salt is worth a pound of precept.” What is needed now are more data, not speculation. ACKNOWLEDGMENTS The author is grateful to Dr. the Hon. Gerald Choa, Director of the Medical and Health Services of Hong Kong, for his permission to publish this paper; to Dr. J. K. Craig for helpful information; to Dr. C. C. Lin for histological diagnosis; to the medical staff of the Medical and Health Department Institute of Radiology for their care in obtaining family histories of the cases; to Mr. C. M. Lam for statistical assistance; to Mr. R. Abessor for preparing Figs. 3-7; to Mrs. P. Liu for careful secretarial assistance; to Messrs. K. Fung and H. K . Tam for collection of data, and Messrs. K. W. Leung and A. Lam for photographic assistance. The author is also indebted to the Swedish Cancer Registry and Professor Nils Ringertz, Scientific Surveyor of the Registry, for generous cooperation in supplying Swedish data; to Professor J. Mitchell, F. R. S., and Mr. J. A. Fairfax Fossard of Cambridge University for supplying photographs and radiographs of skull No. 238 kept at Duckworth Laboratory, Cambridge, and to Mr. J. C. Trevor, Director of the Laboratory, for the facilities afforded; to Professor R. Doll of Oxford University for helpful advice; to Professor K. Shanmugaratnam for supplying Singapore data; and finally to Dr. L. Atkinson for helpful information regarding conditions in Australian New Guinea.

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Wells, C. (1963). J . Laryngol. 77, 201-265. Wells, C. (1864). Brit. Med. 3. 1, 1611-1612. Worth, R. M., and Valentine, R. (1967). In “Cancer of the Nasopharynx” (C. 9. Muir and K. Shanmugaratnam, eds.), UICC Monogr. Ser. No. 1, pp. 73-76. Munksgaard, Copenhagen. Wu, C. H. (1921). “The Encyclopaedia of Chinese Medical Terms,” Vol. 1, p. 756 (in Chinese). Commercial Press, Shanehai. Yeh, 9. (1967). In “Cancer of the Nasopharynx” (C. S. Muir and K. Shanmugaratnam, eds.), UICC Monogr. Ser. No. 1, pp. 147-152. Munksgaard, Copenhagen. Zippin, C., Tekawa, 1. S., Bragg, K. U., Watson, D. A., and Linden, G. (19a2). 3. Nat. Cancer h a t . 2Q, 485-990.

TRANSCRIPTIONAL REGULATION I N EUKARYOTIC CELLS A. J. MacGillivray, J. Paul, and G. Threlfall Beatron Inrlituto for Cancer Research, Glargow, Scotland

I. Introduction . . . . . . . . . . . . . . A. Molecular Biology and Cancer . . . . . . . . B. Changes in Transcription during Differentiation . . . . C. General Aspects of Transcription and Its Control in Prokaryotes . 11. Eukaryotic Chromosomes . . . . . . . . . . . A. General Structure of Chromosomes . . . . . . . B. DNA in Eukaryotes . . . . . . . . . , . C. Chromosomal Proteins . . . . . . . . . . D. Chromosomal RNA . . . . . . . . . . . E. Miscellaneous Components of Chromatin . . . . . . F. Role of Chromosomal Constituents in the Structure of Chromatin 111. Control of Transcription in Eukaryotic Cells . . . . . . A. Template Properties of Chromatin . . . . . . . . B. Chromosomal Proteins in the Control of Transcription . . . C. Eukaryotic RNA Polymerases . . . . . . . . . IV. Theories of Transcriptional Regulation in Eukaryotic Cells . . . A. Free DNA in the Genome . . . . . . . , , B. Derepression by Polyanions . . . . . . . . . C. Other Mechanisms . . . . . . . . . . . References . . . . . . . . . . . . . .

.

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.

. . .

. . ,

. .

. . . .

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93 93 95

97

101 101 104 106 118 119 120 124 124

129 141 146 146 147 149 150

1. Introduction

A. MOLECULAR BIOLOGY AND CANCER The rapid development of basic ideas and new techniques in molecular biology have only recently made it possible to pose very precise questions about the nature of the abnormalities which characterize cancer cells. Some of the most meaningful questions emerge from studies of developmental biology, particularly from attempts to understand the nature of the differentiated state. It is now well established that, although the differentiated cells in an animal may all have the same genetic information within them (Gurdon and Laskey, 1970), the differentiated state may nevertheless be highly stable and survive clonal cultivation in vitro (Coon, 1966; Cahn and Cahn, 1966; Konigsberg, 1963) or lengthy serial transplantations (Hadorn, 1965). Like normally differentiated cells, cancer cells very often preserve characteristic features of the differen93

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tiated cells from which they were derived, again even in conditions of prolonged culture in vitro (Yasamura et al., 1966). On the other hand, it is a characteristic of cancer cells that they diverge from normality. This is, of course, frequently seen as a loss of differentiated features, but it has recently become clear that the acquisition of functions which are not normally exhibited by cells of the type from which the cancer cells developed is not uncommon. For example, fetal antigens may appear in tumors from adult tissue (Gold and Freedman, 1965), and hormones may be produced by nonhormone-secreting cells which have become neoplastic (Lipsett, 1965). Accordingly, the old idea has reemerged that cancer might be a disorder of cell differentiation. Not only is this a concept which provides a useful framework for the scientific investigation of the disease, but there is the suggestion that some tumors can undergo spontaneous cure through differentiation (Nelson, 1962 ; Smithers, 1962). Current ideas about the mechanisms involved in differentiation have been very much influenced by studies on the regulation of protein synthesis both in prokaryotes and eukaryotes. It is now clearly recognized that in mammalian cells the same basic rules as in bacteria probably apply in that genetic information for protein synthesis is included in DNA; this is transcribed to messenger RNA, and this in turn is translated into protein by a machinery involving ribosomes, activating enzymes, transfer RNA and other factors. I n eukaryotic cells there may be additional mechanisms not identified in prokaryotes. The chromosomes are much more complex and are segregated from the cytoplasm in the cell nucleus. Many of the new species of RNA made in the nucleus never leave it but are rapidly broken down, and their function is not known. Whereas ribosomes become attached to messenger RNA while it is still being transcribed from DNA in bacteria, in eukaryotes the RNA has to leave the cell nucleus and enter the cytoplasm before it is translated into protein. It is possible therefore to identify many levels a t which controls of protein synthesis could act in the course of cell differentiation. Changes could occur in DNA itself: regulatory steps could occur a t the level of transcription; the breakdown of RNA in the nucleus and its transport to the cytoplasm could provide a controlling point; and finally there could be control a t the level of translation of messenger RNA to protein. I n fact, there is good evidence that all these mechanisms can operate in the establishment of the differentiated state. I n this review we wish to consider regulation a t one level only, that of transcription, because there is much evidence to suggest that some very important controls operate there which are particularly relevant to normal differentiation and possibly to the distorted differentiation which is seen in cancer.

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B. CHANGES IN TRANSCRIPTION DURING DIFFERENTIATION The evidence that transcriptional controls play an important role in differentiation comes mainly from studies using the technique of RNAJ DNA hybridization. McCarthy and Hoyer (1964) hybridized rapidly labeled RNA from a variety of mouse tissues to DNA in competition with samples of unlabeled RNA from the same tissues. These studies indicated differences in RNA populations from one tissue to another. Later Whiteley et al. (1966) performed similar experiments on the rapidly labeled RNA in developing sea urchin embryos. They obtained evidence for changes in the RNA populations of the cells a t different stages of development. Similar experiments were carried out by Glisin et a2. (1966). Denis (1966) carried out investigations of the same kind on rapidly labeled RNA from unfertilized eggs and developing embryos of Xenopus laevis, and showed the same behavior. Daniel and Flickinger (1971) obtained similar findings for developing frogs. These studies and many others seem to indicate rather strongly that different species of RNA are made in different cells, and, since there is good reason to believe that the cells have the same DNA contents, it is a reasonable conclusion that transcriptional controls play an important part in differentiation. There are also many studiev which indicate that alterations of the physiological state of organs results in changes in transcription. Again most of these studies have used RNA/DNA hybridization. Church and McCarthy (1967a) found that changes in RNA occur during embryonic liver development. They also found (Church and McCarthy, 1967b,c) that changes in RNA synthesis could be detected in the liver following partial hepatectomy. In their studies they obtained evidence that some of the RNA’s were restricted to the nucleus in normal liver but were permitted to appear in the cytoplasm in regenerating liver. Wyatt and Tata (1968) studied RNA synthesis during induced metamorphosis in Xenopus and showed changing patterns of RNA synthesis (which they interpreted as reflecting preferential synthesis of ribosomal RNA) in response to thyroxine. Drews and Brawerman (1967) also found that new species of RNA were synthesized in the liver after treatment with cortisol. Turkington (1970) has reported changes in RNA during differentiation of mammary cells. There is thus a large body of evidence from DNA/RNA hybridization experiments to suggest that transcriptional controls operate in eukaryotes. The main limitation to interpretation of these experiments resides in the fact that it is now known that only RNA transcribed from repetitious DNA was studied (see Section 113). Evidence for transcriptional control comes from another direction

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in experiments carried out by Borun et al. (1967). These authors studied the synthesis of a messenger RNA which has been provisionally identified as histone messenger. This makes its appearance in the cytoplasm shortly before histone synthesis commences in the cell cycle, and disappears shortly before histone synthesis ceases. Evidence for intermittent transcription of RNA also comes from studies by D. Martin et al. (1969) on the induction of tyrosine aminotransferase during the cell cycle. These lead to the conclusion that a repressor molecule is synthesized only during certain parts of the cell cycle. Both of these studies, and other similar studies, can be explained in other ways, but the most likely interpretation is that transcriptional control results in the synthesis of different RNA molecules a t different times during the cell cycle. Observations of this kind have led several groups to study the patterns of RNA synthesis in normal and tumor cells with a view to determining whether differences in RNA can be identified. Some of the earlier studies were directed to identifying the presence of viral messenger RNA in cells transformed by tumor viruses, and these led, for example, to the demonstration of polyoma messenger RNA in cells transformed with polyoma virus (T. L. Benjamin, 1966). Kidson and Kirby (1964) were the first to look a t the patterns of RNA synthesis in a transplanted spontaneous mouse hepatoma, as compared with normal mouse liver. They used the technique of countercurrent distribution and obtained highly reproducible patterns from normal liver but progressive alterations in the messenger RNA profile of hepatomas during progression of the tumor. They concluded that many regions of the hepatoma genome which were undergoing transcription were different from those transcribed in normal liver. Jackson and Sels (1968) expressed doubts about the validity of studies of this kind because of their failure to obtain reproducible results. S. Jacob and Busch (1967) compared the template activity of chromatin from hepatoma cells with that from normal liver and reported that it was greater. Subsequently several RNAJDNA hybridization studies have been done to compare the rapidly labeled RNA from cancer and normal cells. Drews et al. (1968) reported a deficiency of some RNA classes in hepatoma nuclei as compared with nuclei from normal rat liver. Chiarugi (1969) compared nuclear RNA from rat liver, Yoshida ascites hepatoma cells, and Morris 5132 hepatoma cells and found them all to differ. Church et al. (1969) compared RNA synthesized in mouse liver with that synthesized in the Taper hepatoma. Their studies suggest a more diverse selection of molecules in the hepatoma cells than in normal liver. Similar results were reported by Mendecki et al. (1969) for rat liver hepatomas. Neiman and Henry (1969) compared the RNA from

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normal lymphocytes with that from chronic lymphocytic leukemic lymphocytes and again showed differences between them. Turkington and Self (1970) in a similar way compared RNA from normal mammary tissue with breast cancer cells. They too concluded that new species of RNA were synthesized in cancer cells. All the above experiments were performed by RNA/DNA hybridization under conditions which would permit hybridization only of RNA complementary to repetitious DNA, and they are therefore subject to the same limitations of interpretation as the experiments mentioned earlier. On the other hand, Roche et al. (1969) used polyacrylamide gel separations to compare RNA from nuclei of normal human kidney and several renal neoplasms and reported several additional RNA species in the tumor nuclear RNA. A few other general observations have been made about differences between tumor and normal cells which could be relevant to transcription and may be mentioned here. For example Sluyser (1968) claims that benzopyrene reacts in a fairly specific way with histones, while J. A. Smith et al. (1970) claim that breast cancers can be distinguished from each other and from normal tissue by their acidic nucleoprotein content. Those with the highest content of acidic nucleoproteins included some of the fastest growing tumors. While many of these experimental observations are capable of alternative explanations, taken together they add up to a considerable body of evidence to the effect that changed transcriptional patterns are characteristic both of normal differentiation and of neoplastic change. The case for studying transcription in relation to cancer is, therefore, clear. Before entering into consideration of special features of mammalian cells, some general aspects of transcription, mainly in prokaryotes, will be outlined. C. GENERAL ASPECTSOF TRANSCRIPTION A N D ITS CONTROL IN PROKARYOTES The central role of DNA-dependent RNA polymerase in the synthesis of RNA in bacterial cells was recently reviewed extensively by Richardson (1969). This section will therefore summarize the means whereby the enzyme itself may exert control over RNA transcription, and discuss briefly how it may interact with other controlling elements in the regulation of gene expression. DNA-dependent RNA polymerase catalyzes the synthesis of polyribonucleotide chains from nucleotide precursors by the formation of 3‘,5’-phosphodiester bonds (Hurwitz et al., 1960; A. Stevens, 1960; Weiss, 1960). Apart from its substrate, the 5’-triphosphates of guanosine,

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adenosine, cytidine, and uridine, the enzyme requires a DNA (or synthetic polydeoxyribonucleotide) template. Other requirements include a divalent metal ion (usually MnZ+or MgZ+),and the reaction rate is optimal a t pH 7.8 in a medium of moderate ionic strength. The Escherichiu coli enzyme comprises four main subunits, each being a single polypeptide chain, with a combined molecular weight of about 5 x lo5 daltons in the monomeric form. The two largest subunits have molecular weights of 165,OOO and 155,000 (p’ and p, respectively) and the smallest, a, is about 40,000. Together with u (Burgess et ul., 1969), molecular weight about 90,000 daltons, they form the functional unit of the enzyme (“complete” enzyme), which can be represented as follows: p’p az u. Sigma factor is known to be involved in the selection of correct initiation sites a t which the enzyme binds to the DNA template, whereas p’ is thought to be the template binding site of the enzyme. p is almost certainly involved in initiation of RNA chains, since it binds very strongly with rifampicin (an inhibitor of initiation by bacterial polymerase), and it contains binding sites for u and a. No specific role has yet been allocated to the a subunit, though its presence is required for activity, at least in the “reconstituted” enzyme (Heil and Zillig, 1970). The transcription process involves four well-defined steps (see Chambon, 1968), viz., the binding of the enzyme t o the DNA template, the initiation of polymerization (which involves the binding of the first nucleotide with the enzyme-template complex to give an initiation complex), elongation of the RNA chain on the template and finally, termination, which is accompanied by liberation of the nascent RNA molecule and the enzyme, which can then presumably reinitiate RNA synthesis. A great deal of information has recently accumulated on the role of u factor in the initiation process. It appears that the “core” enzyme (i,e,, complete enzyme minus U ) becomes bound to specific initiation sites on the DNA under the influence of u, which has been shown to associate transiently with the core (Travers and Burgess, 1969). The core enzyme itself can also bind to DNA and catalyze RNA synthesis, though much less efficiently than complete enzyme and with little specificity. For example, if the core enzyme is used to transcribe in vitro from a T 4 DNA template, it transcribes a t a low rate, and the RNA is synthesized randomly from both strands of the DNA. In contrast, when complete enzyme is used with the same template, it transcribes only the “pre-early” phage genes and with high efficiency (Milanesi et al., 1969; Bautz et al., 1969). Similar results regarding the function of u were obtained by Goff and Minkley (1969) in a study of its effect on transcription of the T7 phage genome by E . coli polymerase.

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The importance of u in strand selection is evident from these experiments, since a characteristic feature of in vivo transcription is the predominantly asymmetric nature of the RNA product. Studies of the temperature dependence of the initiation reaction (Hinkle and Chamberlin, 1970; Zillig et al., 1970) suggest that u may be involved in local denaturation of the DNA double strand in the vicinity of initiation sites thus allowing polymerase molecules access to single-stranded regions. It is doubtful whether in each cell type there is a multiplicity of u factors each with specificity for a particular site on the genome, though the appearance of new u factors has been reported in bacteriophageinfected cells. I n particular, the synthesis of RNA from a group of phage genes (“delayed early”) in T4 infected E . coli cells can be strongly correlated with the disappearance of the host u factor and the appearance of a new specificity factor (Travers, 1969, 1970a). This is accompanied by sequential modification of the a and p’ subunits of the host RNA polymerase (Walter et al., 1968; Bautz e t al., 1969; Travers, 1970b), presumably to allow interaction with the new sigma factor. A change in polymerase subunit structure has also been observed in sporulating Bacillus subtilis cells. Early in the sporulation process the template specificity of the enzyme is markedly altered so that it will no longer transcribe phage DNA (Losick and Sonenshein, 1969). The polymerase of vegetative cells requires a u factor for activity on phage DNA, but this factor is ineffective with enzyme from sporulating cells, indicating a change in the core enzyme. It was found that one of the @ subunits of the core enzyme from sporulating cells was considerably smaller than either of the p subunits of the vegetative enzyme (Losick et al., 1970). It is predictable that a new u factor is required for the modified polymerase to be able to transcribe genes associated with the sporulation process, although this has not yet been reported. Finally, it has been shown recently that when T7 bacteriophage infects E . coli cells, among the first phage genes to be transcribed is one which specifies a completely new RNA polymerase, required for transcription of the “late” genes (Chamberlin et al., 1970). A phagespecified RNA polymerase has also been recently identified in T3infected E . coli cells (Dunn et al., 1971). Both of these enzymes seem to be much simpler than E . coli polymerase, comprising a single polypeptide chain of molecular weight about 110,OOO daltons. It is apparent then that there are a t least two ways of directly changing the specificity of transcription in phage-infected cells by modification of the core enzyme in one or more of its subunits so that it will interact only with a new specificity factor, and by the appearance of a completely new polymerase molecule. These are examples of positive control mediated by

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the enzyme itself and factors which influence its ability to bind and initiate transcription a t specific sites in the genome. The studies by F. Jacob and Monod (1961) of the synthesis of E. coli lactose-metabolizing enzymes provide a clear example of negative control of gene transcription. Jacob and Monod postulated that a repressor molecule, the product of a “regulatory” gene for the lactose operon, can inhibit the synthesis of messenger RNA on the structural genes by binding strongly to an operator region of the lac operon. The structural genes are transcribed (“switched-on”) only when the repressor molecule is prevented from interacting with the operator region for example by combining instead with an inducer molecule such as lactose. A protein molecule having the properties expected of such a repressor has been isolated from E. coli cells (Gilbert and Mueller-Hill, 1966). It binds strongly to a specific region of the DNA, and even more strongly with specific inducer molecules (Gilbert and Mueller-Hill, 1967). This kind of mechanism is known to operate in the control of other E . coli operons and also in the mechanism whereby, in E. coli cells infected by phage A, the protein specified by the C, gene of the phage switches off the expression of other phage genes, enabling the phage genome to remain dormant within the host cell (Ptashne, 1967a,b). Recently a further control over gene activation in the E. coli lac operon was detected. During catabolite repression of induction of the lactose enzymes by glucose, a precipitous drop in cyclic AMP content of the cells was observed (Makman and Sutherland, 1965). The repressive effect of glucose was reversed by addition of cyclic AMP (Perlman and Pastan, 1968; Ullman and Monod, 1968). A protein factor was purified from the cells which has a strong affinity for cyclic AMP. It is thought that a combination of cyclic AMP with this protein (known as the CAP protein) exerts a positive control over the production of lac mRNA, though its mode of action remains unknown (Zubay et al., 1970). It could presumably interact with RNA polymerase on the promoter site for the lac operon, in some way enhancing the synthesis of the lac messengers (Perlman e t al., 1970). There is also a role for inhibitory factors in the control of RNA biosynthesis, and an instance of this is the inhibition of RNA synthesis in stringent strains of E . coli starved of an amino acid. An unusual nucleotide, guanosine tetraphosphate (ppGpp) accumulates rapidly in the cells (Cashel, 1970), and there is some evidence that it may specifically inhibit the production of ribosomal RNA. Less is known at present regarding the termination of RNA synthesis and release of nascent RNA molecules, i.e., the process which ensures that RNA molecules of the correct size are produced and released from

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the transcription complex. It can be shown that RNA made on a X phage DNA template by E . coli polymerase has a broad range of sizes, from 20 to 40s. However, when a protein factor isolated from E . coli cells is added, there are two main peaks, 7 S and 12 S, which correspond to early gene products found in &infected E . coli (Roberts, 1969). The protein has been designated p factor and presumably acts in conjunction with core enzyme and u to ensure synthesis of molecules of the correct length specified by the genes being transcribed. Furthermore, in the presence of p factor, RNA is released from the RNA-enzyme-DNA complex (transcription complex) at the end of the reaction. Richardson (1970) has shown that in the presence of 0 . 2 M KC1 (and absence of p) termination of RNA chains also occurs with the release of RNA and enzyme from the transcription complex. The polymerase molecules can reattach to the DNA template and reinitiate the synthesis of more RNA molecules. However, the RNA molecules formed under these conditions are on average 1.3 times larger than those made under lower salt (0.05M KCl) conditions. It would appear that the mechanism of chain termination by high salt conditions is probably not the same as that mediated by p factor, though it is possible that reinitiation in vitro in the presence of 0 . 2 M KC1 may have some relevance to reinitiation carried on under the physiological conditions in E . coli and other cells. A novel aspect of transcription has recently emerged with the demonstration of RNA-dependent DNA polymerase activity in many RNA tumor viruses (Baltimore, 1970; Temin and Mizutani, 1970). This could provide a mechanism by which viral genetic information encoded in an RNA strand can be transcribed into DNA, with subsequent insertion into the genome of the host cell resulting in transformation. Evidence has now accumulated to support this idea, including the identification of a hybrid RNA:DNA molecule in newly infected cells, which is presumably an intermediate in the formation of double-stranded DNA (Spiegelman et al., 1970a). II. Eukaryotic Chromosomes

A. GENERAL STRUCTURE OF CHROMOSOMES Chromosome structure has been reviewed recently by Hearst and Botchan (1970) and by Ris and Kubai (1970). The chromosomes of eukaryotic cells consist of DNA, an assortment of proteins and RNA. Only one major group of proteins, the histones, has been well characterized. Contracted metaphase chromosomes can be isolated fairly easily. During interphase the chromosomes are much more extended and hydrated. They cannot usually be studied micro-

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scopically, but they can be isolated as “chromatin,” the nucleoprotein complex obtained after the removal of cytoplasmic and soluble nuclear components. This may be prepared in a number of ways, e.g., by repeated extraction of the tissue with saline buffers followed by density gradient centrifugation (Bonner et al., 1968a) or by extraction of purified nuclei (Paul and Gilmour, 1968; Shaw and Huang, 1970). The general composition of chromatins prepared by such procedures is given in Table I. TABLE I CHEMICAL COMPOSITION OF CHROMATINS Source

DNA

RNA

Histone

Pig cerebellum Pig pituitary Rat liver Rat kidney Chicken liver Chicken erythrocyte Pea bud Calf thymus

1 1 1 1 1 1 1 1

0.13 0.108

1.6 1.56 1.15 0.95 1.17 1.08 1.10 0.89

0.04 0.06

0.03 0.02

0.05 0.05

Nonhistone protein 0.5 0.45 0.95 0.70 0.88

0.54 0.41 0.21

Reference

Shaw and Huang (1970) Shaw and Huang (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Elgin and Bonner (1970) Paul and Gilmour (1968)

Two types of chromosomes which can be readily studied microscopically during interphase are the giant chromosomes of insects and the lampbrush chromosomes of maturing oocytes. Most studies on the marphological aspects of interphase chromosome structure have been done with these, but chromatin has been commonly used for biochemical or physical studies. The details of chromosomal structure have not yet been worked out, but it has been recognized that the basic unit is a fiber 100-200 A. in diameter. Ris (1961) originally suggested that this was composed of a double fiber which itself contained two DNA molecules, but the suggestion made by DuPraw (1965) that there existed a basic 30 A. nucleoprotein fiber which formed a supercoiled strand of slightly more than 200 A. is now more generally accepted. Structures of this kind have been described by Solari (1968) in sea urchin sperm and by Abuelo and Moore (1969) in human lymphocytes. A similar structure has also been proposed by Wilkins et al. (1959) and by Pardon et al. (1967) based on X-ray diffraction studies of chromatin which indicated that the primary nucleoprotein fiber had a diameter of 35 A. and was coiled into a helical structure, More direct observation of structures with the dimensions predicted from these studies has been obtained in electron microscopical studies which have revealed equispaced electron dense and less dense bands lying adjacent and parallel to the envelope of nucleated erythrocytes (Davies, 1968; Davies and Small, 1968).

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When either isolated chromatin or intact interphase nuclei are studied by electron microscopy, no evidence of a higher order of organization can be discerned. However, since chromosomes segregate exactly like DNA during mitosis (Taylor et al., 1957), there must be a very highly ordered arrangement which has so far defied recognition. One particular fundamental question which remains to be resolved satisfactorily concerns the strandedness of individual chromatids. These behave genetically as though they are single and clearly regulatory mechanisms,for transcription would be easier to understand if they were. Evidence to support this idea has been advanced by Callan and MacGregor (1958) and Gall (1963), on the basis of studies with lampbrush chromosomes in amphibians in which it is possible to examine the single chromatid and to observe the way in which i t responds to enzymes such as proteases and DNA. On the other hand, some genetic findings are difficult to explain on the theory that chromatids are single-stranded and, although ingenious mechanisms have been advanced to explain how single-stranded chromosomes could behave in a way compatible with the occasional unusual patterns of segregation obtained in ascospores of Neurospora, this phenomenon would be more readily explained on the basis of doublestrandedness of the chromosome. Evidence for multistrandedness or a t least double-strandedness is by no means negligible (Wolff, 1969). One of the most important pieces of evidence is the appearance of isolabeled chromosomes a t the second mitosis after labeling (LaCour and Pelc, 1958; Peacock, 1963) and of subsequent mitoses (Deaven, 1968). Halfchromosomes have been observed in living cells (Bajer, 1965) and in disrupted chromosomes (e.g., Trosko and Wolff, 1965). Moreover, the evidence for single-strandedness based on the kinetics of digestion by DNase (Gall, 1963) has been challenged (Hecidle and Bodycote, 1968). These remarks refer to the interphase chromosomes of most eukaryotes. There is, of course, little doubt that the polytenic chromosomes of diptera are multistranded to a high degree. The subject is reviewed in a fairly recent paper (Heddle, 1969). What has been discussed above refers to the general structure of chromosomes. In interphase cells it can be recognized that some chromatin is more compact than the rest; this is referred to as heterochromatin; in general it is much less active in RNA synthesis than euchromatin, which is less densely packed. No precise information is available about the molecular differences between these two ; in particular very little is known about the ultrastructure of areas being transcribed in cells from vertebrates. The only hard information we have about this comes from the dramatic pictures by Miller and Beatty (1969) showing transcription of genes in Triturus oocytes. The examples they have

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studied involve sites which are being very actively transcribed indeed, and in which the chromosome is covered with a procession of polymerase molecules, to most of which is attached a strand of nascent messenger RNA. Since these increase in length from the initiation site to the termination site, the appearance has been likened to a “Christmas tree.” Obviously, there are enormous gaps in our knowledge of the general structure of eukaryotic chromosomes which will have to be filled before we have a good understanding of the control of transcription in these cells. The situation is somewhat better when we turn to look a t some of the details of the molecules which make up chromosomes. B. DNA IN EUKARYOTES In those prokaryotic genomes which have been studied, evidence has been obtained that certain sequences are reduplicated. However, the extent of reduplication is rather small and does not usually amount to more than two or three copies of a given gene. In eukaryotic cells there is much more DNA. For example, the human genome is about 1000 times bigger than that of Escherichia coli and there is evidence for a very high degree of reduplication of some sequences in it. This evidence comes not from genetic studies, but from physicochemical studies of the reannealing of denatured DNA. When DNA is heated it separates into two complementary strands. If this denatured material is then maintained at a temperature a little below the melting temperature, the strands reassociate, each strand of DNA with its complementary partner. Wetmur and Davidson (1968) showed that the rate-limiting reaction in the reannealing process behaves as a second-order reaction, It is characteristic of second-order reactions that the half-life of the reaction is inversely proportional to the concentration of reactant. Hence the more dilute the DNA solution, the longer the reaction will take. In the reannealing reaction the reacting component can be defined as being the minimum length of nucleic acid in which no long sequence of nucleotides is repeated. I n a relatively simple DNA molecule (such as a bacterial genome) a unique sequence of nucleotides might amount to 42 million daltons whereas in a complex DNA the unique nucleotide run might be 1000 times larger. If one took a given concentration by weight of the two, then the first would anneal very much faster than the second, since it would have a higher concentration of “unique” sequences. The rate of annealing by itself can therefore be used as an index of complexity. If one makes the simple assumption that the genome of an organism consists of a unique set of nucleotides without any reduplication, then the complexity of a DNA should be proportional to genome size; this is what is found for viral and bacterial DNA’s. By this criterion one

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would expect a mammalian DNA to be about 1000 times more complex than the DNA of Escherichia coli, and consequently one would expect that in the same conditions a given concentration of mammalian DNA would anneal about 1000 times slower. When experiments of this kind were first done with mammalian DNA, it was appreciated that there was an anomaly (McCarthy, 1967) in that a large part of the mammalian DNA annealed about as fast as bacterial DNA. The situation was analyzed in great detail by Britten and Kohne (1968), who introduced the use of the “Cot curve,” i.e., a plot in which reannealed DNA is measured against the product of the initial concentration of DNA (C,) X time ( t ) , usually placed on a logarithmic scale. Application of this analysis to eukaryotic DNA’s revealed discontinuities in reannealing. It became apparent that, besides DNA which was not repeated in the genome (unique or nonrepetitious DNA), there was a large amount of DNA which was highly repetitious in that it annealed very much more rapidly. The amount of this repetitious DNA varies from one organism to another. The degree of repetitiousness also varies but is often of the order of 10,OOO to 100,000; not infrequently, there are several groups of repetitious sequences. Britten and Kohne further showed, by studying the thermal stability of renatured DNA that the individual components of a given set of repetitious genes were not identical. Although they were similar enough to anneal with each other, there was sufficient mispairing of bases to lower the melting temperature of the hybrid. Accordingly, Britten and Kohne considered that these repetitious sequences occurred in “families” of related sequences. The distribution of repetitious DNA throughout the genome is of considerable interest. Britten’s group have tried, by shearing, to determine how long the unique and repetitious sequences are and have come to the conclusion that, except for a very highly repetitious component, the two are very intimately intermixed (Britten and Smith, 1970). A similar conclusion can be drawn from the results of Thomas et al. (1970), who found that when eukaryotic DNA was sheared and reannealed, either after degradation with exonuclease or after chain separation, a high proportion of the DNA occurred in circular structures. This provided evidence for linear repetition of nucleotide sequences a t relatively short distances. The function of the repetitious sequences is not, in general, known but there is some special information about some of them. When DNA is equilibrated in a cesium chloride density gradient, in addition to the main peak there occur in some species a small, heavy satellite, now known to contain the genes for ribosomal RNA, and a small, light satellite. The small, light satellite has been found to contain a partic-

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ularly highly repetitious class of DNA. This DNA has a base composition which suggests that it could never give rise to a messenger RNA which would be translated into protein (Southern, 1970). It is associated with the constitutive heterochromatin of the cell (Yunis and Yasmineh, 1970; Yasmineh and Yunis, 1970), and in in situ hybridization experiments it is found to be associated with the constitutive heterochromatin of the chromosome (Gall and Pardue, 1969). This DNA, then,probably has a structural role associated with the centromeric region of the chromosome. A degree of redundancy is also observed in the ribosomal genes, although it is not of nearly such a high order. There is now a considerable accumulation of evidence which indicates that the DNA sequences corresponding to the precursors of 2 8 s and 1 8 s RNA are separated by spacer sequences the whole complex being repeated several hundred times. There is also some evidence that sequences coding for messenger RNA may be highly repetitious. Williamson et al. (1970) found that the messenger RNA for globin hybridized with a fraction of DNA sufficient to account for about 1000 copies. Very recently A. Kedes and Birnstiel (1971) have obtained evidence that the sequences coding for histone messengers are similarly repetitious. These discoveries have, of course, to be taken into account in interpreting experimental findings, especially those based on RNA/DNA hybridization. C. CHROMOSOMAL PROTEINS 1. Histones

Much of the background to histone chemistry, characterization, and fractionation has been covered in recent reviews (Hnilica, 1967; Georgiev, 1969a; Stellwagen and Cole, 1969; Fambrough, 1969; E. L. Smith et al., 1970; Elgin et al., 1971; Johns, 1971). Hence for this discussion it is only necessary to summarize the situation and describe recent and relevant work. Many of the recent advances in histone chemistry have been due to the development of satisfactory methodology for their preparation and characterization. For example, the differential solvent extraction methods of Johns (1964) and ion-exchange chromatography procedures (Rasmussen et al., 1962; Kinkade and Cole, 1966a), together with reliable methods of electrophoresis (Johns, 1967a; Panyim and Chalkley, 1969a,b) have led to histones being characterized from many species and tissues (see Johns, 1971). It would appear that (a) histones occur only in eukaryotes and ( b ) there is little evidence for species or tissue specificity. They appear to consist of five main groups of proteins of rela-

TRANSCRIPTIONAL REGULATION IN EUKARYOTIC CELLS

107

tively low molecular weight (12,000-22,OOO) but of varying basicity. The main characterist'ics of these proteins are given in Table 11. Cell specificity of histones has been found in relatively few instances. Nucleated erythrocytes possess a specific serine- and lysine-rich histone (Neelin et al., 1964) [see Table 11, F2c(V)] which largely replaces the very lysine-rich F1 in these cells (Dick and Johns, 1969b), and may be connected with the lack of chromat'in template activity in mature erythrocytes. Certain other tissues have been shown to possess a specific very lysine-rich histone (Panyim and Chalkley, 1969b,c). In addition, in certain spermatozoa histones are replaced by arginine-rich protamines (Bloch, 1969). Although the proportions of histones and DNA are roughly equal (Table I), only a few studies have been carried out on the relative amounts of the various histone fractions in different tissues. In the chick embryo the proportions of the histone components vary during development until the adult pattern is reached (Lindsay, 1964; Agrell and Christensson, 1965; Kischer and Hnilica, 1967). In the embryo of the newt, however, no histones were observed until the early gastrula stage, when a constant amount of arginine-rich histones first appeared, followed by increasing amounts of slightly lysine-rich histones from late gastrula and finally by the lysine-rich fraction at the tail bud stage (Asao, 1969). A somewhat similar situation was found during embryogenesis in the sea urchin (Orengo and Hnilica, 1970). This sequence of events has been found to be associated in the newt with the appearance of the various histones in different regions of the embryo during development (Asao, 1970). Fambrough et a2. (1968) reported that the same pattern of histone fractions was found during development of the pea cotyledon except that the amount of histone F1 increased as maturation progressed. A few studies indicate that considerable variation may exist in the relative proportion of the histone fractions in various adult tissues (Hnilica et al., 1966; Johns, 1967a; MacGillivray, 1968). With the exception of the very lysine-rich histone F1, individual histone fractions probably consist of one molecular species (Fambrough and Bonner, 1969; Johns, 1971). The F1 histone fraction has been shown to be heterogeneous in electrophoresis (Panyim and Chalkley, 1969b) and ion-exchange chromatography systems (Kinkade and Cole, 1966a; Bustin and Cole, 1969a). Electrophoresis shows differences in the F1 components of mouse tissues, the complexity of the patterns being associated with the extent of phosphorylation of a parent histone molecule (Sherod et al., 1970). Chromatography on IRC-50, on the other hand, has revealed differences in the F1 complement from different species, but no convincing tissue differences in any one animal (Bustin and Cole,

? ?

TABLE I1 ~OMFQSXTION OF ~ ~ R O M APEOTEINS~ TIN ~

~

~~

~~~

Histon& Component

ASP (A)(P) (P) Ser (PI Glu @)(PI Pro (ap) GlY Ala Val (ap)

F1

F2b

F2a2

F2sl

2.5 5.6 5.6 3.7 9.2 7.2 24.3 5.4

5.0 6.4 10.4 8.7 4.9 5.9 10.8 7.5 Trace 1.5 5.1 4.9 4.0 1.6

6.6 3.9 3.4 9.8 4.1 10.8 12.9 6.3 Trace Trsce 3.9 12.4 2.2 0.9

5.2 6.3 2.2 6.9 1.5 14.9 7.7 8.2 Trace 1.0 5.7 8.2 3.8 2.1

t-Crs

0

Met ( w )

0 1.5 4.5 0.9 0.9

ne (ap)

Leu (ap) TYr Phe ( w )

F3

F2C(V)

4.2 6.8 3.6 11.5 4.6 5.4 13.3 4.4 1.0 1.1 5.3 9.1 2.2 3.1

1.7 3.2 11.9 4.3 4.7 5.3 16.3 4.2 0 0.4 3.2 4.7 1.2 0.6

A

9

Nonhistone proteins

B

pH5=

B+a’

HAP.

CSCl’

9.4 4.8 5.0 15.4 4.5 7.5 8.3 6.7 0.8 2.1 5.1 8.5 2.7 3.7

8.4 4.3 9.9 10.9 4.4 14.7 8.3 5.2 1.1 1.4 3.7 7.7 3.3 2.6

9.2 4.9 6.1 13.5 5.5 8.0 7.4 6.1 0.6 1.8 4.3 8.0 3.7 4.9

10.9 3.6 8.5 12.2 5.5 14.0 5.5 4.6

B r

5

”$

4

*d w

r 9

3

-

2.1 2.9 4.9 4.2 3.9

8

w

F! %i 9 F

r

26.8 0 Trace 1.8 3 6.2 28.6 4.6 46.0 21.5 2.1 Acetyl

LYfj

14.1 0 2.3 6.9 6 13.7 23.3 1.7 51.5 25.8 2.0

Pro Lys

10.2 0 3.1 9.4 7 16.4 22.7 1.4 43.3 27.6 1.6 Acetyl

Lys

0 Amino acid analysea are given as moles per 100 moles. *Johns (1971). Wang (1967a). 1 Elgin and Bomer (1970). MacGillivray e2 ol. (1971). W.Benjamin and GelIhorn (1968).

10.2 1.2 2.2 12.8 5 12.1 26.4 2.2 44.8

25.7 1.7 Acetyl Gly

9.0 1 .o 1.7 13.0 7 15.7 24.7 1.6 49.4 27.6 1.7 Ala

Ala

23.6

k! 3

7.7

4.8

-

6.7 -

6.2

3d

1.9 12.4

2.1 5.7

3.9

3.1

-

6.7

-

1.5 9.3

$

6.0 37.9 6.3 57.1 17.8 3.2

24.8 15.5 0.54 48.0 30.6 1.6

19.3 14.6 0.76 44.2 25.0 1.8 -

22.7 16.5 0.73 47.1 30.6 1.5

23.1 17.0 0.74 50.7 23.9 2.1

-

-

-

-

-

-

-

-

5.9

-

-

-

$ !a

f8! @

2

110

A. J . MACGILLIVRAY, J . PAUL, A N D G . THRELFALL

1968; Stellwagen and Cole, 1968; Kinkade, 1969). The F1 complement appears to be dependent on the physiological state of the animal (Stellwagen and Cole, 1968; Hohmann and Cole, 1969). Structural studies have been carried out on a purified component of rabbit thymus F1 (Bustin and Cole, 1970). These studies indicate an asymmetric distribution of amino acid residues such that the C-terminal portion of the molecule is highly basic (and possibly the site for binding to DNA) whereas the N-terminal portion has a more balanced composition. Other studies suggest that the C-terminus of F1 histone is invariant in structure, unlike the N-terminal region which may vary from one subfraction to another and possibly also depends on the species (Kinkade and Cole, 1966b; Bustin and Cole, 196913, 1970). Greenaway and Murray (1971) have suggested that these variations in structure may be genetically linked so that individual animals have different forms of F1 histones. Structural studies on the chick erythrocyte specific histone have shown microheterogeneity due to one genetically determined amino acid substitution near the N-terminus (Greenaway and Murray, 1971). It should be pointed out in this context that this specific erythrocyte histone varies considerably in analysis from one species to another (Edwards and Hnilica, 1968). The slightly lysine-rich histone fraction F2b has been obtained in purified form and its primary structure has been determined (Hnilica et al., 1970; Iwai et al., 1970). In this case the N-terminal region is the most basic part of the molecule and hence the most likely site for attachment to DNA. Many recent structural studies of histones have centered on the F2al histone. These have shown structural similarity of this fraction in various species and in both normal and tumor tissue (L. Desai et al., 1969; Leclerc et al., 1969; L. S. Desai and Foley, 1970; Sautiere et al., 1970a,c). The most significant studies, however, have resulted from the determination of the primary structure of the F2al histone of calf thymus, pig thymus, and pea seedling (Sautiere et al., 1970b; DeLange et al., 1969). The amino acid sequence of the calf and pig histones are identical, the majority of the basic residues being situated in the Nterminal region of the protein as in fraction F2b. However, it is of outstanding interest that the F2al histone of the more distant species, the pea, should contain the same sequence apart from two conservative substitutions (2% difference) (DeLange et al., 1969). The only similar comparison which has been made between plant and animal proteins concerns cytochrome c in which 30% of the residues differ between the two species (F. C. Stevens e t al., 1967; Yasunobu et al., 1963). Hence it would appear that during evolution the structure of this histone has been rigidly conserved, presumably because it has a highly specific

TRANSCRIPTIONAL REGULATION IN EUKARYOTIC CELLS

111

function to perform. Despite these manifestations of homogeneity, electrophoresis in systems of high resolution shows the F2al histone to be present in some tissues as a single component and in others as multiple components (Panyim and Chalkley, 1969b; Sung and Dixon, 1970). These tissue differences are probably associated with the varying degrees of acetylation and phosphorylation of lysyl residues in this histone (DeLange et al., 1969; Sung and Dixon, 1970). It is also interesting from the point of view of cell specificity that cow, but not pea, F2al histone should possess a methylated lysyl residue (DeLange et alJ969). Little structural work has been carried out on the F2a2 histone although it appears to have the same N-terminal peptide as F2al (Phillips, 1968; Sung and Dixon, 1970). It appears electrophoretically homogeneous (Johns, 1967a; Leclerc et al., 1969; Panyim and Chalkley, 1969a,b), but in high resolution systems it runs as a doublet due to microheterogeneity associated with phosphorylation (Sherod et al., 1970). Fraction F3 is the only histone to contain cysteine, the number of residues present depending on the species (Fambrough and Bonner, 196813). Since changes in the oxidoreduction state of this histone have been observed (see Section II,B,3), heterogeneity of this fraction could result from polymerization through disulfide bond formation. As before, microheterogeneity can be postulated since F3 histone possesses internal lysyl residues which are capable of being acetylated and methylated (Section II,B,3). Limited sequence studies have been performed on this histone (DeLange et al., 1970); these indicate little homology with F2al fraction. However, it is interesting to note that, unlike the other histones, Fraction 3 (and F2al) prepared from several species has constant electrophoretic mobility ; this may indicate some maintenance of structure during evolution (Panyim et al., 1970). I n summary, the characteristic properties (and possibly also the structure) of histones have been conserved during evolution in eukaryotes. This would tend to indicate that they have a specific function which cannot tolerate major changes in primary structure. Since they comprise a rather simple group of proteins, it is difficult to ascribe to them a complex role such as that of specific gene regulation; hence their actual function, although specific, may be of a much more general nature, e.g., as structural chromosomal protein. Modification of the structure of histones by phosphorylation, acetylation, etc., does introduce the possibility of a more varied role and this will be discussed later. 2. Nonhistone Proteins Several recent reviews describe the background and early work in this field (Hnilica, 1967; Georgiev, 1969a; Elgin et al., 1971) ; the study

112

A. J . MACGILLIVRAY, J. PAUL, AND G . THRELFALL

of nonhistone proteins is only now gaining momentum. They are defined as those proteins which remain associated with DNA after the histones have been removed. Owing to their ability to aggregate with themselves, histones, and nucleic acids, they have until recently been difficult to handle and characterize. Unlike histones, the amount of these proteins in chromatin varies with the tissue source and may be correlated with the level of RNA synthesis. For example, the nonhistone protein complement of maturing spermatozoa diminishes as the DNA template activity decreases (Marushige and Dixon, 1969). Conversely, the nonhistone proteins increase as the chromatin template activity increases in developing sea urchin embryos (Marushige and Ozaki, 1967). The results of Dingman and Sporn (1964) indicated that the amount of nonhistone chromatin proteins was less in mature than in immature chick cells. They also observed that treatment with certain carcinogens and hormones affected the level of these proteins in rat liver chromatin (Sporn and Dingman, 1966). Several methods are now used to characterize nonhistone proteins. These studies will be reviewed, and the implication of the results discussed. Wang (1966, 1967a) distinguished between chromosomal acidic proteins extracted from washed rat liver nuclei along with histones and DNA by 1 M NaCl and the nuclear residual proteins, which are insoluble under these conditions. The chromosomal acidic proteins are obtained in solution when the DNA and histones precipitate on reduction of the salt concentration to 0.14M. Of the four initial fractions obtained by ammonium sulfate and acid precipitation, three can be further separated into numerous components by chromatography on DEAE-cellulose and starch gel electrophoresis (Wang and Johns, 1968). The amino acid analysis of one fraction is shown in Table I. The presence of basic proteins in these fractions is indicated by protein unretained by DEAEcellulose and proteins electrophoretically cationic a t p H 8.3. The residual proteins can be solubilized by sodium deoxycholate and by procedures similar to those described for chromosomal acidic proteins. Pate1 et al. (1968) showed them to be highly heterogeneous as well. These two groups of proteins can be distinguished metabolically since the residual proteins show the highest specific activity in leucine-l'c labeling experiments (Kostraba and Wang, 1970). There is some evidence to suggest that the residual proteins are nucleolar in origin (Kostraba and Wang, 1970). Application of these techniques to the tissues of a number of species has shown tissue variations and differences between normal and tumor cells (Kostraba and Wang, 1970). Since the development of Wang's procedures, other workers have found a second fraction of nonhistone

TRANSCRIPTIONAL REQULATION IN EUKARYOTIC CELLS

113

proteins which can be extracted from chromatin by NaCl (0.35M) (Johns and Forrester, 1969; X. Wilhelm and Champagne, 1969; Spelsberg and Hnilica, 1971a). It would appear that these 0.35M NaCl soluble proteins are derived from the nuclear residual proteins (Johns and Forrester, 1969). Other authors have also claimed tissue differences in chromosomal acidic proteins prepared according to Wang’s procedure (19674 but separated by electrophoresis in an acrylamide-agarose system (Kruh et al., 1969,1970; Loeb and Creuzet, 1969, 1970; Dastugue et al., 1970). W. Benjamin and Gellhorn (1968) treated saline-washed rodent nuclei with dilute acid to remove histones. After the residue had been dispersed in CsCl at pH 11.6 the nonhistone proteins were separated from DNA by equilibrium density centrifugation and then applied to a polyacrylamide gel electrophoresis system run a t the same pH. Both mouse and rat proteins gave similar heterogeneous electrophoresis patterns, These preparations gave an acidic amino acid analysis (Table 11) and by 3*P labeling some of the electrophoresis bands appeared to be phosphoproteins. The work of Wang (1967a) and W. Benjamin and Gellhorn (1968) showed that some of the nonhistone proteins were phosphoproteins ; now a number of procedures have been developed for their isolation from chromatin. Langan (1967), Gershey and Kleinsmith (1969a), and Kleinsmith and Allfrey (1969) all used procedures based on that of Wang with a final purification step using calcium phosphate. Although these phosphoproteins appeared to be difficult to handle, solubilization in sodium dodecyl sulfate (SDS) followed by electrophoresis in an SDSacrylamide system has shown them to be highly heterogeneous and tissue specific (Platz et al., 1970). Shelton and Allfrey (1970) and C. T. Teng et al. (1970) have adopted a somewhat different procedure whereby phosphoproteins are extracted from dehistoned chromatin by phenol, a procedure used earlier by Steele and Busch (1963). Electrophoresis in an SDS-electrophoresis system has confirmed the tissue specificity of chromatin phosphoproteins. Using chromatins prepared according to Bonner et al. (1968a), Elgin and Bonner (1970) removed histones with dilute acid and then solubilized the residue in SDS a t pH 8. The DNA was removed by ultracentrifugation, and the SDS-nonhistone proteins were applied to an SDS-acrylamide gel electrophoresis system. The proteins from a number of tissues and species were found to be heterogeneous, the majority having molecular weights of the order of 5000-100,000, and to be remarkably similar from one organ to another. Very few tissue differences were found although pea bud nonhistone proteins appeared to possess only half of

114

A. J . MACGILLIVRAY, J . PAUL, AND G . THRELFALL

the vertebrate complement. The amino acid analyses of some of these components are given in Table 11. I n this laboratory we have attempted to develop a method of isolating the nonhistone chromatin proteins without exposing them to extreme conditions of pH, etc. (MacGillivray et al., 1971). Following upon the observations of Faulhaber and Bernardi (1967) and Hacha and Fredericq ( 1968), that salt-dissociated nucleoproteins could be chromatographed on hydroxylapatite (HAP) , we solubilized chromatins in 2 M NaCl-5 M urea-1 mM phosphate, pH 6.8 (Bekhor et al., 1969a) and applied the solution to HAP columns run in the same solution. The histones are unretained and the bulk of the nonhistone proteins are recovered from the column by increasing the phosphate concentration to 0.05 M . Table I1 gives the amino acid analysis of one such preparation. Since aggregation problems prevented the electrophoresis of desalted preparations in conventional systems, we then treated the nonhistone proteins with SDS as well as urea prior to electrophoresis in an SDS-urea-acrylamide system. Figure 1 shows that, in agreement with Elgin and Bonner (1970), the nonhistones are very heterogeneous and, compared with histones, are of a wide molecular weight range. However, the patterns of these proteins from different tissues and species are generally similar. Many of the proteins are of high molecular weight, but each chromatin extract has a discrete number of low molecular weight components which do not appear to be contaminating histones. A similar procedure for solubilizing cerebellum and pituitary chromatins in salturea was employed by Shaw and Huang (1970). After removal of the DNA by centrifugation or gel filtration, the proteins were separated on polyacrylamide gels at pH 2.7 in urea. Surprisingly, despite even freeze-drying such a mixture of proteins, an electrophoretic separation of histones and nonhistone proteins was obtained. Although a considerable amount of protein appeared to be unable to enter the gels, the patterns of the nonhistone proteins from both tissues were remarkably similar. Shirey and Huang (1969) attempted a separation of chromatin proteins using SDS alone. Sperm chromatin was solubilized in 1% SDS prior to removal of the DNA by ultracentrifugation. The ultracentrifuge supernatant contained most of the chromatin protein which was recovered by freeee-drying followed by removal of the SDS by urea dialysis and BaCl, precipitation procedures. Electrophoresis showed that all of the histone fractions had been recovered, together with one acidic protein fraction. However, the presence of nonmigratory material indicated that aggregation of histones and acidic protein had taken place to some extent during the fractionation procedure. Thus, in comparison with histones, the nonhistone proteins of chro-

TRANSCRIPTIONAL REGULATION I N EUKARYOTIC CELLS

115

FIQ. 1. Electrophoresis of nonhistone proteins from mouse tissues in a sodium dodecyl sulfate-acrylamide system. Curve A, Mouse kidney; B, mouse liver; C, mouse spleen. The complexity of the proteins may be compared with the pattern given by histones run in the same system (curve D). From MacGillivray et al. (1971), by permission fram the Federation of European Biochemical Societies.

matin appear to be more heterogeneous and of higher molecular weight. The degree of their complexity is difficult to estimate at present. The conditions employed by Wang (1967a), W. Benjamin and Gellhorn (1968), and Shaw and Huang (1970) may lead to aggregation, thus making the interpretation of electrophoresis data difficult to assess. In addition Sonnenbichler and Nobis (1970) have described the binding of histones to DNA in acid solutions so that by some criteria they appear as acidic proteins. This could complicate the results of the fractionation procedures which utilize acid extraction of histones as a first step (W. Benjamin and Gellhorn, 1968; Elgin and Bonner, 1970; Shelton and Allfrey, 1970). The use of detergent systems appears to avoid some of these problems, but this procedure separates proteins only on the basis of molecular weight differences (Shapiro et al., 1967; Weber and Osborn, 1969). Hence other forms of heterogeneity-e.g., differences in primary structure-will not be seen. If, however, we take the results of the SDS

116

A. J . MACGILLIVRAY, J . PAUL, A N D G . THRELFALL

procedures at face value, it appears that the nonhistone proteins show “limited heterogeneity” (Elgin and Bonner, 1970) in various species and tissues. This result is easy to rationalize since most chromatins are likely to possess similar enzymes (e.g., polymerases) , structural proteins, and perhaps contaminating membrane proteins. Demonstration of cell specificity may only come from differential extraction procedures similar to those described above for phosphoproteins. At this stage it should be pointed out that the distinction between histone and nonhistone proteins has been made entirely on the basis of chemical extraction procedures and from some aspects of their analysis. Thus dilute acid or strong salt will liberate histones, but not nonhistone proteins, from chromatin. Such a separation may be entirely arbitrary or reflect differences in binding to DNA since nonhistone proteins freed of DNA are soluble in acid (Wang and Johns, 1968; Shaw and Huang, 1970) and salt solutions (Wang, 1967a; Bekhor et al., 1969a; Shaw and Huang, 1970; MacGillivray et al., 1971). Likewise amino acid analysis shows histones to be basic, and nonhistone proteins to be acidic, proteins; hence, on the basis of isoelectric point data they also appear to be separate groups of proteins. If, however, their composition is assessed with respect to their content of polar and apolar residues, then the two groups of proteins appear similar except that some preparations of nonhistone proteins contain more apolar amino acids than the histones (Table 11).In fact, owing to the asymmetric distribution of basic amino acids, some portions of histones appear to be similar to enzymes and globular proteins (Bradbury and Crane-Robinson, 1971). 3. Synthesis and Turnover of Chromosomal Proteins

Early work suggested that the site of histone synthesis was within the nucleus since thymus nuclei were found to be capable of incorporating amino acids into histones by a puromycin-sensitive pathway (Allfrey et al., 1964; Reid and Cole, 1964). In support of this general impression, such nuclei were shown to contain ribosomes (Allfrey, 1963). However, more recent investigations with other cells have shown histonelike polypeptides on cytoplasmic ribosomes (Borun et al., 1967; Gallwitz and Mueller, 1969; Gurley et al., 1970b). In addition a 7-9 S RNA has been observed immediately before DNA synthesis and during histone synthesis in HeLa cells (Borun et al., 1967; Gallwitz and Mueller, 1969, 1970) and in sea urchin embryos at cleavage (L. W. Kedes and Gross, 1969). Nemer and Lindsay (1969) have also isolated slowly sedimenting polysomes which may be the sites of histone synthesis in sea urchin embryos. Hence, it seems very likely that histones are synthesized on cytoplasmic ribosomes in the orthodox way.

TRANSCRIPTIONAL RHXJLATION I N EUKARYOTIC CELLS

117

Although there appears to be little translational control of histone synthesis during the cell cycle (Pederson and Robbins, 1970), the major synthesis of histones occurs a t or near the time of DNA replication. There is some slight difference of opinion about the exact timing of these two events; e.g., Butler and Cohn (1963) and Orlova and Rodionov (1970) indicated that histone synthesis precedes that of DNA in regenerating liver whereas Takai et al. (1968) found concomitant synthesis of DNA and histones in the same cells. The synthesis of histones along with DNA in the S phase of the cell cycle has been confirmed in experiments using ascites (Yarbo, 1967), HeLa (Robbins and Borun, 1967), salivary gland (G. Stein and Baserga, 1970a), and cultured hamster ovary cells (Gurley and Hardin, 1968). Little is known concerning the site of synthesis of the chromosomal nonhistone proteins, and details of the timing of their synthesis during the cell cycle varys. G. Stein and Baserga (1970a) found maximum synthesis of these proteins prior to that of DNA, whereas in ascites cells their synthesis appeared to follow that of DNA and histones (Ingles, 1971). Halliburton and Mueller (1971) on the other hand, found the synthesis of nonhistone proteins in HeLa cells to come after DNA and histone synthesis. G. Stein and Baserga (1970b) have also reported that synthesis of nonhistone proteins takes place even during mitosis in HeLa cells. If either the histone or nonhistone proteins perform a regulatory function it might be expected that this would be revealed by a rather higher turnover than other proteins. However, Hancock (1969) found that labeled histones were not appreciably lost from chromatin during several cell cycles and Byvoet (1966) showed a similar turnover of both histones and DNA in rat tissues. This evidence would tend to indicate a somewhat general metabolic stability of the DNA-histone complex. However, in addition to the wave of histone synthesis near the time of DNA replication turnover of histones does occur to some extent during the rest of the cell cycle. Some workers indicate that all histone fractions turn over at the same rate (Dick and Johns, 1969a), whereas other workers have reported differential turnover of histone components (Chalkley and Maurer, 1965; Spalding et al.,1966). I n a study of histone synthesis in cultured Chinese hamster ovary cells, Gurley and Hardin (1969, 1970) found that during exponential growth only histone F1 turned over, the other histones being conserved for several generations. Moreover, part of the F1 histone complement appeared to be synthesized a t least 1 hour before being complexed with DNA; this indicates the probable existence of nonchromatin pools of histone in these cells (Gurley and Hardin, 1970; Gurley et al., 1970a). Interference with cell division

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A. J . MACGILLIVRAY, J. PAUL, AND G . THRELFALL

causes abnormalities in histone synthesis. Thus, according to Gurley et al. (1970a), after irradiation histones dissociate from chromatin but reassociate after some delay. I n preparation for the ensuing replication of nuclear constituents, histone F3 is synthesized some hours before reassociation in such a way that a large excess of this histone is ultimately complexed with DNA. Histone synthesis has been reported during inhibition of DNA synthesis (Spalding et al., 1966; Gurley and Hardin, 1968). On the other hand, in HeLa cells, the synthesis of lysine-rich histones has been found to be more dependent on DNA replication than that of the arginine rich fractions (Sadgopal and Bonner, 1969). Apart from experiments in which stimuli, such as hormones, induce the synthesis of specific acidic proteins in target tissues (Shelton and Allfrey, 1970; C. S. Teng and Hamilton, 19701, only a few studies have been carried out on the turnover of the nonhistone proteins. It appears that these proteins are synthesized during the entire cell cycle at a level higher than that of histones (Ingles, 1971; Halliburton and Mueller, 1971; G. Stein and Baserga, 1970b). Also, Holoubek and Crocker (1968) observed that the specific activity of DNA-associated acidic protdins was higher in ascites cells than that of histones, and Hancock (1969) found that there was a higher proportion of labeled nonhistone proteins in the chromatin of mitotic than in interphase cells. In HeLa cells Halliburton and Mueller (1971) found that nonhistone proteins turned over faster than histones, but in ascites cells Ingles (1971) observed similar turnover rates for both groups of proteins.

D. CHROMOSOMAL RNA When total chromosomal proteins are isolated, they contain a considerable amount of RNA. Huang and Bonner in 1965 first described a special class of RNA containing a high content of dihydrouridylic acid which they considered to be bound to histones covalently. They postulated that the histones were subunits of larger structures linked by this nucleic acid. W. Benjamin et al. (1966) described a similar RNA which was bound to histone, and was ribonuclease insensitive. Subsequently Bonner and Widholm (1967) found that chromosomal RNA hybridizes with a large amount of DNA, and they were able to demonstrate organ specificity between pea cotyledon and pea bud chromosomal RNA. The properties of calf thymus chromosomal RNA were described more recently by Shih and Bonner (1969). They found that it was bound not to histones but to the nonhistone proteins, contained 40 nucleotides, and had approximately 3-4 dihydrouridylic acid residues per chain. Dahmus and McConnell (1969) described similar material from rat ascites cells. This had an s value of 3.3 S and a high content of dihydroribothymidine;

TRANSCRIPTIONAL REGULATION I N EUKARYOTIC CELLS

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it hybridized with about 4% of nuclear DNA. They found that the turnover rate of this RNA was not high, and was similar to that of transfer RNA. A further unusual property of this RNA was reported by Bekhor et al. (1969b). They described observations which suggested that chromosomal RNA hybridized to double-stranded native DNA a t a low temperature (4°C.) in urea. The hybrid molecule formed was very stable and did not dissociate until the nucleic acid became denatured. The function of this RNA has been a matter for speculation. Bonner’s group (1968b) has repeatedly made the suggestion that it may provide a simple DNA recognition mechanism. Evidence for this will be discussed in a later section. On the other hand, Heyden and Zachau (1971) have evidence that a large part of the chromosomal RNA from calf thymus chromatin is simply tRNA.

E. MISCELLANEOUS COMPONENTS OF CHROMATIN The histone fraction of chromatin has already been described as consisting of relatively few components (Section II,C,l) whose functions appear to be structural (Section II,F) or to be involved in masking DNA (Section 111,B13).It is therefore quite likely that the few reports of the presence of enzymatic activity in histones, e.g., nucleases and proteases (Leslie, 1961; S.J. Martin et al., 1963; K. B. Tan e t al., 1969) are due to contamination by other proteins. The most probable source of these is the nonhistone fraction, the heterogeneity of which has already been referred to (Section II,C,2). Wang (1967a) showed that his chromosomal acidic protein fraction contained several enzymatic activities, e.g., dehydrogenase, transaminase, and ATPase. Since then the presence of enzymes more directly associated with nucleic acid metabolism has been described. An aggregate RNA polymerase enzyme was described in chromatin in the early 1960’s (Weiss, 1960; Huang and Bonner, 1962), DNA polymerase was subsequently isolated from several tissues by means of procedures based on Wang’s methodology (Pate1 et al., 1967; Wang, 1967b, 1968a; Howk and Wang, 1969). Such preparations have also been found to contain nucleotidy1 transferase activity (Wang, 1968b). DNase activity has been reported to be present in chromatin (Swingle e t al., 1969) and has been isolated from Wang preparations of nonhistone proteins (O’Connor, 1969). A number of other enzymes have been found in chromatin whose activities affect chromosomal proteins. Thus Langan (1967), Kleinsmith and Allfrey (1969), and King and Gordon (1969) demonstrated protein phosphokinase activity in chromatin nonhistone proteins, including phosphoproteins. A number of authors have described proteolytic enzymes

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in chromatin (Phillips and Johns, 1959; Furlan and Jericigo, 1967a,b; Panyim et al., 1968). Elgin et al. (1971) have suggested that these proteases may play a specific role in control mechanisms in assisting in the removal of histones from chromatin (cf. during the replacement of histones by protamine in sperm; Section II,B,3). I n this respect it is interesting to note that the susceptibility of histones to the proteolytic activity described by Bartley and Chalkley (1970) should depend on whether or not the histones are bound to DNA. Enzymes which methylate chromatin proteins have also been observed in nonhistone protein preparations (Comb et al., 1966; Burdon and Garven, 1971). Chromatin has also been found to contain enzymes which catalyze the methylation of its DNA (Burdon, 1971) and the acetylation of its histones (Racey and Byvoet, 1971). Nonhistone proteins have also been shown to contain receptor proteins for a number of compounds which stimulate target organs. Haussler and Norman (1969) demonstrated that a metabolite of vitamin D was localized among certain nonhistone proteins of intestinal mucosa. Likewise receptor proteins for some steroid hormones appear to be present in extracts of nonhistone proteins of intact nuclei, e.g., androgens (Fang et al., 1969; Mainwaring, 1969), estrogens (Jensen et al., 1968; King et al., 1969), and progesterone (O’Malley et al., 1970). As judged from the investigations using estrogen (Maurer and Chalkley, 1967; Rochefort et al., 1969; Shyamala and Gorski, 1969; Alberga et al., 1971), these hormone receptors are actually part of the nonhistone proteins of chromatin. It is interesting that these receptors should be extracted from chromatin (along with other nonhistone proteins) with 0.3 M salt, a procedure which does not alter the template properties of the chromatin (Spelsberg and Hnilica, 1971a). Since the nuclear hormone receptors may be cytoplasmic in origin (King et al., 1969), this might be thought to support the suggestion of Johns and Forrester (1969) that the proteins extractable from chromatin by this procedure are cytoplasmic contaminants. Finally Mondal et al. (1970) have reported that the nonhistone fraction of coconut chromatin contain factors which affect the indigenous RNA polymerase. These include initiation, rifampicin inhibitor and polymerase inhibitory activities.

F. ROLEOF CHROMOSOMAL CONSTITUENTS IN THE STRUCTURE OF CHROMATIN In the preceding sections of this chapter we reviewed aspects of the biological function of chromosomal proteins. Since strands of DNA double helix of considerable length are condensed into tightly packed

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metaphase chromosomes or into the looser structure of interphase chromatin, it is highly probable that some chromosomal proteins play a structural role. Attempts have been made to obtain information about the chemical structure of giant chromosomes and lampbrush chromosomes by eneymatic digestion and histochemical methods. With regard to the lampbrush chromosomes, in which all loci seem to be active in RNA synthesis, Izawa et al. (1963a,b) showed that the protein:DNA and RNA:DNA ratios were much higher than in other chromosomes. The nature of the proteins associated with nucleic acids in giant chromosomes has not been studied in any detail. More extensive work has been done with giant chromosomes. Both Swift (1964) and Gorovsky and Woodard (1967) produced evidehce that the histone:DNA ratio remained constant throughout the length of the giant chromosome, both in the condensed regions of heterochromatin and in the less condensed regions in the puffs. On the other hand, most of the nonhistone protein that could be detected occurred in Balbiani rings and puffs. Similarly, all the RNA could be detected in Balbiani rings and puffs. Pelling (1964) showed by autoradiography that RNA was in fact being synthesized or accumulated there, while Edstrom and Beermann (1962) found that dRNA was locusspecific on the basis of base compositions. Further studies on the characteristics of the RNA from puffs by Daneholt e t al. (1969a,b) showed that this RNA is of high molecular weight and is heterogeneous in size. It turns over rapidly with a complete turnover time of less than 45 minutes in puffs and of less than 30 minutes in Balbiani rings. In view of the speculation that acetylation of proteins might play an important part in gene activation, Clever and Ellgaard (1970) have studied acetylation of protein using autoradiography. They found no evidence for preferential incorporation of acetate in preexisting or in newly induced puffs. The information gained from these morphological studies is limited, and a much more detailed picture has now begun to emerge from biochemical and physical studies on chromatin. Much of the current work has been dealt with in recent reviews (Bradbury and Crane-Robinson, 1971; Fredericq, 1971). Using biochemical parameters, Murray et al. (1970) found little evidence for stretches of free DNA in sheared chromatin. However, some phosphate groups of DNA in chromatin are free to react, as shown by binding studies wit,h a number of reagents, e.g., Azure A, polylysine, toluidine blue, and specific anti-DNA sera. According to these studies, the fraction of the phosphate groups of DNA in chromatin which can react is of the order of 50% (Klein and Szirmai, 1963), 38-500/0 (Itzhaki, 1970; R. J. Clark and Felsenfeld, 1971), 28-60% (Miura and Ohba,

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1967; Salganik et al., 1969; Kurashina et al., 1970), and 1-5% (Stollar, 1970), respectively. Itehaki (1970) showed by polylysine titration that these phosphates occurred in lengthy stretches, a finding confirmed by R. J. Clark and Felsenfeld (1971). Frenster et al. (1963) described a technique for fractionating chromatin into “heterochromatin” and “euchromatin.” Both fractions contain similar amounts of histones, but euchromatin contains more nonhistone and phosphoproteins than heterochromatin (Frenster, 1965a ; Salganik et al., 1969). Spectroscopic and physical chemical techniques have been used to determine the structure of DNA-protein complexes, but most of these have involved nucleohistones ; i.e., little or nonhistone protein has been present. Some workers, with the exception of Permogorow et al. (1970), have indicated from circular dichroisim studies that the conformation of DNA is changed in nucleohistone (Fasman et al., 1970; Simpson and Sober, 1970) and chromatin (Henson and Walker, 1970a; Shih and Fasman, 1970), the effect being related to the presence of protein in the complex. Similar changes have been found in the optical rotatory dispersion spectra of the DNA of chromatin (Tuan and Bonner, 1969), and these have been ascribed to a slight alteration in the DNA double helix when it is part of the chromatin superstructure (Sponar et al., 1970). Henson and Walker (1970b) have shown that DNA is more asymmetrical than nucleohistone, histones F2b, F2a2, and F3 being involved in this effect. X-ray diffraction, infrared, and optical rotatory dispersion spectroscopy studies have shown that isolated or DNA-bound histone fractions possess both helical and random coil configurations, the amount of the former being estimated a t 20-50% in some fractions (Bradbury and Crane-Robinson, 1971). Nuclear magnetic resonance studies show that regions of histones F1, F2a, and F2b can undergo conformational changes and are responsible for histone-histone interactions. Such regions appear to be not the basic parts of the histone molecule, which probably bind to DNA, but the remainder of the polypeptide chain, whose composition is similar to that of enzymes and globular proteins (Bradbury and Crane-Robinson, 1971). This technique has further confirmed the impression that the basic region is the only part of histone F2b to bind to DNA, whereas all of the polypeptide chain of histone F1 is apparently capable of such an interaction (Boubuk et al., 1971). It is interesting to note that histone F1 possesses significant amounts of proline (Table 11) and has been reported to have an extended structure with little a helix content (Bradbury et al., 1967; Haydon and Peacocke, 1968). Studies using interphase and metaphase chromosomes (Littau et al., 1965; Mirsky et al., 1968; Sluyser and Snellen-Jurgens, 1970) indicate that this histone

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fraction may link DNA-containing fibrils together. Although Olins (1969) has provided evidence that the lysine-rich histones lie in the large groove of the DNA helix, the most tightly bound histones (arginine-rich) show an extended chain form which appears to lie parallel to the helix rather than to grooves themselves (Bradbury and Crane-Robinson, 1971). DNA still retains its native B-form in nucleohistone, but several studies already mentioned indicate that other aspects of its conformation are altered when it is part of the nucleohistone complex. The physical studies which indicate that histones cause DNA to assume a compact and symmetrical structure in nucleohistone have recently been reviewed by Fredericq (1971). Likewise Bradbury and Crane-Robinson (1971) have discussed the evidence that nucleohistone has a coiled or folded structure. Based on X-ray diffraction work in Wilkins’ laboratory (Pardon et al., 1967; Richards and Pardon, 1970) using stretched fibers, the structure of nucleohistone currently considered most likely is a “supercoil” in which the DNA double helix is folded upon itself to form a coil of diameter 100A and pitch 120 A. Bradbury and Crane-Robinson (1971) point out some discrepancies in the evidence and emphasize that the supercoil is a t present only a convenient working model. Other evidence also favors the supercoil structure and X-ray diffraction studies on reconstituted nucleohistones show that histones are required for this effect (Zubay and Wilkins, 1964; Palau e t al., 1967). Since heterologous nucleohistones prepared from DNA and histones from very different sources still show evidence of supercoiling, it seems unlikely that there are specific base sequence associations between DNA and histones (Garrett, 1968). Electron microscopic studies also support the concept of supercoil formation (see review by Bradbury and Crane-Robinson, 1971). Experiments using nucleohistone partially depleted of histones or reconstituted from DNA and histone fractions show that neither the F1 histone nor the erythrocyte-specific histone is involved in forming supercoils and that more than half of the histone complement has to be removed before the supercoil properties of chromatin are lost (Murray et al., 1970). Attempts have thus been made to determine which of the individual histone fractions is responsible for the supercoiled formation. Specific removal of the F2al and F2a2 histones has resulted in the loss of supercoil in nucleohistone, a result which has been reversed in some experiments by replacement of these histones (Bradbury and CraneRobinson, 1971). Moreover, nucleohistones reconstituted from DNA and histones show supercoiling properties using either a mixture of F2a1,2, and F3 histones or a mixture containing F2a2 and F3 histones (Richards and Pardon, 1970; Bradbury and Crane-Robinson, 1971). Bradbury and Crane-Robinson (1971) regard the formation of the supercoil-type of

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structure as resulting from histone-induced constraints “arranged both around the DNA molecule and displaced one from another in a regular manner along the molecule.” These constraints might be due to the basic regions of histones straining the DNA helix into a supercoil form or be associated with interactions between the portions of histone molecules which do not bind to DNA. Further degrees of condensation of nucleohistone fibers might then come about through a succession of histonehistone interactions or could be associated with cross-linking caused by F1 histones (Littau et al., 1965). The problem of how specific sequences of DNA are made available for transcription will be discussed in Section 111, but in the present context it may be remarked that ,Johns (1969) has suggested that supercoiling, by itself, may inhibit transcription by preventing progression of RNA polymerase dong DNA. Ill. Control of Transcription in Eukaryotic Cells

A. TEMPLATE PROPERTIES OF CHROMATIN 1. Heterochromatinization and Puffing

The first information to suggest that transcription might occur in localized regions of interphase chromosomes came from studies on “puffs” and “Balbiani Rings” in the giant chromosomes of Diptera. The field has been extensively reviewed elsewhere (Beermann, 1966; Clever, 1968; Berendes and Beermann, 1969), and it will suffice here only to mention the more important aspects. The terms “puffs” and “Balbiani rings” are used in a loosely interchangeable way, but more particularly the term “Balbiani ring” describes some of the larger swellings whereas the term “puff ,” although used rather generally, refers to smaller swellings. Early in the study of these structures it was observed that they were inconstant, but that the appearance of certain Balbiani rings and puffs was characteristic of certain stages of development in the larva. Beermann (1952) described differential puffing patterns in different organs. These observations led to the suspicion that puffs might be sites of gene activity, and this was much strengthened by the observation of Pelling (1964), who showed by autoradiography that puffs were sites of RNA synthesis. Edstrom and Beennann (1962) isolated RNA from individual puffs and obtained evidence for locus specificity. These studies in giant chromosomes have provided the best direct demonstrations of RNA synthesis of a presumably organ-specific nature occurring in localized sites in chromosomes. Some other observations of a similar nature have come from studies on heterochromatinization (see Brown, 1966). There is both

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genetic and autoradiographic evidence that transcription is much reduced, if it occurs at all, in heterochromatic (condensed) regions of chromosomes. Some of these regions, described as “constitutive” heterochromatin are always condensed whereas “facultative” heterochromatin may be condensed in some circumstances but uncondensed and active in RNA synthesis in others. Balbiani rings and puffs can be induced in giant chromosomes by treatment of the whole insect or isolated salivary glands with ecdysone. This work is reviewed by Clever (1966a, 1967, 1968). Juvenile hormone can also stimulate a Balbiani ring in Chironomus (Lezzi and Gilbert, 1969; Laufer and Holt, 1970). Izawa et al. (1963a) had shown that actinomycin D caused regression of lampbrush chromosome loops, and this suggested that similar experiments should be carried out with giant chromosomes. Clever (1966b, 1968) found in such experiments that actinomycin D interfered with certain puffs but not others; he suggested that the puffs appeared in sequence and that RNA synthesis by the primary puffs was essential for the occurrence of secondary puffs. Beermann (1963) had already reported that low doses of actinomycin D prevented puffing in Chironomus tentans whereas high doses prevented reduction in the size of the puffs. Clever (1964, 1966b) and also Ritossa and Pullitzer (1963) found that puromycin did not prevent puff formation; indeed, Clever found that certain puffs could be induced by cycloheximide, a n induction which could be prevented by actinomycin D. It is not easy to explain all these findings in a unified theory. Clever has inclined to the view that the appearance of secondary puffs in some way depends on RNA synthesis by primary puffs. Kroeger (1963) has interpreted the findings differently, since he has demonstrated that a rejuvenation pattern of puffing can be induced by changing the sodium/ potassium balance in the medium. He postulates that a primary effect of agents inducing puffing is an alteration of the ionic balance within the cells, to which the appearance of puffs is secondary. The arguments for this hypothesis are discussed in review papers by Kroeger and Lezzi (1966), Lezzi and Gilbert (1970), and Lezzi (1970). Berendes (1968) has shown that one of the earliest events in the formation of a puff is the accumulation of acidic proteins (previously synthesized) in the locus. This precedes RNA synthesis. 2. Activation of Nuclei

Probably associated with the activation of special sites in the genome is the general activation of chromatin which is observed when condensed facultative chromatin becomes less condensed. Very little RNA synthesis occurs in condensed heterochromatin; Hotta and Stern (1966) showed

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that in meiotic plant cells RNA synthesis was inversely related to the state of condensation of the chromosomes. Renewed interest in this problem has arisen from studies of nuclear transplantation and cell fusion. Gurdon (1968) has shown that when frog blastula nuclei are injected into oocytes they swell and the chromatin becomes more dispersed. This is associated with cessation of DNA synthesis and an increase in RNA synthesis. Zetterberg and Auer (1968) studied phytohemagglutinin-stimulated lymphocytes by cytochemical methods and deduced that a reduced number of arginine residues in histones was bound to DNA phosphate groups. Bolund et al. (1969) have investigated the behavior of chick erythrocyte nuclei introduced into the cytoplasm of HeLa cells (by fusion in the presence of inactivated Sendai virus). After fusion the volume of the nucleus and its dry mass increased until DNA synthesis began. During this time acridine orange binding increased and the temperature stability of DNA decreased. Rigler et al. (1969) developed methods for measuring heat denaturation of DNA in individual cell nuclei and showed that both activation of erythrocyte nuclei by fusion with HeLa cells and activation of lymphocytes with phytohemagglutinin resulted in striking changes in the melting profiles of nuclear DNP, the changes being evident as a reduction of melting temperature. Ringertz and Bolund (1969) showed that swelling of erythrocyte nuclei could be produced in intact red cells or red cell ghosts by washing and incubation in salt solutions or tissue culture media devoid of serum proteins. The changes which occurred in these nuclei were similar to those observed in nuclei of erythrocytes fused with HeLa cells. I n general these studies lead to the conclusion that quite major changes can occur in chromatin; they strongly suggest that the reduction in density of chromatin is a prerequisite for the resumption of RNA (and possibly DNA) synthesis. 3. Priming of R N A Synthesis b y Chromatin

Morphological studies on giant interphase chromosomes are made possible by their high degree of polyteny. Such studies cannot be performed on mammalian interphase chromosomes because they are not visible by ordinary microscopy. However, the possibility of exploring the occurrence of similar phenomena in mammalian cells emerged from studies of the RNA synthesized from isolated nuclei, or chromatin, with either endogenous or exogenous polymerases. Experiments to compare the template activity of chromatin with that of DNA were first carried out by Huang and Bonner (1962), Bonner and Huang (1963), and Frenster et al. (1963). They found that, while

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it was possible to obtain some RNA synthesis using a bacterial polymerase and labeled triphosphate as precursors, chromatin was clearly a much less effective primer than DNA. On the basis of this observation it was speculated that there might be a specific restriction to priming, but this was questioned by Sonnenberg and Zubay (1965), who demonstrated that by shearing chromatin to make it more soluble, the priming activity could be greatly increased. They therefore suggested that the inefficiency of chromatin as a primer was simply a measure of its insolubility in the buffers used in the enzyme assay. A more critical analysis was then carried out by Marushige and Bonner (1966), who used rat liver chromatin as a template for RNA synthesis and performed an absorption analysis of Michaelis-Menten type to study the interaction of template and RNA polymerase. Their evidence suggested that while RNA polymerase bound equally well to DNA and chromatin, transcription was less with the latter as primer. This conclusion and later studies by Shih and Bonner (1969) are based on the assumption that RNA polymerase binds only to DNA and not to any of the chromosomal proteins. However, there is some evidence that RNA polymerase is also bound by histones (Spelsberg and Hnilica, 1969a). Hence, interpretation of the observations is uncertain. The problem was clarified by a quite different approach. Paul and Gilmour (1966a) and Georgiev et al. (1966) performed hybridization experiments with the RNA produced in the reaction and found that chromatin-primed RNA was complementary to a smaller fraction of DNA than DNA-primed RNA. From this Paul and Gilmour (1966a) concluded that many of the DNA sequences in chromatin were not transcribed, and that this restriction of transcription was of a specific nature (i.e., the same sequences were transcribed from all molecules of chromatin present in the preparation). Subsequently Paul and Gilmour (1966b) showed that the RNA transcribed from chromatin was effectively competed out by natural RNA isolated from the tissue from which the chromatin was prepared whereas it was not effectively competed out by E . coli RNA. They went on to perform experiments with chromatin from different tissues and claimed to be able to demonstrate organ specificity. Subsequently, other groups have made similar observations. Ursprung et al. (1968) and K. D. Smith et al. (1969) prepared chromatins from mouse liver, kidney, and brain and showed that the RNA synthesized from these had a high level of tissue specificity, as determined by competition experiments. It may be noted that in the experiments which Paul and Gilmour had performed they used a Micrococcus lysodeikticus polymerase. Smith et al. found that the results obtained with a bacterial polymerase and mouse polymerase were identical. C. H.

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T a n and Miyagi (1970) have also shown differences in priming by chromatin from adult rat, liver, and kidney. They confirmed that RNA synthesized in vitro from chromatin was indistinguishable from natural RNA from the corresponding organism, using a double saturation technique to demonstrate organ-specific differences between the transcription products. Some somewhat similar experiments have been done with whole nuclei. For example, Sullivan (1968) isolated RNA made from bovine thymus nuclei (without adding exogenous polymerase). This RNA was hybridized and competed out by unlabeled RNA’s from thymus and other organs. Evidence of organ specificity was obtained. The main shortcoming of these experiments is that the conditions of RNA/DNA hybridization used could be expected to detect only hybridization between RNA and repetitious DNA. With the publication of Britten and Kohne’s work (1968) this became clear, and Melli and Bishop (1969) presented some direct evidence for it. Nevertheless, the RNA/DNA hybridization experiments do seem to provide evidence that some kind of restriction of transcription occurs in chromatin, that, a t least so far as repetitious sequences are concerned, this is organ specific, and that the template properties of isolated chromatin are related to the priming behavior of the intact nucleus in vivo. I n other words, the data provide evidence for a pattern of control of transcription in the whole cell which is perpetuated in isolated chromatin. These observations stimulated studies of the properties of chromatin itself. 4. Hormone Action and Chromatin

A useful review of this field is contained in O’Malley (1969). The observation that ecdysone and juvenile hormone can induce puffing in the giant chromosomes of insects has stimulated a great deal of work on the effects of hormones on the priming behavior of chromatin. Means and Hamilton (1966) reported that an early derepression of protein synthesis occurred in all uterine subcellular fractions within 30 minutes of estrogen action. They found that actinomycin D abolished this effect and reduced nuclear RNA-synthesis, but that cycloheximide did not do this and indeed resulted in an increased incorporation of uridine in nuclear RNA compared with control or estrogen-stimulated uteri. These observations are of course very similar to observations which have been made with giant chromosomes. At about the same time, K-H. Kim and Cohen (1966) reported that the administration of thyroxine to tadpoles resulted in the modification of chromatin in liver nuclei, so that it became a more efficient template for RNA synthesis, as measured by incorporation of nucleotides. C-S. Teng and Hamilton (1968) found that estrogens

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increased the amount of genetic material available for transcription in the uterus. They reported that the RNA and total protein content of chromatin increased while the relative concentration of histone decreased. They also found that the binding of the hormone to chromatin was increased. O’Malley and McGuire (1968) and O’Malley et al. (1968) showed that progesterone and estrogen treatment resulted in changes in the hybridizable RNA in chick oviduct cells. Hamilton (1968) reviewed the field at that time and drew the conclusion that estradiol bound to chromatin in the nucleus, that this resulted in the stimulation of the synthesis of chromosomal and ribosomal RNA and an acceleration in the rate of formation of ribosomal particles, and that this ultimately resulted in an accumulation of new polysomes. Hahn et a2. (1969) also found that estrogens induced the appearance of a new hepatic RNA species in immature fowls. At the same time, a new species of RNA was formed in the oviduct, but the species induced in the oviduct and the liver were not identical. There are, therefore, a number of reports which seem to indicate that one effect of estrogens and progesterone is the appearance of new species of RNA and that this is associated with changes in chromatin. The review by O’Malley (1969) summarizes much of this evidence. King et al. (1969) reported that the nuclear estradiol receptor was an acidic protein containing protein-bound phosphate, while C-S. Teng and Hamilton (1970) demonstrated that estrogen stimulation of the uterus of ovariectomized adult rats caused incorporation of tryptophan into a specific band of nuclear acidic proteins. A somewhat similar observation was made by Shelton and Allfrey (1970), who observed that the injection of cortisone into rats specifically enhanced the synthesis of a nonhistone protein of molecular weight 41,000. This was prevented both by puromycin and by low doses of actinomycin D ; the authors concluded that synthesis of certain chromosomal proteins was necessary for the induced synthesis of RNA following cortisone administration. There are, therefore, a number of reports which suggest that the effects of estrogen, and progesterone, and possibly cortisol, may be mediated through a nonhistone chromosomal protein which modifies the transcription pattern from the chromosomes. At least superficially, these phenomena seem to resemble in many respects the phenomena following stimulation of salivary glands with ecdysone.

PROTEINS IN THE CONTROL OF TRANSCRIPTION B. CHROMOSOMAL A number of recent authors have reviewed various aspects of this subject (Georgiev, 1969a; Stellwagen and Cole, 1969; Hearst and Bot-

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chan, 1970; Lezzi, 1970; Elgin et al., 1971). We shall examine the evidence for transcriptional control by chromosomal proteins in the following order: ( 1 ) effect of histones on DNA template activity; (9) result of partial deproteinization on transcription of chromatin; (3)reconstitution of chromatin from its components ; and ( 4 ) control through modification of histones. 1. Histones

The observation that bacterial RNA polymerase would transcribe mammalian DNA in vitro led a number of workers in the early 1960’s to examine the effect on DNA template activity of histones, long suspected to be gene regulators (Stedman and Stedman, 1950). In these experiments template activity was simply measured by estimating the rate of incorporation of labeled nucleotides into RNA in the presence of the template and a bacterial RNA polymerase. It was found that histones would inhibit the ability of DNA to act as a primer for RNA synthesis in vitro (Huang and Bonner, 1962; Butler and Chipperfield, 1967), but the efficiency of each histone fraction to do so varied from one laboratory to another (Allfrey et al., 1963; Barr and Butler, 1963; Hindley, 1963; Huang et al., 1964). Although Bonner and Huang (1966) reported that their nucleohistones were soluble in the RNA synthesis medium, others have found out that the insolubility of the DNA protein complex could render the primer unavailable to the polymerase enzyme (Sonnenberg and Zubay, 1965; P. R. Clark and Byvoet, 1970; Moskowitz et al., 1969, 1970). Johns and Hoare (1970) have suggested that the different DNA-histone ratios used in several of these experiments could result in precipitation of different amounts of DNA from the incubation mixture. Hence, different histones might appear to inhibit template activity. However, in the experiments of Butler and Chipperfield (1967) the reduction of template activity did not precisely parallel the amount of DNA precipitated, indicating that histones do have some real effect on DNA template. The inhibition of RNA polymerase itself by histone fractions has also been reported (Spelsberg et al., 1969). Moreover, finely dispersed deoxyribonucleoprotein is a more efficient primer for RNA synthesis than fibrous prepartions (Sonnenberg and Zubay, 1965; Roy and Zubay, 1966; Hoare and Johns, 1970). Thus it appears that technical problems can cause discrepancies in the results of the template assay used in these experiments. Apart from the studies of Skalka et al. (1966), little or no attempt was made to characterize RNA produced i n vitro or even to show that the product was in any way complementary to the DNA acting as primer. Characterization by DNA-RNA hybridhation of such RNA’s made from

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DNA-histone templates indicates that histones prevent most of the DNA sequences from being transcribed (see Section III,B,3). 2. Partial Deproteinization Experiments Another approach to this problem was to remove the protein components of chromatin, selectivity or otherwise, so that the release of this restriction might lead to the identification of the specificity-bearing molecules. Conveniently a number of procedures were developed to selectively remove histone fractions (e.g., Hindley, 1964; Johns, 1964, 1967b; Murray, 1966, 1969; Ohlenbusch et al., 1967; Fambrough and Bonner, 1968a; Murray et al., 1968; Johns and Diggle, 1969) and certain nonhistone proteins (Johns and Forrester, 1969) from chromatin. Such procedures should be carefully controlled (especially when rate of RNA synthesis is used to estimate template activity) so that alteration in the physical state of chromatin does not lead to apparent change in transcription. I n the first instance, salt and acid extraction procedures demonstrated that the removal of histone F1 caused no change, but the elimination of groups F2b and F2a,3 considerably increased the template activities of chromatin from a number of tissues (Hindley, 1964; Seligy and Neelin, 1970; Spelsberg and Hnilica, 1971a). In the chick erythrocyte much of the inactivity of the chromatin was abolished on removal of the serine-rich specific histone (Seligy and Neelin, 1970), thus implicating this protein in shutting off genes as red cell maturation progresses. Other workers, however, reported that dehistoning chromatin caused a gradual increase in template activity from the time fraction F1 was removed (Georgiev et al., 1966). Kurashina et al. (1970) demonstrated a similar effect using a nucleohistone purified from calf thymus and also judged that histone molecules were distributed randomly along the DNA chains. The amount of RNA synthesized in vitro by their partial nucleohistones was proportional to the number of free phosphate groups in the template, not to the amount of histone removed. DNA-RNA hybridization techniques were also used to further this approach. A considerable amount of nonhistone proteins can be removed from chromatin with 0.35M NaCl (Johns and Forrester, 1969). This step does not affect the specificity of the template restriction (Spelsberg and Hnilica, 1971a). Bonner et al. (1968b) showed that RNA synthesized on pea chromatin would saturate pea DNA to the extent of 2.5%. Removal of histone F1 increased this to 776, and the level continued to increase as more histones were progressively removed. Using both saturation and competitive hybridizations, Spelsberg and Hnilica (1971b) reported that removal of histone F1 caused no change in the

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type of DNA sequences transcribed from rat chromatins. Removal of the F2b or the F2a-3 groups of histones only partially derepressed the DNA, so that chromatin completely devoid of histones still possessed a considerably restricted template (Spelsberg and Hnilica, 1970, 1971b). The results of Paul and Gilmour (1968) and Seligy and Neelin (1970) using chromatins dehistoned by acid and salt also agree with the latter finding, but Marushige and Bonner (1966) claimed that the DNA in their rat liver chromatin when complexed only with nonhistone proteins possessed no such restriction. Seligy and Neelin’s and Marushige and Bonner’s studies involved rate measurements only. In contrast, Georgiev et al. (1966) reported that when F1 histone was removed by salt extraction, ascites cell chromatin was so derepressed as to resemble pure DNA. On the basis of a study of the growth of RNA chains synthesized in vitro, Koslov and Georgiev (1970) claimed that histone F1 molecules act as stop points and prevent the movement of RNA polymerase along DNA. Clearly there is some conflict in the conclusions reached by different workers, and this may be due in part to the use of different criteria. A serious shortcoming of some of these experiments, especially those depending on salt extractions in the 0.6-1.0M range is that in these conditions a state of mass equilibrium is established for a t least some DNAhistone complexes. They can, therefore, theoretically bind to regions to which they were not originally attached. A number of important issues arise through these studies. First, in general it appears that removal of the 0.35 M NaCl nonhistone proteins and histone F1 causes little or no alteration in the template specificity of chromatin. Removal of the arginine-rich histones, F2a3 in particular, causes some alteration of this property, but the significant residual restriction of the template appears to be associated with the nonhistone fraction remaining with the DNA. Second, as indicated earlier in this review, the conditions of hybridization used by, for example, Spelsberg and Hnilica (1970, 1971b) and Paul and Gilmour (1968) indicate the degree of restriction applied only to the repeated or redundant DNA sequences. Little is known concerning control over the unique type of DNA sequences. In this respect the results of Georgiev’s group are obviously a t variance with many of these points. There is considerable difficulty in comparing their results with those of others since in Georgiev’s hybridization experiments low RNA to DNA ratios are used. However, Georgiev (1969a) claims that his hybridization conditions give an estimate of the amount of RNA synthesized in vitro which has been transcribed from the redundant DNA sequences, Hence he suggests that these portions of the genome are masked by the F1 histones.

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3. Reassembly of Nuclear Proteins

The third strategy used to identify the components responsible for the specific restriction of chromatin template is the reconstruction of chromatins and nucleoproteins from previously isolated components. I n most of these studies DNA/RNA hybridization has been used to characterize RNA’s made in vitro on reconstituted templates. I n their original studies Paul and Gilmour (1968)dissolved components in 2 M sodium chloride and reconstituted nucleoproteins by a stepwise dialysis down to 0.2 M sodium chloride, after which the insoluble nucleoprotein was centrifuged and washed repeatedly with distilled water. Subsequently the technique was modified according to a recommendation by Bonner (1968). The main difference was that the components were taken up in 2 M sodium chloride, 5 M urea, 0.01 M Tris a t pH 8.3, preparatory to stepwise dialysis and washing as before. Using the former method, Paul and Gilmour (1968)showed that RNA transcribed from a nucleoprotein reconstructed from isolated DNA, histones, and a nonhistone fraction saturated the same amount of DNA as RNA transcribed from natural chromatin. Later, Gilmour and Paul (1969) showed that RNA transcribed from a nucleoprotein reconstituted from more highly purified component using the salt-urea method was indistinguishable from RNA transcribed from normal chromatin and that both were extensively similar to natural RNA from calf thymus, from which the chromatin and individual components were derived. I n other experiments Bekhor et al. (1969a) and Huang and Huang (1969) dissociated chromatin in salt-urea and reconstituted it by dialyzing out the salt and urea. They too obtained evidence that the reconstituted material behaved like the original chromatin. Paul and Gilmour (1968) exploited this reconstitution technique to investigate the functions of individual components. When they combined histones alone with DNA they found that most of the DNA in the reconstituted nucleohistone was not transcribed. On the other hand, when they dissociated chromatin into DNA and total proteins and then recombined these, they found that the normal transcriptional pattern was restored. Subsequently they separated the total protein fraction into an acid-soluble fraction (histones) and an acid-insoluble fraction (nonhistones and RNA). They found that if these fractions were added to DNA together, the original pattern of transcription could be restored. Material reconstructed in this way yielded RNA which, by competition experiments, was not distinguishable from RNA transcribed from native chromatin. If serum albumin was used instead of the nonhistone fraction, effective reconstruction was not obtained. In a further investigation

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Gilmour and Paul (1970) prepared fractions from several rabbit tissues and experimented with combinations of nonhistone proteins, DNA, and histones from different organs. These results suggested that the nonhistone fraction directed the organ specificity of template restriction. Paul and Gilmour (1968) also found that the residual material left after extracting histones from chromatin with acid had much greater template activity than chromatin (although it did show some restriction). When histones were added back to this, the reconstructed material behaved like natural chromatin, again suggesting that the nonhistone material remaining bound to DNA after acid extraction prevented the restriction of transcription which histones alone produced. Similar conclusions have been reached by Spelsberg and Hnilica (1970) using a slightly different approach. They found that, on extracting chromatin with 2 M sodium chloride a t pH 6, most of the histones were removed but most of the nonhistone proteins remained attached to DNA. When the residual nucleoproteins prepared in this way from two different organs were then combined with histones, the RNA synthesized from each template behaved like RNA transcribed from chromatin from the organ providing the DNA-nonhistone protein complex (which had been shown to exhibit organ specificity). I n both these experiments and the experiments of Paul and Gilmour (1968) there is evidence that dehistoned chromatin did not have the full template activity of pure DNA. This suggests that the nonhistone fraction possesses different components with properties of both inhibiting and promoting transcription of repeated sequences of DNA. Analysis shows that the nonhistone fractions prepared by both PaulJs and Hnilica’s groups are largely protein, with a small amount of RNA present. Since the proteins are retained by anion exchange resins at nearly neutral pH, they are probably acidic; recent preparations of our fractions showed an RNA content of approximately 1%. Some findings by Marushige and Bonner (1966) and Marushige et al. (1968) showed that the rate of RNA synthesis on dehistoned rat liver chromatin and on a nucleoprotein complex prepared from DNA and nonhistone proteins was not significantly different from that on DNA. These results are not necessarily in conflict with those of the groups of Paul and of Hnilica, since RNA/DNA hybridization was not used to test the nature of the RNA. I n the experiments of Marushige et al. (1968) the nonhistone proteins were prepared by an SDS-extraction procedure, and this could have led to denaturation of some of the components. Although there is general agreement that the nonhistone fraction of chromatin is of importance in establishing specificity of transcription, there are some differences concerning the nature of the molecules actually

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involved in DNA recognition. Bekhor et al. (1969a) and Huang and Huang (1969) reported findings which suggest that RNA plays an important part in this. Bonner has expressed the view that the requirement for 5 M urea in the reconstitution experiments may exist because some kind of hydrogen-bonding of RNA to DNA has to occur. [In this connection it should be noted that urea was not used in Paul and Gilmour’s (1968) original experiments.] As more direct evidence for the role of RNA, Bekhor et al. (1969a) treated chromatin with RNase; when this was used as a template, they showed that the RNase treatment had abolished specificity of transcription. The main problem in this kind of experiment is the complete removal of RNase; in their paper Bekhor et al. (1969a) merely stated that the chromatin was “relatively free of RNase.” Huang and Huang (1969) performed a similar kind of experiment; they attempted to degrade RNA by treating chromatin with zinc nitrate. Again they found that this destroyed the ability of the chromatin to reassemble accurately. Again the question which arises is whether treatment with zinc nitrate is sufficiently specific to enable the conclusion to be drawn that RNA is an essential component of the reassembly reaction. The results of other workers have tended to favor the protein components of the nonhistone fractions as being involved in controlling DNA template activity. Wang (1968~)demonstrated that a fraction of rat liver nonhistone proteins would reverse the inhibitory effect of histones on the rate of transcription. The addition of nonhistone proteins to chromatin increased the template activity, so that the RNA synthesized saturated DNA a t twice the level obtained with RNA from untreated chromatin (Wang, 1970). Kamiyama and Wang (1971) showed that a similar effect occurred when nonhistone proteins were added in vitro to heterochromatin. They reported that the new RNA so produced differed in base composition and coded for proteins which were different from that produced by the untreated chromatin. As this RNA also differed in analysis from that of controls, it appears that this form of activation of chromatin opens up new sites for transcription. Spelsberg and Hnilica (1969b) reported that acidic nuclear proteins, including phosphoproteins, would only prevent histones from inhibiting DNA template activity if the protein fractions were allowed to react before combing with the DNA. In a similar series of experiments C-S. Teng and Hamilton (1969) found that in order to restore histone inhibited template activity of endometrial chromatin, nuclear acidic or chromatin nonhistone proteins had to be added to the synthesis mixture before histones. Thus in some of these experiments the order of addition of components to the reaction mixture is important.

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Other findings which implicate nonhistone proteins in regulation of chromosomal function are concerned with evidence for differential synthesis and differential binding to DNA. c-S. Teng and Hamilton (1970) observed that estrogen stimulated the synthesis of nonhistone proteins in uteri, but not in livers. As judged by electrophoresis in SDS-acrylamide gels, this synthesis was largely confined to one species of protein. Similarly Shelton and Allfrey (1970) found that cortisol stimulated the synthesis of a protein of molecular weight 41,000 in rat liver chromatin. Berendes (1968) also reported that, during puff formation Drosophila larvae brought about by ecdysone or temperature shock, acidic proteins accumulate prior to the onset of RNA synthesis. Nonhistone proteins prepared from coconut chromatin have been found to contain activities which resemble the factors which control bacterial RNA polymerase (Mondal et al., 1970). The demonstration that repressor proteins can bind specifically to DNA of bacterial systems opens up the possibility that such a technique might be useful in identifying which of the chromatin nonhistone proteins control the mammalian template. Thus Kleinsmith et al. (1970) showcd that a small proportion of such proteins (prepared according to Wang’s procedure) would bind to DNA in a species specific manner. Likewise, C. T. Teng et al. (1970) found that chromatin phosphoproteins would similarly bind to DNA, tissue specificity being demonstrated by differences in the electrophoretic and 32P-labeling patterns of the proteins. Although it remains to be seen whether these proteins affect transcription in any way, the concept of small amounts of the nonhistone proteins binding specifically to DNA is an attractive one. In summary, the nonhistone fraction of chromatin appears to be biologically complex, since its components not only can restrict and promote transcription but also can determine organ specificity of the template. Evidence has been presented above that both the nonhistone proteins and chromosomal RNA may be responsible for this. It is by no means clear whether either of these is uniquely responsible for opposing the inhibitory effects of histones, and much further experimentation is needed. 4. Chemical Modification of Chromosomal Proteins

At present we must assume that the binding of chromatin proteins to DNA is associated with the function they perform, whether it be a structural or regulatory role. The observation that some of the chromatin proteins posscsscd modificd amino acids, e.g., methyllysine (Murray, 1964), and the finding that in some systems such groups were metabolically unstable (Kleinsmith et al., 1966a) have led to a series of investigations to determine whether such changes could affect

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the association of protein with DNA and so provide a mechanism for releasing areas of DNA unavailable for transcription. For example, it might be possible for derepression to take place by removal of histones from DNA as a result of the introduction of groups to the peptide chain which weakened the ionic links between DNA and protein. Modifications to be described here are acetylation, phosphorylation, methylation, and the reversible oxidation of thiol groups. Recently, ethylation and acylation of nuclear proteins have also been reported (Orenstein and Marsh, 1968; Friedman et al., 1969) ; Ramponi and Grisolia, 1970). a. Acetylation. Acetyl groups occur in histones as a result of two processes. The N-terminal acetyl groups of histone fractions F1 and F2a1,2 are incorporated into the protein a t synthesis (Liew et al., 1970; Marzluff and McCarty, 1970). These groups are apparently stable, unlike the metabolically unstable acetyls which are enzymatically added to 90% tumor cells) will provide a stronger test of the hypothesis that genetically determined tumors have a multiple cell origin.

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2. Multiple Neurofibromatosis Neurofibromatosis (von Recklinghausen’s disease) is characterized by numerous yellow-brown cutaneous macules (caf6-au-lait spots), multiple tumors (neurofibromas), and an autosomal dominant pattern of inheritance. Typically the neurofibroma consists of spindle-shaped cells resembling fibroblasts in a matrix of either ground substance or fibrillar material similar to loose connective tissue. Special staining may reveal the presence of small numbers of nerve fibrils and mast cells, but most cells are spindle-shaped. Fialkow et al. (1971d) studied tumors and the overlying skin from seven noncontiguous sites in each of two unrelated G-6-PD heterozytotes. Double enzyme phenotypes were found in all 14 tumors, and in each instance the phenotype of the tumor was identical to that of the overlying skin. Contamination with normal cells as a cause for the double enzyme phenotype was excluded histologically. The number of nuclear chromatin positive cells in the tumors and the overlying dermis was similar, making it unlikely that two X-chromosomes were active in the tumor cells. Furthermore, if both X chromosonics were active in a single cell, the electrophoretic band intermediate in migration between A and B enzyme (presumably a hybrid molecule of A and B subunits) might have been observed. Thus, it seems likely that the hereditary neurofibroma has a multiple cell origin. The number of cells giving rise to the neurofibroma was estimated to be a t least 150 and quite probably several thousand (Fialkow et al., 1971d). Multiple cell origin involving this number of cells could come about in a t least two ways: ( 1 ) a relatively large number of cells may be simultaneously affected by the tumorigenic process, as, for example, might occur under the influence of a hormone; or ( 2 ) the oncogenic mechanism might initially alter only a single cell with subsequent influence on the pattern of growth in neighboring cells.

E. OTHER BENIGNTUMORS Single enzyme phenotypes were found in a lipoma (Linder and Gartler, 1965b), in an adenoma arising in an ectopic salivary gland, and in a colloid adenoma of the thyroid (Fialkow et al., 1971b) and in single thyroid nodules from three patients (Linder, 1969). Implications of these findings are discussed in Section VI1,A. A double enzyme phenotype was reported in a granular cell myoblastoma of the tongue (Linder, 1969).

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VI. Study of Genetic Markers in Established Tissue Culture lines

A. HETEROPLOID EPITHELIAL-TYPE CELLLINES

G-6-PD variants also prove useful as cell markers in tissue cultures derived from tumors. Gartler (1967, 1968) used the G-6-PD and phosphoglucomutasc-1 (PGM,) systems in a study of 18 established heteroploid human cell lines. All of these presumably different human cell lines, some derived from Caucasians, had PGM, 1 and G-6-PD A phenotypes. Type A G-6-PD has thus far becn observed only in Negroes. The HeLa cell line was derived from a carcinoma of the cervix arising in a Negro, and it was the first reported established human cell line. Thus, it was suggested that the 18 human ccll lines had become contaminated with HeLa cells. It is unlikely that the 18 unrelated subjects assumed to have given rise to these cell lines all had PGM, 1 phenotypes. However, the contamination hypothesis has becn debated by other workers, who reported finding differences between the G-6-PD types of some of these cell lines and that of HeLa, and between HcLa G-6-PD and type A or A- (Hathaway and La Rock, 1969; Rattazzi and Siniscalco, 1969; Steele, 1970). The question can be definitively resolved by more refined examination of the G-6-PD proteins and by employing a larger number of genetic markers.

B. BURKITT LYMPHOBLASTOID CELL LINES Fialkow et al. (1971e) used G-6-PD and PGM, enzyme systems as cell markers in a study of 19 tissue cultures derived from Burkitt tumors. The G-6-PD and/or PGM, phcnotypes in six of the 19 cultures were discordant with those found previously in the tumors from which the cultures were derived. Presumably, if additional markers with similarly favorabIe gene frequencies were utilized, w e n more discordant cultures would be found. I n searching for an explanation of these changes in phenotype, three general possibilities were considered: laboratory contamination, in vitro genetic accidents, and derivation of the cultures from foreign cells already present in the host’s tumor at the time of sampling. Laboratory contamination of this magnitude could not be entirely excluded, but if the discordant phenotypes resulted from in vitro contamination, at least three contaminating lines would have to be involved. However, during the two years of study only two cultures were grown in large amounts and for long periods of time in the same laboratory. These lines had the same G-6-PD and PGM, phenotypes (B and 1, respectively). Therefore, contamination by them could account for only one of the three putative

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contaminating lines. Similarly, an in vitro genetic accident, such as somatic mutation, seemed improbable since several of the discrepancies between tumor and culture involved both of the markers and simultaneous mutation a t two loci would be an extremely unusual event. A more interesting possibility was favored, viz., that the lymphocyte progenitors giving rise to the tissue culture with a discordant phenotype were present in the tumor in vivo but were genetically foreign to the host (i.e., they originated in another human subject). Precedent for this type of reasoning was recently provided by Manolov et al. (1970), who reported finding that the cells of a Burkitt tumor and the cells of cultures derived from that tumor had typical female sex chromosomes even though the tumor arose in a phenotypic male. Unfortunately, only two 46,XY cells were found in the bone marrow, but barring the unlikely possibility that this boy had a preexisting chromosomal abnormality such as 47,XXY or 46,XY/46,XX mosaicism, the only possible source for the female cells would be another human subject. The means by which the genetically foreign cells gain access to the host with Burkitt lymphoma is unknown. Manolov et al. (1970) suggested that the female cells in the tumor of their male patient might have been introduced by transplacental passage from his mother or by transmission from a mosquito, a putative vector of Burkitt lymphoma. The former possibility was rendered very unlikely in their case because different histocompatibility allotypes were found in the patient’s tumor cells and the cells of his mother (Bremberg and Klein, 1971). Another potential source of genetically foreign cells is transfused blood which is often given to patients with Burkitt lymphoma. I n the study reported by Fialkow et al. (1971e), three of the six discordant cultures were derived from patients who were transfused within 4 weeks of the biopsy. Only one patient with a discordant culture had no transfusion history. I n contrast, 7 of 13 concordant cultures were obtained from patients who had apparently never been transfused, and only 2 of the 13 received a transfusion within 4 weeks before the biopsy. The short-term persistence of donor lymphocytes in recipients with Burkitt tumors might be favored by the patients’ reduced immunological responsiveness (Stjernsward et al., 1970) and by prior reduction of the hosts’ lymphocyte pools (Doenhoeff et al., 1970) due to chemotherapy or irradiation. Nonetheless, the single untransfused patient from whom discordant cultures were derived and the untreated boy reported by Manolov et al. (1970) must have acquired their foreign cells via another mode of transmission, conceivably the bite of an arthropod. The observations reported by Nadkarni and associates (1969) could also be interpreted as evidence for the presence of foreign cells in subjects

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with Burkitt lymphoma, since they found morphological alterations in tumor cells after chemotherapy. Such changes could reflect a change in cell line. Furthermore, these workers also detected an unusual cellular trait, IgM surface reactivity, in three biopsies of one tumor and the tissue cultures derived from the biopsy cells. In contrast, a fourth biopsy and culture taken after the patient had received cytosine arabinoside and blood transfusions exhibited little or no IgM specificity. Moreover, the chromosomal constitution of this cell line was different from the one found in a reactive line taken before chemotherapy. More recently it was shown that the first culture (IgM-reactive) and fourth culture (nonreactive) had different G-6-PD phenotypes. This demonstrates that one of these two cultures (presumably the nonreactive one) was derived from cells genetically foreign to the host. I n the series reported by Fialkow et al. (1971e), all 5 patients with discordant cultures had active disease a t the time of biopsy and three were receiving chemotherapy. Among the possible explanations for the growth of foreign cells in the established lymphoblastoid lines are that these cells became “transformed” by allogenic or tumor-associated antigenic stimulation, or they were “transformed” into malignant cells within the host tumor. Fialkow et al. (1971f) used chromosomal markers to demonstrate a similar phenomenon in a patient with another lymphoblastic disease, acute lymphoblastic leukemia. This patient, a 16-year-old girl, received 1000 rad of whole-body irradiation followed in 1 day by a marrow graft from her HL-A matched brother. When recurrent leukemia was evident about 60 days later, cytogenetic studies showed that the donor cells were now leukemic. I n summary, determinations of genetic markers in lymphoblastoid cell lines derived from Burkitt tumors indicate, a t the very least, that caution must be exerted in the interpretation of studies based on the assumption that these cells are genetically identical to the hosts’ cells. More importantly, the cell line investigations may lead to studies which could provide important clues about the processes involved in the establishment of cell cultures and perhaps about the development of tumors

in vivo. VII. Concluding Remarks

A. IMPLICATIONS OF CLONAL VERSUS MULTICELLULAR ORIGIN

The data already reported have demonstrated that applying the G-6-PD system to the study of tumors can provide important insight into the initiation and progression of neoplasms (e.g., stem cell origin of CML). The difference between uni- and muticellular origin is likely to

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be based on the nature of the various factors responsible for the initiation and progression of the tumors, and on the number of susceptible target cells. Thus far, the majority of neoplasms studied seem to have a clonal origin. This would be expected if tumor initiation is dependent upon a rare, more or less random event, such as somatic mutation. C M L may arise in this way. However, the fact that the cells in a fully developed neoplasm have a clonal origin does not necessarily imply that the event which started the tumor occurred in only one cell. A single enzyme phenotype would also be observed if the “progression” of the tumor prior to the time of testing were dependent upon a number of steps, at least one of which was random. A clonal origin may also occur under other circumstances. For example, even if a whole organ were exposed to the oncogenic environment, as might occur with a virus or endocrine alteration, only one or a very few cells in the target tissue might be susceptible. Susceptibility could depend upon the cell’s genotype, age, state of differentiation, cytoplasmic environment, or some other factor. Thus, although the development of thyroid adenomas may be under hormonal influence, these neoplasms appear to have a unicellular origin. Similarly, Burkitt lymphoma and verruca vulgaris, the two presumably virally induced neoplasms thus far examined, apparently have a clonal origin. Presumably, then, development of these tumors does not depend on spread of the virus from cell to cell. However, there could be virions in the tumor cells which replicate with the host’s DNA and are passed on to daughter cells during mitosis. It may be further speculated that either the virus induces the tumorigenic change in a single cell or the presence of the virus is only one of two or more factors required for tumor development. Multiple cell origin implies that either the initial tumorigenic event occurs in only a single cell and that this alteration subsequently influences the pattern of growth in neighboring cells, or that tumor initiation as well as all the steps in tumor progression involve more than one cell. This type of origin might occur if the target tissue for the oncogenic factors contained a large number of susceptible cells. Inherited tumors such as multiple trichoepithelioma and neurofibroma are examples of tumors with multicellular origin; all cells in the tissue of origin have an innate tumorigenic potential. On the other hand, the nature of the oncogenic agent itself may be the primary factor in determining the number of cells affected. Thus, tumors induced by highly infectious viruses may be found to have a multiple cell origin. However, the fact that most tumors thus far studied in man appear to have a unicellular origin suggests that the development of human tumors is not generally dependent upon cell-to-cell spread of infectious agents. Hormones could

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also bring about the development of tumors from more than a single cell. Of possible relevance is the fact that all of the three reported carcinomas of the breast had double enzyme phenotypes compatible with multiple cell origin. Information of this type about more cases of breast carcinomas and other endocrine-influenced tumors would be of considerable interest.

B. PROSPECTS FOR FUTURE STUDIES With the exception of genetically determined tumors, warts, and possibly Burkitt lymphoma, the etiologies of human tumors studied with G-6-PD are unknown. If X-linked genetic marker studies could be done in tumors with known etiology, thc results might have implications for neoplasms with unknown cause. Unfortunately, this approach is limited by the small number of human neoplasms for which an etiologic agent has bten defined, and by the present inability to detect markers a t the cellular level for frequently occurring X-linked variant alleles other than G-6-PD. Until more markers are discovered, continued study of human tumors can generally be done only in black populations. These studies might best be directed toward tumors which originate in defined circumstances, such as neoplasms associated with radiation exposure, immunologic abnormalities, inherited gene mutations (e.g., multiple polyposis of the colon), organic chemicals, and hormonal changes. Another potentially profitable approach is to study induced tumors in lower organisms. Once suitable X-linked markers are discovered in these animals, problems such as selection can be definitively explored. It is to be hoped that this information will have bearing on the causes and, utimately, the therapy and prevention of human malignancies. ACKNOWLEDGMENTS I wish to thank the many colleagues who assisted me in various aspects of the preparation of this manuscript. Special acknowledgment is made to Drs. E. R. Giblett and G. Klein for their critical reviews of the paper. The author’s studies were supported by Grant No. GM 15253 from the National Institutes of Health of the U. S. Public Health Service.

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Gartler, S. M. (1967).Nat. Cancer Inst., Monogr. 26, 187-195. Gartler, S.M. (1968).Nature (London) 217, 750-751. Gartler, S. M., and Linder, D. (1964). Cold Spring Harbor Symp. Quant. Biol. 24, 253-260. Gartler, S. M., Ziprkowski, L., Krakowski, A., Ezra, H., Szeinberg, A., and Adam, A. (1966). Amer. J. Hum. Genet. 18, 282-287. Gorman, J. G., Di Re, J., Treacy, A. M., and Cahan, A. (1963).J. Lab. Clin. Med. 61, 642-649. GunvBn, P., Klein, G., Henle, G., Henle, W., and Clifford, P. (1970). Nature (London) 228, 1053-1056. Hathaway, P., and La Rock, J. F. (1969). Abstr. Amer. SOC.Hum. Genet. Meet., 1969 p. 25. Hellstrom, I., Hellstrom, K. E., Pierce, G. E., and Bill, A. H. (1968).Proc. Nat. Acad. Sci. U.S. 60, 1231-1238. Kelley, W. N., Greene, M. L., Rosenbloom, R. M., Henderson, J. F., and Seegmiller, J. E. (1969).Ann. Intern. Med. 70, 155-206. Klein, E., Klein, G., Nadkami, J. S., Nadkarni, J. J., Wigzell, H., and Clifford, P. (1968). Cancer Res. 28, 1300-1310. Klinger, H. P., Davis, J., Goldhuber, P., and Ditta, T. (1968). Cytogenetics 7, 39-59. Lawler, S . D.,and Sanger, R. (1970).Lancet 1, 584-585. Lee, G . R., MacDiarmid, W. D., Cartwright, G. E., and Wintrobe, M. M. (1968). Blood 32, 59-70. Linder, D. (1969).Proc. Nat. Acad. Sci. U . S. 63, 699-704. Linder, D., and Gartler, S. M. (1965a).Science 150, 67-69. Linder, D., and Gartler, S. M. (1965b).Amer. J. Hum. Genet. 17, 212-220. Linder, D., and Gartler, S. M. (1967). Proc. Berkeley Symp. Math. Statist. Probability, 6th, 1966 pp. 625-633. Linder, D., and Power, J . (1970).Ann. Hum. Genet. 32, 21-30. Lyon, M. F. (1968).Annu. Rev. Genet. 2, 31-52. McCurdy, P. R. (1968).In “Hereditary Disorders of Erythrocyte Metabolism” (E. Beutler, ed.), City of Hope Symp. Ser., Vol. I, pp. 121-125. Grune & Stratton, New York. Manolov, G., Levan, A., Nadkarni, J. S., Nadkarni, J., and Clifford, P. (1970). Hereditas 66, 79-100. Mirtensson, L. (1963). Lancet 1, 946-947. Mintz, B.,and Sle.mmer, G. (1969).J. Nat. Cancer Inst. 43, 87-95. Morton, D. L., Malmgren, R. A., Holmes, E. C., and Ketchan, A. S. (1968). Surgery 64, 233-240. Motulsky, A. G. (1960). Hum. Biol. 32, 28-62. Murray, R. F., Jr., Hobbs, J., and Payne, B. (1971).Nature (London) 232, 51-52. Nadkarni, J. S., Nadkarni, J. J., Clifford, P., Manolov, G., Fenyo, E. M., and Klein, E. (1969).Cancer 23, 64-79. Nance, W. E . (1964). Cold Spring Harbor Symp. Quant. Biol. 29, 415-425. howell, P. C., and Hungerford, D. A. (1960).Science 132, 1497. Nowell, P. C., and Hungerford, D. A. (1961).J. Nat. Cancer Znst. 27, 1013-1035. Nyhan, W. L.,Bakay, B., Connor, J. D., Marks, J. F., and Kelle, D. K. (1970). Proc. Nat. Acad. Sci. U . S. 65, 214-218. Old, L..J., Boyse, E. A., Oettgen, H. F., de Harven, E., Geering, G., Williamson, B., and Clifford, P. (1966). Proc. Nat. Acad. Sci. U . S. 56, 1699-1704.

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Oni, S. B., Osunkoya, B. O., and Luzaatto, L. (1970).Blood 36, 145-152. Park, I. J., and Jones, H. W., Jr. (1968).Amer. J. Obstet. Gynecol. 102, 106-109. Penis, B., and Chiappino, G. (1964).Immunology 7, 500-506. Polani, P. E.,Angell, R., Giannelli, F., de la Chapelle, A., Race, R. R., and Sanger, R. (1970).Nature (London) 227, 613-616. Rastrick, J. M., Fitzgerald, P. H., and Gunz, F. W. (1968). Brit. Med. J. 1, 96-98. Rattazzi, M. C.,and Siniscalco, M. (1969). Abstr. Amer. Sac. Hum. Genet. Meet., 1969 p. 25. Richart, R. M. (1967). Clin. Obstet. Gynecol. 10, 748-784. Robbins, 5.L. (1967).“Pathology,” 3rd ed. Saunders, Philadelphia, Pennsylvania. Sandberg, A. A., Ishihara, T., Crosswhite, L. H., and Hauschka, T. S. (1962). Blood 20, 393-423. Sigman, B., and Gartler, S. M. (1966).Humangenetilc 2, 372-377. Silagi, S., Darlington, G., and Bruce, S. (1969). Proc. Nut. Acad. Sci. U. S. 62, 1085-1092. Smith, J. W., Townsend, D. E., and Sparkes, R. S. (1971a).Clin. Genet. 2, 160-163. Smith, J. W., Townsend, D. E., and Sparkes, R. S. (1971b).Cancer 28, 52S537. Stamatoyannopoulos, G. (1971). Unpublished observations. Stamatoyannopoulos, G., Papayannopoulou, T., Bacopoulos, C., and Motulsky, A. G. (1967). Blood 29, 87-101. Steele, M . (1970). Biockem. Genet. 4, 25-40. Stjernswiird, J., Clifford, P., and Svedmyr, E. (1970). In “Burkitt’s Lymphoma” (D. Burkitt and D. Wright, eds.), pp. 164-171. Livingstone, Edinburgh. Straub, D. G., Lucas, L. A., McMahon, N. J., Pellett, 0. L., and Teplitz, R . L. (1969). Cancer Res. 29, 1233-1243. Tough, I. M., Court Brown, W. M., Baikie, A. G., Buckton, K. E., Harnden, D. G., Jacobs, P. A., King, M. J., and McBride, J. A. (1961). Lancet 1, 411-417. Tough, I. M., Jacobs, P. A., Court Brown, W. M., Raikie, A. G., and Williamson, E.R.D. (1963).Lancet 1, 844-4346, Townsend, D. E., Sparkes, R. S., Balude, M. C., and McClelland, G. (1970). Amer. J . Obstet. Gynecol. 107, 1168-1173. Trujillo, J. M., and Ohno, S. (1964).Presented at Int. Cong. Znt. Sac. Hematol., 9th, 1962, Mexico City, Mexico. Wegmann, T. E. (1970).Nature (London) 225, 462463. Weitkamp, L. R., Guttormsen, S. A., Shreffler, D. C., Sing, C. F., and Napier, J. A. (1970a).Amer. J. Hum. Genet. 22, 216220. Weitkamp, L. R., Guttormsen, S. A., and Greendyke, R. M. (1970b). Clin. Bes. 18, 396. Whang, J., Frie, 111, E., Tjio, J. H., Carbone, P. P., and Brecher, G. (1963). Blood 22, 664-673. Yoshida, A. (1967).Proc. Nut. Acad. Sci. U.8.57, 831-833. Yoshida, A., Stamatoyannopoulos, G., and Motulsky, A. G. (1967a). Science 155, 97-99. Yoshida, A., Steinmann, L., and Harbert, P. (1967b). Nature (London) 216, 275-276. zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, W., Henle, G., Clifford, P., and Santesson, L. (1970).Nature (London) 228, 1056-1068.

ELECTRON SPIN RESONANCE STUDIES OF CARCINOGENESIS Harold M. Swartz Departments of Radiology and Biochemistry, The Medical College of Wisconsin, Milwaukee, Wisconsin

I. Introduction . . . . . . . . . . . . . . 11. The ESR Technique . . . . . . . . . . . . A. Theoretical Basis . . . . . . . . . . . . B. Experimental Aspects of Importance to ESR-Carcinogcnesis Studies 111. Experimental Results . . . . . . . . . . . . A. Experimental Problems and Approaches . . . . . . B. Free Radicals and Carcinogenesis . . . . . . . . C. ESR Spectra of Malignant Tissues . . . . . . . . D. Dynamic Studies . . . . . . . . . . . . IV. Summary and Conclusions . . . . . . . . . . . References . . . . . . . . . . . . . .

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227 228 228 231 233 233 234 239 243 248 248

I. Introduction

Interest in free radicals as possible carcinogens arose from several lines of related speculations. Free radicals are usually quite chemically reactive, especially with SH groups and also with nucleic acids, and so might be logical initiators of chemical changes that result in malignant behavior of cells. Many chemical carcinogens readily form free radicals, some with unusual stability. Some physical carcinogenic processes, most notably ionizing radiation, have free radicals as important intermediates. It is not surprising then, that soon after the development of electron spin resonance (ESR) spectroscopy (a technique that permits sensitive detection and characterization of paramagnetic species including free radicals), this technique was applied to the study of carcinogenesis. The purpose of this review is to summarize the current status of ESR studies of carcinogenesis. Although ESR-carcinogenesis studies were initiated to study free radicals, such studies inevitably also provided information on other types of paramagnetic species. Changes in paramagnetic trace elements have turned out to be as prominent and interesting as free radical changes and therefore many studies have emphasized this type of unpaired electron species rather than free radicals. The free radical changes themselves are quite varied and complicated 227

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and do not closely correspond to the types of changes anticipated in the theoretical speculations that led to ESR studies of carcinogenesis. Thus much of the current literature covers experimental problems that Were not considered prior to the first ESR-carcinogenesis studies. To date such ESR-carcinogenesis studies have probably raised more questions than they have answered and their value has not been settled. An understanding of the literature and some of the important experimental problems requires an understanding of the ESR technique itself. Some elements of ESR spectroscopy are presented in the following section. More detailed considerations, also directed toward the background and intcrests of the biologically oriented investigator, are available in two recent textbooks (Ingram, 1969; Swartz et al., 1972). II. The ESR Technique

A. THEORETICAL BASIS 1. Magnetic Properties Associated with Electron Spin Electron spin resonance (ESR) spectroscopy is based on the magnetic properties of the electron associated with its spin and charge. The spin of an electron can be considered to be a motion like the spinning of the earth on its axis, Electron spin, however, is subject to quantum mechanical restrictions such that only two spin directions, each of the same magnitude, are possible. This restriction, combined with the fact that a moving (or spinning) charge generates a magnetic field, means that electrons act as magnets (the electronic magnetic strength and direction is described as its magnetic moment) and these magnets can have one of two orientations. ESR spectroscopy is based on inducing and detecting transitions between two spin orientations. Most molecules have all their electrons paired, because this is a more stable state. Two electrons form a pair when they are identical except for having oppositc spins. Paired electrons cannot change their spin states because this would violate the Pauli principle which states that no two electrons in a system can be identical. Thus most molecules cannot be detected by ESR spectroscopy.

2. Nature of Unpaired Electron Species

If a molecule has an unpaired electron, it is termed a free radical. Free radicals are usually quite reactive and always have a net magnetic moment and so are detectable by electron spin resonance. Figure 1 illustrates the usual dissociation of water into paired electron products

ESR STUDIES O F CARCINOGENESIS A

.. ..

H:@H -:o:H

0

+H

229 0

FIG. 1. Dissociation of water into (A) usual ionic components and (B) free radical components. The eight (four pairs) “valence” electrons are indicated. Reaction A is spontaneous, Reaction B requires several electron volts of external energy and is seen most typically after exposure to ionizing radiation. The small open circle used for the products of reaction B is the usual symbol indicating that a molecule or fragment contains an unpaired electron and is, therefore, a free radical.

(1A) and the corresponding dissociation into unpaired electron species or free radicals (1B) ; hydroxyl radicals and hydrogen atoms are much more reactive than hydroxyl ions and protons. Most, but not all free radicals are neutral. If an ionic species has an unpaired electron it is also a free radical, either an anion radical or a cation radical. The other type of unpaired electron species discussed in this review is that associated with certain states of transition elements such as iron, copper, manganese, molybdenum, cobalt, and vanadium. The unpaired electron in this case is usually from an incompletely filled d shell and there is usually considerable interaction with the other electrons in the atom and surrounding molecules. The result is an ESR spectrum that may be quite complicated and whose shape is sensitive to the environment the paramagnetic atom is in; this environment is usually a macromolecular complex. (The term paramagnetic can be applied to any species with an unpaired electron, it is most commonly used for inorganic atoms with unpaired electrons.) 3. E S R Spectroscopy The ESR technique takes advantage of the net magnetic moment associated with unpaired electrons. If a species with an unpaired electron is placed in a magnetic field it will become oriented by the field, just as any small bar magnet will be affected by a nearby large magnet. However, as discussed above, the possible spin states and hence the magnetic moments of the unpaired electrons are restricted and so each unpaired electron can assume only one of two orientations. Thc two possible orientations with respect to the applied magnetic field differ slightly in energy. The larger the external magnetic field, the greater the energy difference, but even a t the relatively high magnetic fields ordinarily employed experimentally (about 3000 G) this difference is still only fractions of an electron volt, and thus ESR spectroscopy rarely, if ever, causes chemical changes. If electromagnetic energy is applied that is

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just equal to the energy difference between the two possible orientations of the unpaired electrons, some of the unpaired electrons may absorb this energy (“resonant” energy) and go to the higher energy state. So in a given magnetic field (H), there will be an energy ( h v ) that will be resonantly absorbed by the unpaired electrons. The energy of the absorption (hv) depends on the magnetic moment (p, a constant that indicates what “size” magnet an electron is) associated with all unpaired electrons and on a constant ( 9 ) that is characteristic of the particular species of unpaired electrons. The basic equation then, for resonant absorption of energy by an unpaired electron in a magnetic field is:

hv

=

gpH

Experimentally, it is simpler to vary the magnetic field ( H ) than the frequency ( h v ) , and so a constant frequency is used (most typically in the X-band microwave range, with v ~ 9 , 2 0 0 , 0 0 0 , 0 0 0cycles per second). As the magnetic field is increased the resonant condition will be met and then passed, so if the absorption of the microwave is monitored, the presence and g-factor of unpaired electron species can be determined. I n addition, some information on the environment of the unpaired electron can be obtained. I n the case of transition elements, the g-factor and shape of the observed absorption line may be affected by its surrounding atoms. I n free radicals, the lines may be further split up by the phenomenon of hyperfine structure. This is due to the fact that some nuclei (principally hydrogen and nitrogen in compounds of biological interest) also have charge and spin and so have net magnetic moments. The nuclear magnetic moments are smaller than electron magnetic moments so they do not absorb the exciting microwave radiation, but they may modify the magnetic field that the unpaired electron experiences. For example, if an unpaired electron is located near a hydrogen nucleus, the magnetic moment of the hydrogen nucleus will either line up with or against the external magnetic field. If it adds to the external field, then the unpaired electron will experience a slightly greater total magnetic field, and it will undergo resonance absorption a t a slightly lower value of the external magnetic field than it would have had there been no hydrogen nucleus present. Similarly, an unpaired electron located near a hydrogen nucleus opposing the external field will have its point of resonance absorption occur a t higher values of the external field. The overall result is the splitting of the absorption line into two lines, occurring above and below the position where absorption would have occurred in the absence of the hydrogen nucleus (Fig. 2 ) . The number of lines and the magnitude of the splitting depends on the number and type of magnetic nuclei that the unpaired electron interacts

231

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:?

Applied magnetic field

2990

3000

3010

Magnetic field due to nearby nucleus

10

0

-10

3000

3000

Total magnetic field

3000

FIG.2. Effect of a magnetic nucleus on an electron spin resonance (ESR) spectrum. Schematic ESR absorption spectrum of an unpaired electron in which the center of resonance occurs at 3000G. Spectrum A (dashed line) indicates the spectrum seen in the absence of an adjacent magnetic nucleus. Spectrum B shows the effect of a nearby hypothetical nucleus with a spin of 1/2 which generates a local field equivalent to 10G at the unpaired electron. Depending on the orientation of the nucleus, its field of 10G will add to or subtract from the applied field changing the net magnetic field the electron experiences. This type of splitting of an ESR absorption line is termed hyperfine splitting. with. Thus, in favorable cases, considerable information can be obtained on the molecular environment of the unpaired electron and hence on the structure of the free radical.

B. EXPERIMENTAL ASPECTSOF IMPORTANCE TO ESR-CARCINOGENESIS STUDIJCS 1. Discrimination between Free Radicals and

Paramagnetic Trace Elements Selective experimental observation of either free radicals or paramagnetic transition elements in tissue is possible by varying the intensity of the microwave field (Swartz and Molenda, 1965; Swartz, 1972). This fact was not fully realized in many early carcinogenesis studies and some recent studies, resulting in confusion as to whether observed ESR changes represented changes in free radicals or trace elements. When it is not clear whether a particular ESR signal arises from a free radical or another type of paramagnetic entity such as a paramagnetic trace element, a nonspecific term such as “ESR signal” or “unpaired electron” is frequently employed, especially in this review. I n reading the literature, the reader should bear in mind that some authors (incorrectly) may use the term free radical in this same, nonspecific sense. Experimental detection of unpaired electrons depends on there being more unpaired electrons in the lower energy level so that they can absorb the microwave radiation in being raised to the higher energy state. If

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the excited electrons do not promptly return to the lower energy level, energy absorption can no longer occur and so the apparent intensity of the ESR signal will diminish. This phenomenon is termed power saturation and it occurs quite readily with free radicals in tissues. Unpaired electrons associated with transition metal ions, on the other hand, do not power saturate very readily and so, although their concentration is generally less than that of free radicals, they will dominate high-power, about 200 milliwatts (mW.), ESR spectra of tissue. The free radicals can be observed by using relatively low microwave power (1mW. or less) ; this intensity will still detect free radicals, but the concentration of transition elements is often so low that they are not observed a t this low microwave power. (In the absence of power saturation, the intensity of the ESR signal is proportional to the square root of the microwave power, so that a reduction of power from 200 mW. to 1 mW. decreases the signal by a factor of about 14.) Free radicals can usually also be differentiated from paramagnetic trace elements on the basis of their g-factor. Most organic free radicals have g-factors quite close to that of the free electron, about g = 2.003. If there is a significant unpaired electron density on an oxygen or sulfur atom this value may shift as high as g = 2.06. Paramagnetic trace elements, on the other hand, have g-factors ranging from less than 1.9 to greater than 4.0, depending on the individual element. This range includes the g = 2.00 area or “free radical area” so the presence of a peak near g = 2.00 is not conclusive evidence of the presence of a free radical. Careful power saturation studies, however, will usually permit the differentiation of signals in the g = 2.00 area due to overlapping ESR peaks from trace elements and free radicals. 2. Preparation Techniques and the Problem of Lyophilization

The method of sample preparation can have a considerable effect on the ESR spectrum. The ideal is to study the ESR spectrum as it occurs in living, functional tissue but each method of sample preparation that is currently available requires some compromise of this ideal. Observation of wet “surviving” tissues in the form of thin slices can be accomplished, but the sensitivity for such preparations is low, the tissues rapidly become anoxic and the spectrum is liable to change during the prolonged observation period required to get a sufficiently large signal output (Swartz, 1972). An acceptable alternative is to quickly freeze the sample to very low temperatures. This method gives greater sensitivity and stability without appreciably altering the ESR spectrum, providing corrections are made for temperature and saturation effects (Swartz et al., 1970; Kent and Mallard, 1969). A very poor alternative

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is the use of lyophilized preparations. Heckley (1972) has recently reviewed in detail the problems associated with this technique. Briefly, the problem is that lyophilized samples react very rapidly with trace amounts of oxygen to generate new g = 2.00 signals that are larger and unrelated to those originally in the samples. The original g = 2.00 signal may also be reduced or eliminated by the lyophilization process and other, nonfree radicals may also be affected. The use of lyophilized samples therefore leads to the study of changes in the factors that lead to generation of the oxygen dependent artifact and do not reflect changes in cellular free radicals. Unfortunately many very critical studies of the role of free radicals in carcinogenesis have been done with lyophilized samples. The interpretation of such studies is very difficult. Usually the authors thought that they were studying tissue free radicals, but the observed signals actually depended on factors that they were not aware of, and so may not have controlled. These factors include exposure to oxygen, exposure to moisture, and the physical state of the sample preparation. I n spite of the fact that we now know that the ESR spectra seen in lyophilized tissues do not reflect unpaired electron species existing prior to lyophilization, if all samples are treated identically they can reflect true differences in the capacity of samples to interact with oxygen to form free radicals. This capacity may be related to significant changes in the tissues. With this possibility in mind, and because some potentially valuable concepts have been tested using lyophilized preparations, some experimental results using these preparations will be discussed in this review. In summary, ESR is a technique that specifically and sensitively detects unpaired electron species ; for carcinogenesis studies these are principally free radicals and paramagnetic transition elements. ESR can determine the number of unpaired electron species present and, in favorable cases, the chemical nature of the molecule in which they are located. Ill. Experimental Results

A. EXPERIMENTAL PROBLEMS AND APPROACHES The initial ESR studies of carcinogenesis were inspired by theoretical speculations on the carcinogenic potential of free radicals (e.g., Commoner et al., 1957, Brues and Barron, 1951). The first ESR studies of tissues revealed that instead of the increased number of free radicals that might have been anticipated from these theoretical considerations, most neoplastic tissues had decreased free radical concentrations (Commoner et al., 1954). However, many neoplasms also have different, and

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in some cases increased, numbers of paramagnetic transition elements (e.g., Nebert and Mason, 1963). Later studies indicated that there were some ESR signals that were detectable during the process of carcinogenesis before any histological evidence of carcinogenesis, but these disappeared by the time the malignant changes were evident by classical criteria (e.g., Vithayathil et al., 1965). Thus, ESR studies of carcinogenesis involve three somewhat different experimental problems: ( 1 ) free radicals as carcinogens and visa versa; (2) the ESR spectra of malignant tissues, and (3) ESR signals occurring during the process of carcinogenesis. Keeping these separate problems in mind will aid the reader of ESR carcinogenesis papers, especially in view of the fact that some of the authors may not clearly state these objectives. Although some papers touch on two or more of these topics, the topics will be considered separately in this review to emphasize the current state of knowledge in each problem area. ESR studies of carcinogens will not be covered exhaustively because of the volume of papers in this field and the fact that many of them deal with chemical generation of free radicals under conditions that have no apparent relationship to events in vivo. The latter studies may some day, contribute to our understanding of the physicochemical properties of carcinogens, but it would appear better to cover this subject in a broader review that is not limited to ESR studies. The ESR characteristics of malignant tissues will be reviewed in detail. This will be followed by a consideration of experiments dealing with ESR changes during the process of carcinogenesis and factors that modify these ESR changes. These last studies probably offer the greatest opportunity for ESR to make an important contribution to the understanding of carcinogenesis.

B. FREERADICALS AND CARCINOGENESIS 1. Carcinogens as Free Radicals

One of the prominent chemical characteristics of free radicals is their chemical reactivity. The existence of an unpaired electron in a molecule is a relatively unstable state, so such a molecule will tend to undergo reactions that lead to a pairing of electrons. This high reactivity has led a number of workers to suggest that free radicals may be involved in the carcinogenic process, which presumably involves some unusual reactions that might require unusually reactive reagents. The fact that free radicals can initiate chain reactions SO that a few molecules can cause a considerable effect has also been cited as a factor favoring their participation in carcinogenesis (Fitzhugh, 1953). Support for this type of speculation is found in the fact that some physical carcinogenic

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processes such as ionizing radiation and ultraviolet light, produce free radicals (Commoner et al., 1957; Fitzhugh, 1953) and the observations that there are large numbers of free radicals in a common carcinogen, tobacco smoke (e.g., M. J. Lyons et al., 1958). A number of studies have indicated that many carcinogens do readily form free radicals and that one can frequently correlate carcinogenicity and ease of free radical formation (e.g., Kensler et al., 1942; Park, 1950; Brues and Barron, 1951; Butler et al., 1950; Ross, 1950; Lipkin et al., 1953 ; Szent-Gyorgi et al., 1960). Free radical formation has been demonstrated in a variety of carcinogenic chemical types including polycyclic hydrocarbons, aromatic amines, and azo compounds. Several theories, some of them related, have evolved that more specifically attempt to link free radicals and chemical carcinogens. These include the postulate that the active forms of many carcinogens are free radicals and in these forms react directly with target molecules a t the site of free radical formation (Park, 1950; Syrkin, 1960; Brues and Barron, 1951); that carcinogens or their metabolic products are unusually stable free radicals and can therefore diffuse to critical sites readily (Commoner et al., 1957; Kensler et al., 1942) ; that carcinogens, in free radical forms, act via reactions with SH groups (Brues and Barron, 1951); that the free radical nature of some carcinogens is a reflection of the carcinogenic requirement of high electron mobility (Ingram, 1960), and that free radical formation is important because it leads to charge-transfer complexes which initiate the carcinogenic changes (Szent-GyGrgi, 1941 ; Szent-Gyorgi et al., 1960; Mason, 1958, 1959). Many of these theories obviously overlap, but several distinctive concepts can be separated from them and pertinent experiments designed to attempt to answer them. Is carcinogenesis associated more with very reactive short-lived free radicals or relatively stable long-lived free radicals? Is the association with carcinogenesis one of cause and effect, or do the free radical properties of carcinogens simply reflect a more fundamental requirement, such as electron mobility? If free radical reactions are the key events, are these reactions that lead directly to the critical chemical changes (e.g., reactions with DNA) or do they cause their effects more indirectly (e.g., by reacting with SH groups, and then the changes in SH groups lead to the alteration of the critical molecules)? Some of these questions have been answered in part for certain carcinogens, but unequivocal answers applicable to all or most carcinogens are not yet available and possibly never will be, because carcinogenesis is most, likely a diverse process involving different mechanisms with different carcinogens and different cell types. Suggestions

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that free radical reactions might form a common unifying concept that would explain many aspects of carcinogenesis (e.g., Boenig, 1966; Harman, 1962) are probably overly simplistic in view of the diverse nature of carcinogenesis. Recent reviews of, and conferences on, chemical carcinogenesis have emphasized its diverse nature (e.g., Miller, 1970; Arcos and Argus, 1968; Bergman and Pullman, 1969). The work cited in reviews of chemical carcinogenesis appear to rule out the possibility that all carcinogenesis involves free radicals, but they do offer grounds that free radicals may be involved with certain types of chemical carcinogens, especially in view of the demonstrated interactions between free radicals and nucleic acids (e.g., Akasaka and Dearman, 1969). For instance, Miller (1970) concluded that many carcinogens are strong electrophiles ; such compounds should readily form radical ions when they do attract electrons. In addition, neutral free radicals are also strong electrophiles. Studies by the Pullmans (e.g., Pullman and Pullman, 1969) indicating that many carcinogens have the property of an active “K” region may also be related to ease in forming free radicals according to Szent-Gyorgi et al. (1960). The possible role of free radicals as sulfhydryl reagents has been mentioned previously, and to the extent that such reactions are important in carcinogenesis, frec radicals may also play a role in this mechanism (Harrington, 1967). Borg (1972) recently reviewed some of the evidence linking free radicals and carcinogenesis and concluded that free radicals probably play an important role in certain types of carcinogenesis, but they do not provide a basis for understanding all types of carcinogenesis. The current status of the role of free radical forms of specific classes of carcinogens is best considered within the context of the overall physicochemical properties of each class of compounds. Such detailed considerations are available in the extensive reviews available on particular classes of compounds. Some of the specific evidence on free radical intermediates of particular carcinogens has been recently reviewed by Borg (1972). The most plausible general mechanism emerging from such considerations is that relatively inert compounds become converted (intracellularly ) under mild oxidizing or reducing conditions into reactive free radicals which attack DNA, forming covalent bonds. The carcinogenic change might result directly from this covalent addition or it may result from an additional, later event in which energy is deposited in the covalently attached carcinogcn and then transferred to DNA. 2. Free Radicals as Carcinogens

One possible and apparently “direct” approach to the role of free radicals in carcinogenesis is to study the carcinogenic ability of free

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radicals administered directly to experimental animals. Only a few such experiments have been done, perhaps because of reasoning similar to that expressed in the final paragraph of this section. Peacock and Spence (1967) investigated the tumor-inducing ability of free radicals using a pulmonary adenoma-prone strain of mice. They did not find a significant increase in the incidence of lung adenomas. The technique they used for free radical generation, however, may not have produced free radicals sufficiently long-lived to interact with the pulmonary tree. The stable free radical, D P P H (apdiphenyl-p-picrylhydraayl) was also studied as a possible carcinogen in mice (subcutaneous, 0.01 M ) with negative results (Boyland and Sargent, 1951). Fractions of tobacco smoke with high concentrations of stable free radicals were also found to be ineffective carcinogens on mouse skin (Wynder and Hoffman, 1964). Oppenheimer e t al. (1953) reported carcinogenesis by implantation of plastics in rodents; because these materials contained free radicals the carcinogenesis may have been related to free radical reactions (Fitzhugh, 1953). However, there did not appear to be a relationship between free radical content and carcinogenicity in a series of plastics embedded under similar conditions (Oppenheimer e t al., 1955). It is difficult to draw conclusions on the role of free radicals as carcinogens from these experiments because, by experimental necessity, only quite stable free radicals could be studied. These are stable because they are less reactive, but the high reactivity of free radicals is what originally led workers to suspect a role of free radicals in carcinogenesis. It seems that if free radicals are involved in some types of carcinogenesis they are most likely generated near their site of action; “direct” experiments are therefore unlikely to shed much light on this problem.

3. Tobacco Smoke This common carcinogen has been extensively investigated by ESR including studies of major components. A complete review of these studies will not be attempted here, but the general problems and findings will be indicated as a modcl of what can be expected in studies of free radicals in some types of carcinogenesis. Wynder and Hoffman’s (1964) review of experimental tobacco carcinogenesis includes an excellent discussion of ESR studies to that date. Borg (1972) has a more recent but brief discussion of this subject. Important individual references include Bluhm e t al. (1971), Cooper et al. (1969), Forbes e t al. (1967), Forbes and Robinson (1967, 1968), Ingram (1960), M. J. Lyons e t al. (1958), M. J . Lyons and Spence (19601, and Tully e t al. (1969). I n the sub-

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sequent discussion, which covers general aspects of the experimental problems, individual works will not be cited. ESR signals are readily detected in all types of tobacco smoke. The usual procedures have been to trap the smoke in a cold trap a t -77°C. or lower. Some of the most reactive, shortest-lived free radicals are usually lost in these experiments; the extent of the loss is a very sensitive function of the experimcntal procedures employed. After trapping, the condensate can be separated into various fractions by solvent extraction, sometimes with further loss of short-lived, reactive components. Individual experiments must therefore be scrutinized carefully to determine what types of radicals may have been lost in that experiment, and results between different laboratories cannot always be directly compared. It is difficult to estimate the biological role of the very short-lived components that are lost in these trapping experiments. Their short lifetimes reflect their high reactivity, and thus these components are the most capable of initiating profound chemical changes. On the other hand, if they are too short-lived they may not get to the tissues or critical intracellular sites. An additional potential complication is the possibility that within tissues short-lived free radicals may generate other free radicals from tissue components that diffuse to critical sites; simple cigarette smoke trapping experiments cannot reflect the existence of such intermediate species. Under some conditions considerable hyperfine structure can be resolved from tobacco smoke condensates, suggesting that a limited number of types of free radicals predominate. (If many different types of free radicals were present their hyperfine structures would so overlap that individual lines could not be resolved.) Individual components of cigarette smoke can then be studied to determine the incidence and hyperfine structure of free radicals created when these components are heated as they are in cigarette smoke. Several cigarette smoke components do give rise to characteristic free radicals when heated. 3,4-Benzopyrene, frequently considered to be the most important carcinogen in tobacco smoke, has been extensively investigated and found to readily form semistable free radicals. Several other components also form radicals under similar conditions, but there does not appear to be a straightforward relationship between the known carcinogenicity of smoke components and their capacity to form free radidals. The amounts and types of free radicals generated and recovered depend very much on the experimental conditions, strongly suggesting that interactions between components tnay be important in whole tobacco smoke. An attempt was made to attack some of the experimental problems

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discussed above by impinging tobacco smoke on lungs, smoke being drawn into the lungs directly by expanding and contracting isolated animal lungs connected to a lighted cigarette, and observing the ESR spectrum of the lungs (Rowlands et al. 1967, 1968). A characteristic ESR signal was generated in the lungs, but subsequent studies revealed that the important components were not free radicals but nitrogen oxides. It is also unlikely the ESR signal generated in the lungs is directly related to carcinogenesis because it is a typical “triplet” which does not appear to be a carcinogenic initiator (see Section III,C,4). This excellent study again points out the hazards of ‘(simple, direct” approaches in ESR-carcinogenesis studies (see Section III,B,2). C. ESR SPECTRA OF MALIGNANT TISSUES 1. Free Radical Content of Tumors In view of the postulated role of free radicals in carcinogenesis, it is not surprising that the earliest reports on the ESR spectra of tissues included a listing of the “free radical content” of a mouse hepatoma (Commoner et al., 1954). This lyophilized specimen had almost the same amount of radicals as normal mouse liver, but more detailed subsequent studies, utilizing better preparation techniques soon demonstrated that most mature tumors have a decreased free radical content (Truby and Goldzeiher, 1958; Kolomiitseva et aZ., 1960; Commoner and Ternberg, 1961; Nebert and Mason, 1963). Subsequent studies have confirmed the fact that, in general, tumor tissues have fewer free radicals (Saprin et al., 1966a,b,c,d, 1967a,b; Sentjurc et aZ., 1970; Vithayathil et al., 1965; Mallard and Kent, 1964, 1966; Duchesne and Van de Vorst, 1970). These studies included spontaneous human as well as spontaneous and experimental animal tumors. Some human tumors, however, do not show a decreased free radical content (Swartz, unpublished data). Some benign tumors also show decreased free radical contents, indicating that this change is probably characteristic of tumors in general and not only malignant tumors (Duchesne and Van de Vorst, 1970). Some exceptions have been reported in which tumors had increased amounts of free radicals. These were mainly in lyophilized tissues, but the tumor tissues appear to have been treated comparably to the normal tissues. Mulay and Mulay (1967) reported increased free radical content in melanotic mouse melanomas. Pavlova and Livenson (1965) noted increased signals in leukocytes of patients with chronic lymphoid leukemia and normal levels in leukocytes of patients with acute leukemia or chronic myeloid leukemia. Saprin’s group (see Section III,D,l) has reported increased free radicals in a variety of tumors a t very early stages

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of carcinogenesis, which later fell to below normal levels as the tumors matured. Cole and Hodgkinson ( 1965), using quick-frozen, nonlyophilized samples, reported that about one-third of human carcinomas of the cervix (9/27) had significantly increased free radical contents, but the other two-thirds had about the same concentration as normal cervices. Positive papanicolau smears also showed increased free radical concentrations. Cole and Hodgkinson also noted that the concentration of free radicals varied within individual tumors, the highest concentrations being found at the edges of the tumor and decreased concentrations near the center. Commoner and Ternberg (1961) suggested that the decreased free radical signal seen in neoplastic tissues might be a reflection of the decreased number of mitochondria in most neoplastic tissues. This suggestion is based on the assumption that most of the free radical signals seen in tissues originate in mitochondria. This assumption is not universally accepted; a recent text reviews current concepts on the origin of the free radical signals seen in tissue (Swartz, 1972). 2 . Paramagnetic Trace Elements Another important difference between normal and neoplastic tissues is the number and type of resonances attributable to paramagnetic transition elements. Nebert and Mason (1963) studied 29 types of mouse tumors and found that virtually each tumor type had a different ESR spectrum in regard to the non-free radical part of the spectrum (everything but the g = 2.00 area). Even for the same histological type of tumor, the spectra vary. Nebert and Mason (1963) studied six different mouse mammary adenocarcinomas and found that each had a distinctive spectrum. Emanuel et al. (1969) compared two mouse ascites sarcomas and found different spectra for each. Mulay and Mulay (1967) found that a melanotic mouse tumor (S91) had resonances at g = 2.05 and g = 4 while a nonmelanotic derivative (S91A) had neither of these peaks. There have been no explicit reports comparing ESR spectra of tumors of the same cell line. Some reports which comment on the differences between cell lines imply that the ESR spectra with a given cell line are consistent. Nebert and Mason (1963, 1964) compared ESR spectra of microsomes and mitochondria of normal liver and hepatomas. The mitochondria were quite similar, but differences were noted in the microsomal fractions. Both smooth and rough hepatoma microsomes varied from normal, some peaks being increased and some decreased. Because of the relatively high microwave power utilized in these experiments the ob-

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served differences were probably due primarily to paramagnetic transition elements, especially heme iron-containing compounds; free radicals were probably not responsible for much of the g = 2.0 peak.

3. Changes in g-Factor in the Region of g = 2.00 Another potential type of differentiation between normal and malignant tissues is a change in g-factors in the free radical region (around g = 2.00). Different organic free radicals may have characteristic slightly different g-factors (Swartz et al., 1972) and it seems reasonable to expect that if the type of cellular free radicals changes during carcinogenesis this may be detected as a shift in g-factor even though the line shape does not change (Mallard and Kent, 1969). In addition, changes in the amounts and types of paramagnetic trace elements may also affect the g = 2.00 area, especially if the studies are not performed a t very low microwave power levels. To date there have been few reports of true g-factor shifts in carcinogenesis studies. Cole and Hodgkinson (1965) and Brennan et al. (1965) mentioned some slight g-factor changes between normal and malignant tissues. Wallace et al. (1970) felt that g-factor shifts were very important parameters in differentiating between normal and tumorous breast tissue and suggested that these changes might be reflected in blood as well. However, they used lyophilised preparations, so the origin and meaning of these g-factor shifts is not clear. In unpublished studies I have noted that about 10% of patients with advanced cancers show a g-factor shift in their plasma, but this appears to be due to changes in transition elements rather than free radicals. Driscoll et aZ. (1967b) reported a 10-fold change in g-factor variation between normal tissues and neoplasms, using lyophilised samples. I n most other studies no g-factor shifts have been observed. I n view of the relatively broad lines seen in most tissues, this finding is not unexpected, and it is still quite possible that g-factor shifts are common in malignant tissues. It is unlikely, however, that sufficient resolution can be obtained to detect most of these shifts if they do occur. It is also quite possible that the g-factor shifts noted to date were all due to transition element effects and not to free radicals. Away from the g = 2.00 area, quite prominent g-factor changes are readily observed in malignant tissues (Section III,C,2). 4. “Triplet”

One of the possible practical applications of ESR spectroscopy is as a diagnostic test for the presence of tumors. This would be possible if

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a unique ESR signal could be found in tumors. Brennan et al. (1966) reported such a spectrum in 5 of 13 mice with a reticulum cell sarcoma and in 6 of 6 mice with a JAX C 1300 neurosblastoma. As described below, it is now apparent that the spectrum they reported can occur in both normal and malignant tissues, but it occurs “spontaneously” more in the latter and can also be induced more easily in neoplastic tissues under the proper conditions. Thus, although this is not the unique signal Brennan et al. originally thought it was, it may offer some insight into metabolic characteristics of neoplasms and it may still have some diagnostic value. The signal described by Brennen et al. was a triplet, centered a t about g = 2.012 with a splitting of about 16 G. They presented strong evidence that this triplet arose from the interaction of an unpaired electron with a nitrogen atom and, on spectroscopic grounds, also suggested that a heavy atom, such as iron, sulfur, or calcium, was also involved. Subsequent studies indicate that this is indeed the case, with perhaps both iron and sulfur atoms involved, with the primary interaction being due to a nitrate group. Recently, Maruyama et al. (1971) summarized their extensive work on this signal and concluded that it was due to a NO-hemoprotein complex under hypoxic conditions. They found that ascites tumor cells frequently, but not always, showed the triplet signal. If cells that did not have the triplet were allowed to stand in solution for 2-3 hours the triplet then developed. Normal tissues could also be induced to generate the triplet signal; for liver, heart, spleen, kidney, and muscle 1-2 days were required, while normal gastrointestinal tract tissues developed the triplet quite rapidly. They were able to generate similar signals by forming NO-hemoglobin complexes, although the latter were not affected by oxygen while the triplet seen in tissues could be reversed by flowing oxygen into the solution. They concluded that the essentials for development of the triplet were ( 1 ) anoxia, (2) protein denaturation, (3)heme iron, and ( 4 ) NO. Apparently some tumors and normal tissues denature more readily and/or form NO more readily and therefore rapidly generate the triplet signal. Borg (1972) has summarized the excellent, detailed ESR evidence that supports the conclusions of Maruyama et al. Similar triplets have been reported by other workers in tumors (Emanuel and Saprin, 1969; Emanuel et al., 1969; Saprin et al., 1970; Tazaki et al., 1970; Matsunaga, 1969; Sakagishi, 1968), in irradiated blood (Swartz et al., 1965), in tissues treated with NaNOz (Azhipa et al., 1966; Kayushin and Azhipa, 1969), and in lungs subjected to cigarette smoke (Rowlands et al., 1967, 1968). Tazaki et al. (1970) also reported the presence of the triplet in proliferative, nonmalignant bladder mucosa

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in patients that also had overtly malignant mucosa; bladder mucosa from normal bladders did not show the triplet. Another NO-iron-protein complex ESR signal without the triplet hyperfine structure and having a different g-factor ( g = 2.035) has also been reported under a variety of conditions including experimental carcinogenesis. The iron in this case is nonheme, and a sulfur atom is intimately involved. It is discussed in detail in Section III,D,2. Azhipa et al. (1969) have discussed both types of signals. 5. Changes in Nontumor-Bearing Organs of Organisms with Tumors Most tissues’ ESR spectra are unaffected by the presence of tumors in other organs or even in other parts of the same organ. In one situation, however, an abnormality was reported. Kolomiitseva et al. (1960) found that while liver and brain of sarcoma 45-bearing rats were unchanged, the spleen showed a small increase in the amplitude of its ESR signal. The shape and position of the signals were unchanged. Some changes may also be seen in blood plasma, but these may represent spillovers from tumors (Swartz, 1971).

D. DYNAMIC STUDIES Potentially the most useful types of information obtainable by ESR - carcinogenesis studies are the changes occurring during carcinogenesis and/or modification of the carcinogenic process. If characteristic ESR changes were to be found, these might be useful in understanding the nature of the carcinogenic process and/or as diagnostic acids and/or as prognostic indicators. Some very exciting studies of this nature have been carried out but, most frustratingly, some of the most critical experiments have been performed with lyophilized samples so their meaning is not clear. 1. ESR Changes during Carcinogenesis

Commoner’s group (Vithayathil et al., 1965) studied livers of rats fed three different carcinogens, p-dimethylaminoazobenzene (butter yellow), thioacetamide, and 2-acetylaminofluorence (AAF) . With each carcinogen they noted the development of a g = 2.035 peak a t times ranging from 14 to 40 days from the onset of carcinogen feeding, before any histological or other biochemical indication of malignant change. By the time of overt malignant change, the g = 2.035 signal was no longer present. The g = 2.005 signal seen in normal liver was not observed in overtly malignant hepatic tissue, but did persist through the time of appearance and disappearance of the g = 2.035 signal. Commoner’s group (Wollum and Commoner, 1970; Commoner et al.,

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1970) later elucidated the nature and significance of the g = 2.035 signal. This signal appears to arise from an interaction between NO and Fez+ in a thiol-containing protein in which the thiol interacts with the unpaired electron. The iron is probably not part of a heme group. This signal can be generated in both normal and carcinogen-fed animals if there is sufficient nitrate or nitrite in the diet. The presence of the carcinogen, however, greatly reduces the amount of nitrate or nitrite required to generate the g = 2.035 signal. The generation of the g = 2.035 signal was also enhanced by the feeding of a protein deficient diet. The relationship between the g = 2.035 signal and tumor incidence was studied, and it was found that the same factors that enhanced generation of the g = 2.035 signal (nitrite or nitrate feeding and/or protein deprivation) also decreased the incidence of tumors. The authors concluded that the g = 2.035 might indicate successful anticarcinogenic activity, perhaps by deviating thiol binding effects from more critical sites. Although the g = 2.035 signal is usually considered to be due to a free radical, Hutchison et al. (1969) suggest that it may be due primarily to an unpaired electron localized on a trace element. The occurrence of the g = 2.035 signal resembles the situation seen with the “triplet” discussed in Section III,C,4. The latter, however, arises from an interaction of heme (rather than nonheme) iron with an NO complex and has a lower g-factor as well as a resolved nitrogen hyperfine splitting. The “triplet” may not necessarily involve a thiol group. Saprin et al., using lyophilized preparations, have studied the time course of free radical content in a variety of experimental tumors ( 1966a,b,c,d, 1967a,b, 1970) with remarkably consistent results. The number of free radicals progressively increased above normal at the early stages of tumor development. As the tumor enlarged the radical concentration dropped back toward normal and frequently, by the time the tumor reached appreciable size, the radical content was significantly below normal levels. Petyayev et al. (1967) and Driscoll et al. (1967a) have reported similar results. These findings are very attractive because they are consistent with both the theory that free radicals are involved in the carcinogenic process and with the empirical finding that most tumors have decreased free radical concentrations. Such findings also suggest that the early phases of carcinogenesis may involve an increased metabolic state that could be detected and therefore serve as the basis of an early diagnostic test for cancer. Unfortunately, because all these studies were done on lyophilized specimens, these results must currently be considered as an unconfirmed finding of potential great importance but not susceptible to adequate interpretation. We have recently un-

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successfully attempted to confirm these results using quick frozen instead of lyophilised samples. Some ESR changes were found during tumor development in spleens of AKR mice innoculated with leukemia cells, but the power saturation properties and the g-factors of these changes indicated that these changes were due to inorganic paramagnetic ions. I n addition, these changes increased progressively until death and did not show an early maximum and a subsequent fall to normal or below. I n contrast to the studies of Saprin et al., dynamic studies of Vithayathil et al. (1965) and Duchesne and Van de Vorst (1970) did not demonstrate an increase in free radical content during carcinogenesis due to butter yellow or AAF. A late decrease in free radical content, after the development of overt tumors has been observed (Duchesne and Van de Vorst, 1970; Mallard and Kent, 1964; Duchesne et al., 1969). 2. Modification of ESR Changes Associated with Carcinogenesis Acting on the assumption that free radicals are intimately associated with carcinogenesis, several workers have attempted to alter the course of experimental carcinogenesis by the feeding or injection of substances that react readily with free radicals. Again, most of these studies were performed with lyophilized samples, and therefore the interpretation of the ESR results is subject to the usual reservations, but certainly the accompanying biological data should not be affected by these considerations. Kozlov and Dobrina (1966) studied the effect of acrylamide in rats inoculated with "Cracker" sarcoma and on tissue cultures of monkey heart and human angiosarcoma. They reported inhibition of both free radical development and tumor growth in vivo and in vitro. Saprin et al. (1966b, 1967b) reported that free radical inhibitors decreased in parallel with the rate of tumor development and generation of ESR signals in tumors. On the other hand, Kalmanson et al. (1961) studied free radical inhibitors, such as propyl gallate, which were advocated as antitumor agents and found that these led to an increase in the ESR signal seen in several types of tumor cells. They concluded that, because of the complexities of interactions of these substances with tissues and the effect of environmental parameters such as oxygen, a consistent trend in the effect on free radical concentrations was not to be expected. The results of Woolum and Commoner (1970) and Commoner et al. (1970) that indicated a correlation between inhibition of tumor formation and generation of the g = 2.035 signal were interpreted by the authors as being consistent with inhibition of carcinogenesis by free radical reactions. Burlakova (1967) proposed a theory of free radical regulation of

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cell multiplication based in part on the studies that indicate a decrease in free radicals is associated with carcinogenesis. She proposed that the rate of cell division is regulated by intracellular levels of free radicals, with increased free radical levels leading to decreased cell division rates. She suggested that either a decrease in levels of normal free radical inhibitors or an increase in exogenous free radicals leads to decreased cell multiplication rates. In general free radical inhibitors are also antioxidants and she cited evidence that an increase in antioxidant activity is associated with carcinogenesis (Burlakova et al., 1965 ; Burlakova and Pal’mina, 1967; Burlakova and Molochkina, 1968). 3 . Melanomas

Melanins usually have remarkably stable ESR signals due to unpaired electrons associated with the polymer matrix (Blois et al., 1964). Several investigators have studied melanin-producing tumors by ESR to determine whether the paramagnetic nature of melanin could be utilized to follow treatments designed to affect growth rates of melanomas. Some of the investigators, however, ended up studying a nonmelanin, less stable, free radical that could be induced in the melanomas by blue light or chemical treatments. This less stable free radical was much more susceptible to modification than melanins, which are stable to both boiling and concentrated acids (Blois et al., 1964). The experimental melanomas investigated indicate that the melanin produced by these melanomas has an ESR spectrum similar to that of melanins of other origins (Duke et al., 1966). This is not surprising in view of the known chemical similarity of melanin of tumors to that of normal tissue melanins and the rather nonspecific nature of the ESR spectrum of melanin (Blois et al., 1964). Recently techniques have been described that do demonstrate some fine structure in melanin ESR spectra, and it would be of interest to apply these techniques to melanomas (Grady and Borg, 1968). Duke and co-workers (1966, 1967) studied the effects of cysteine and penicillamine on growth and ESR characteristics of S91 mouse melanomas. They utilized conditions that obscured the ESR signal due to melanin and instead followed a doublet that probably originated in abscorbate, which may have been related to melanin, but was definitely not due to melanin itself (Demopoulos et al., 1966). (Because they changed experimental conditions between the studies of the two drugs, the ESR results cannot be compared directly.) L-Cysteine a t sufficiently high concentrations (10 mg./ml.) inhibited generation of a light-induced signal in melanoma tissue culture while a t lower concentrations it increased the ESR signals. Both concentrations inhibited melanoma growth

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in tissue culture. Blue light itself also inhibited melanoma growth in vitro (Demopoulos et al., 1966). D-Penicillamine was tested both in vivo and in vitro. Its effect was greater in vivo but in both situations it decreased the melanoma ESR signal. Although penicillamine had no selective inhibitory effect on tissue cultures of melanoma, it did inhibit melanoma growth in vivo. The authors interpreted their results as indicating that the inhibitory effects noted were due to copper removal rather than direct free radical interactions with melanin. This was based in part on the known effects of copper removal on melanin signals (Blois et al., 1964) and on the fact that, while cysteine reacts readily with free radicals, is a strong reducing agent, and complexes copper, penicillamine has only the last property. Mulay and Mulay (1967) studied the S91 mouse melanoma and an amelanotic derivative S91A. Unfortunately, they used lyophilized preparations and their results with normal tissues indicate that, in these tissues a t least, their results were affected by the oxygen-generated artifactual signal, as indicated by the large signal they observed in spleen. They did find that the amelanotic variant had a smaller ESR signal and that only the melanotic variant had a g = 2.05 signal, which they attributed to Cu*+.The signal they studied was a singlet, so it may have been melanin, as compared to the signal studied by Duke et al. (1966, 1967), which was a doublet that could not have been melanin. 4, Generation of ESR Signals in Mixtures of Carcinogens and Tissues

This type of experiment may reveal the formation of relatively short-lived free radicals that, cannot be detected by studying either component separately. Only a few such experiments have been reported to date, however, and the results have not indicated any definite trends. Nagata et al. (1966, 1967) studied free radical formation in mixtures of carcinogens with mouse skin homogenates. They used lyophilized samples and found, in addition to the usual lyophilization-induced tissue ESR signals, a large, narrow signal when the carcinogens 3,4-benzopyrene or 3-methylcholanthrenc were present. Several related noncarcinogens gave no such signal, nor did the potent carcinogen 9,lOdimethyl-l,2-benzanthracene.The generation of the additional radical required both the carcinogen and tissue homogenate or albumin. Heating the tissues to 100°C. did not prevent radical formation. Rondia (1967) reported similar results with nonlyophilized skin in acetone and 3,4benzopyrene irradiated with visible light. Rowlands et al. (1967, 1968) studied the effects of cigarette smoke on lung parenchyma. They found a signal was induced in lungs by the

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smoke of 6 cigarettes or less, but additional work indicated that the critical component of smoke was a nitrogen oxide and not a free radical. IV. Summary and Conclusions

Much of the literature reviewed here can be summarized as follows: 1. Characteristic ESR changes are seen in most, but not all, mature malignant and benign tumors, including a decrease in free radical content and a change in the type and amount of paramagnetic trace elements. 2. There is as yet no clear-cut evidence for an early stage during carcinogenesis characterized by an increase in free radical content, although this remains a very attractive hypothesis. 3. Certain ESR signals (“triplet” and g = 2.035 signals) occur spontaneously more frequently in tumors and/or are more easily induced in tumors but are also seen in normal tissues under certain conditions. 4. A role of free radical intermediates in chemical carcinogenesis is not established as a general phenomenon, but the carcinogenic properties of some classes of carcinogens appear to parallel properties related to free radical formation. These findings do not yet lead to broad, useful conclusions on important problems in carcinogenesis. One can conclude that although it is unlikely that ESR studies will lead to a dramatic breakthrough in our understanding of cancer, several studies do indicate that ESR-carcinogenesis investigations may eventually lead to valuable knowledge. Areas of particular promise include a better understanding of intracellular changes that occur during malignant transformation (by understanding the factors that lead t o the observed ESR changes) and, perhaps, the use of ESR changes for diagnostic or prognostic purposes. Most of the possible types of experiments involving simple ESR observation of malignant and premalignant tissues have now been done, but it would be highly desirable to have more systematic data, especially on human material. Further progress would seem to depend on more complex studies which include measurements of other, non-ESR parameters as well as employing the more sophisticated ESR techniques that are now available. (A possible exception is the time course of ESR changes during carcinogenesis utilizing nonlyophilized samples.) Perhaps the delineation of some of the experimental problems in this review will aid in the initiation of such studies.

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Azhipa, Ya. I., Kayushin, L. P., and Nikishkin, Ye. I. (1966). Biofizika 11, 71&713. Azhipa, Ya. I., Kayushin, L. P., and Nikishkin, Ye. I. (1989). Biofizika 14, 852-857. Bergman, E. D., and Pullman, B., eds. (1969). “Physico-Chemical Mechanism of Carcinogenesis.” Isr. Acad. Sci. Humanities, Jerusalem. Blois, M. S., Jr., Zahlan, A. B., and Maling, J. E. (1964). Biophysics 4, 471-490. Bluhm, A. L., Weinstein, J., and Sousa, J. A. (1971). Nature (London) 229, 500. Boenig, H. V. (1966). J. Amer. Geriat. SOC. 14, 1211-1220. Borg, D. C. (1972). I n “Biological Applications of ESR’ (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), Chapter 7. Wiley (Interscience), New York. Boyland, E., and Sargent, S. (1951). Brit. J. Cancer 5, 433440. Brennan, M. J., Cole, T., Singley, J., and Hodgkinson, C. P. (1965). Fed. Proc., Fed. Amer. SOC. Ezp. Bwl. 24, 437. Brennan, M. J., Cole, T., and Singley, J. A. (1966). Proc. SOC.E z p . Biol. Med. 123, 716-718.

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Cole, T., and Hodgkinson, C. P. (1965). Technical Report, Ford Motor Co., Detroit, Michigan. Commoner, B., and Ternberg, J. L. (1961). Proc. Nat. Acad. Sn’. U. S. 47, 13741384.

Commoner, B., Heise, J. J., Lippincott, B. B., Norberg, R. E., Passonneau, J. V., and Townsend, J. (1957). Science 126, 57-63. Commoner, B., Ternberg, J. L., Woolum, J. C., and Senturia, B. H., Jr. (1970). Cancer Res. 30, 2091-2097. Commoner, B., Townsend, J., and Pake, G. (1954). Nature (London) 174, 689-691. Cooper, J. T., Forbes, W. F., and Robinson, J. C. (1969). Nut. Cancer Inat., Monog. 28, 191-197.

Demopoulos, H . B., Landgraf, W., Duke, P. S., and Tai, H. (1966). Lab. Invest. 15,1652-1658.

Driscoll, D. H., Dettmer, C. M., Wallace, J. D., and Neaves, A. (1967a). Cum. Mod. Biol. 1, 275-278. Driscoll, D. H., Wallace, J. D., and Dettmer, C. M. (1967b). Fed. Proc., Fed. Anmer. SOC. Ezp. Biol. 28, 626. Duchesne, J., and Van de Vorst, A. (1970). Bull. C1. Sci., Acad. Roy. Belg. 56, 433-448.

Duchesne, J., Lion, Y., and Van de Vont, A. (1969). C. R. Acad. Sn’. 269, 1562-1563. Duke, P. S., Landgraf, W., Mitamura, A. E., and Demopoulos, H. B. (1966). J . N u t . Cancer Inst. 37, 191-198. Duke, P. S., Hourani, B. T., and Demopoulos, H. B. (1967). J. Nat. Cancer Inst. 39, 1141-1147.

Emanuel, N. M., and Lepatova, L. (1960). Dokl. Akad. Nauk SXSR 130, 221-222. Emanuel, N. M., and Saprin, A. N. (1969). Proc. Int. Biophys. Congr. Srd, 1969. p. 224. Emanuel, N. M., Saprin, A. N., Shabalkin, V. A., Kozlova, L. E., and Kruglyakova, K. Ye. (1969). Nature ( h n d o n ) 22, 165-167.

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Fitzhugh, A. (1963). Science 118, 783. Forbes, W. F., and Robinson, J. C. (1967). Nature (London) 214, 8 W 1 . Forbes, W. F., and Robinson, J. C. (1968). Nature (London) 217, 550-551. Forbes, W. F., Robinson, J. C., and Wright, G. L. (1967). Can. J . Biochem. 45, 1087-1098. Grady, F. J., and Borg, D. C. (1968).J. Amer. Chem. SOC. 90, 2949-2952. Harman, D. (1962). Radiat. Res. 16, 753-763. Harrington, J. 8. (1967).Advan. Cancer Res. 10, 248-310. Heckley, R. J. (1972). Zn “Biological Applications of ESR” (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), Chapter 5. Wiley (Interscience), New York. Hutchinson, J. M. S., Foster, M. A., and Mallard, J. R. (1969). Proc. Znt. Biophys. Congr., Srd, 1969 p. 225. Ingram, D. J. E. (1960).Acta Med. Scand., Suppl. 369,43-61. Ingram, D. J. E. (1969). “Biological and Biochemical Applications of Electron Spin Resonance.” Adam Hilger Ltd., London. Kalmanson, A. E., Lipchina, L. P., and Chetverikov, A. G. (1961). Bhjizika 6, 4 1 W . Kayushin, L. P., and Aehipa, Ya. I. (1969). Proc. Znt. Bwphys. Congr., 3rd p. a25. Kensler, C. J., Dexter, S. O., and Rhoads, C. P. (1942). Cancer Res. 2, 1-18. Kent, M., and Mallard, J. R. (1969).Phys. Med. Biol. 14, 431439. Kolomiitseva, I. K., L’Vov, K. M., and Kayushin, L. P. (1960).Biojizika 5, 636-637. Kozlov, Yu. P.,and Dobrina, S. K. (1966).Biofizika 11, 168-170. Lipkin, D., Paul, D. E., Townsend, J., and Weissman, S. I. (1953). Science 117, 534636. Lyons, M. J., Gibson, J. F., and Ingram, D. J. E. (1958).Nature (London) 181, 1003-1004. Lyons, M. J., and Spence, J. B. (1980).Brit. J . Cancer 14,703-708. Mallard, J. R., and Kent, M. (1964).Nature (London) 204, 1192. Mallard, J. R., and Kent, M. (1966).Nature (London) 210, 588591. Mallard, J. R., and Kent, M. (1969).Phys. Med. Biol. 14, 373-396. Maruyama, T., Kataoka, N., Nagase, S., Nakada, N., Sato, H., and Sasaki, H. (1971). Cancer Res. 31, 179-184. Mason, R. (1958).Nature (London) 181,822-824. Mason, R. (1959). Discuss. Faraday SOC. 27, 129-139. Matsunaga, J. (1969).Jap. J . Urol. 60, 214-230. Miller, J. A. (1970).Cancer Res. 30, 559-576. Mulay, I., and Mulay, L. (1967). J . Nat. Cancer Znst. 39, 735-743. Nagata, C.,Tagashira, Y., Kodama, M., and Imamura, A. (1966). Jap. J. Cancer Res. 57, 437442. Nagata, C., Kodama, M., and Tagashira, Y. (1967).Jap. J. Cancer Res. 58, 493-504. Nebert, D. W.,and Mason, H. S. (1963).Cancer Res. 23, 833-840. Nebert, D. W., and Mason, H. S. (1964). Bwchim. Biophys. Acta 86, 415-417. Oppenheimer, B. S.,Oppenheimer, E. T., Stout, A. P., Danishepky, I., and Eirich, F. R. (1953).Science 118, 783. Oppenheimer, B. S., Oppenheimer, E. T., Danishepky, I., Stout, A. P., and Eirich, F. R. (1966). Cancer Res. 15, 333-340. Park, H. F. (1950). J. Phys. Colloid Chem. 54, 1383-1412. Pavlova, N. Q., and Livenson, A. R. (1965).Biojizika 10, 169-171.

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Vithayathil, A. J., Ternberg, J. L., and Commoner, B. (1965). Nature (London) 207, 12464249. Wallace, J . D., Driscoll, D. H., Kalomiris, C. G., and Neaves, A. (1970). Cancer ‘25, 1087-1090. Woolum, J. C., and Commoner, B. (1970). Biochim. Biophys. Acta 201, 131-140. Wynder, E. L., and Hoffman, D. (1984). Advan. Cancer Res. 8, 249-453.

SOME BIOCHEMICAL ASPECTS OF THE RELATIONSHIP BETWEEN THE TUMOR AND THE HOST V. S. Shapot Institute of Experimental and Clinical Oncology, Academy of Medical Sciences, Moscow, USSR

I. Introduction . . . . . . . . . . . . . 11. Respiration of Tumors in Vivo . . . . . . . . . 111. Glucose Levels in Ascites Cancer Cells and Their Mediim in Vivo IV. Glycolyeis and Destructive Tumor Growth . . . . . . V. Relative Glucose Deficiency of the Tumors Growing in the Body . Effect of Hyperglycemia on Cancer Cells . . . . . . . VI. Tumor as a Glucose Trap in the Body . . . . . . . A. Tumor as a Hypoglycemic Factor . . . . . . . B. On the Deposition of Glycogen by the Liver of the Host . . C. Gluconeogenesis in the Body and Tumor Development . . VII. The Problem of Cancer Cachexia . . . . . . . . VIII. Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

. .

.

. . . . . . . . . .

253 254 257 202 264 264 208 269 275 278 279

281 283

I. Introduction

It is not an exaggeration to assert that there exists a kind of mutual misunderstanding between clinical oncologists and biochemists studying the metabolism of tumors in vitro. The former, interested in the cancer patient, do not see how to apply the results obtained by the latter t o diagnosis and treatment of cancer or to attain a better understanding of the versatile impairments of the body homeostasis caused by the developing tumor. Presumably, undue emphasis on in vitro experiments is the cause of this situation. In vitro experiments are absolutely necessary to approach the problem, but it is quite obvious that such an approach does not illuminate the biochemical behavior of cancer cells in the organism. I n our investigations we have focused on the tumor growing in its host; we tried to find out what conditions affect the metabolism of tumors in the host, to what extent they allow the cancer cells to realize their potential capacity for respiration and glycolysis, and whether these processes in turn are able to implement in vivo their physiological function of energy transduction. We were curious to learn as well what consequences for the host in terms of its metabolism and physiological systems are to be expected if the potential requirements of the developing 263

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tumor for oxygen and glucose could not be met in vivo. We believed that the answers to the above questions might offer a novel approach t o the riddle of cancer cachexia. II. Respiration of Tumors in Vivo

No sound proof of any damage to the respiratory mechanism in cancer cells has been provided so far. On the contrary, their respiration of cancer cells was shown to be normal in terms of both oxygen consumption (Weinhouse, 1955 ; Aisenberg, 1961 ; Eltzina, 1965) and physiological efficiency. According to the in vitro experiments, carried out under optimal 0, concentrations, respiration alone is able to maintain in cancer cells an optimal rate of A T P resynthesis (Eltzina, 1960, 1965) and to ensure energetically amino acid incorporation into their protein to the same extent as glycolysis (Eltzina, 1953). Thus, cancer cells have two efficient processes elaborating energy-respiration and glycolysis. The question arises, however, whether cancer cells in vivo are under the proper conditions to allow them to realize their full capacity of respiration. There is a growing body of evidence to indicate that in the organism the actual conditions for optimal respiration can be far from favorable. According to Urbach (1956), who used the electrode inserted into tissues to be studied, the PO, in various human tumors of skin (squamous cell carcinomas, basal cell epitheliomas, metastatic cutaneous melanoma lesions, lesions of dermatofibrosarcoma, etc.) of more than 200 patients was essentially lower than that in the immediately adjacent skin. I n almost all these malignant lesions the PO, within the tumor mass was found to be 25% or less of that of the simultaneously examined normal adjacent skin. True, these drastic differences might be ascribed to a very low 0, demand of normal skin, since a significant part, consists of metabolically inert collagen and elastk. However, Rampan (1967) measuring the PO, of extremity melanomas in 16 patients with the platinum electrode also reported values that did not exceed 50% of those of normal tissues other than skin. Still lower PO, values were found by him in sarcoma 45 in vivo. A spectacular demonstration of a scarce 0, supply to the solid tumors in vivo was provided by Malmgren and Flanigan (1955). The spores of Clostridium tetani, which were proved to be nonpathogenio for normal mice, were administered to mice bearing mammary tumor C3HBA, fibrosarcoma HE 8971, and hepatoma 98/45 as well as spontaneous mammary tumors. All the animals thus treated died from tetanus within 48 hours after the injection. The microscopic examination revealed the presence of the vegetative form of the bacteria in the cancerous areas

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

255

only. These results clearly indicate that, of all tissues, only in the tumor did the spores find the pOz sufficiently low to allow for germination. Such a scarce 0, supply is thought to be due to the fact that the blood flow and the vascular space of tumors are severalfold smaller than those of normal homologous tissues (Gullino and Grantham, 1961, 1964; Gullino, 1966). As to transplanted ascites tumors, Warburg and Hiepler (1952) were the first to discover the apparent absence of oxygen in the ascitic fluid of Ehrlich carcinoma in vivo. We have confirmed this observation and found that the 0, content in this fluid ranged from 0.5 to 1.7 vol. ”/. (Gorozhanskaya and Shapot, 1964). Similar values were found for effusion fluid of 42 cancer patients (Gorozhanskaya et al,, 1964). Methylene blue, introduced into the abdominal cavity of mice with Ehrlich carcinoma, was immediately converted into the leuco form (Shapot, 1965), an indication that almost anaerobic conditions were maintained in the ascitic fluid in vivo. One could interpret this phenomenon as a result of a discrepancy between a high 0, consumption by the cancer cells and an inadequate O2 supply from the organism. A special series of experiments was undertaken which plainly indicated that this is not true. Respiration of cancer cells in vivo is low and deficient because the hypoxia prevents them from fulfilling their optimal capacities as explored under in vitro conditions. First we tried, by means of the following experiments, to find out whether the Pasteur effect could be observed in cancer cells in vivo (Tagi-Zade and Shapot, 1971). The glucose content in the ascitic fluid of Ehrlich carcinoma is known to be negligible (see below) although the product of glycolysis, lactic acid, accumulates continuously. However, when 0, was introduced intraperitoneally into the mouse bearing the Ehrlich ascites carcinoma, the picture changed drastically-the concentration of lactic acid in the ascitic fluid dropped gradually and glucose appeared, reaching a plateau within 40 minutes’ (Fig. 1). This result implies that, in vivo, respiration of cancer cells under usual conditions is so weak that the Pasteur effect cannot operate. Only an extra 0, supply enabled the cancer cells to realize their potential capacity of respiration and thereby partially block glycolysis, i.e., reduce the glucose consumption. In separate experiments, we determined the rate of glucose influx from the organism into the ascitic fluid and the rate of efflux of lactic acid from the ascitic fluid. Thus, the extent of the in vivo inhibition of glycolysis by respiration on unlimited 0, could be ‘The animals were taken 5-6 days after inoculation, i.e., during the period when cell multiplication is still proceeding (Efimov and Bernstein, 1988).

256

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S. SHAPOT

assessed. It was found to be about 50%. i.e., close to that described for in vitro conditions (Weinhouse, 1955). The above results plainly demonstrate that there is no reason to ascribe to the aerobic glycolysis any biological meaning as a feature characteristic of tumors; in cancer cells in vivo actually an extensive anaerobic glycolysis proceeds, which is not restricted by the Pasteur effect.* Further experiments (Tagi-Zade and Shapot, 1970b) carried out in both ascites and solid mouse and rat tumors have shown that an extra O2supply selectively stimulated by 40-50% the incorporation of labeled amino acids into tumor proteins since the rate of amino acid incorporation into liver proteins remained unchanged. One can infer, therefore, that the respiration of cancer cells under usual in vivo conditions is essentially lower than their potential capacity and that improvement of the oxygen regime ensures a n elevated level of tumor respiration, thus permitting the utilization of extra energy for protein synthesis. These results imply as well that in vivo glycolysis in cancer cells does not operate to the full extent (see also below) ; otherwise, an enhancement of respiration would not have affected the rate of tumor protein synthesis. To verify further the idea that cancer cells in vivo experience oxygen deficiency, we exploited an observation by Eltzina and Engelhardt (1958). It was shown with Ehrlich carcinoma ascites cells in vitro that the utilization of carbon fragments of g l ~ c o s e - ~for ~ c the synthesis of nonessential amino acids, revealed later on in the tumor protein, was enhanced 10- t o 20-fold when both glycolysis and respiration proceed simultaneously under optimal conditions over that in which glucose utilization was solely by anaerobic glycolysis. A similar stimulation under these conditions was noted on the incorporation of 14C-labeled fragments derived from labeled glucose into the pentose moiety of tumor nucleic acids isolated chromatographically. Thus, the oxidation of the 3-carbon fragments produced by glycolysis facilitated their involvement in synthetic processes through the tricarboxylic cycle. Presumably, this is the reason why the multiplication of Ehrlich carcinoma ascites cells in vitro was arrested under strictly anaerobic conditions, although a very low content of 0,(1.5-3.0 vol. %) was recognized to be optimal for this process (Negelein et al., 1966). We reasoned that additional evidence for tumor hypoxia in vivo would be obtained if we could show that an extra 0, supply (through inhalation for solid tumors and by intraperitoneal administration of

'We are aware that the cells of solid tumors lying close to the vessels might be in a more favorable condition in terms of 0,supply than are ascites cancer cells, However, the average cell of a solid tumor must be hypoxic as well.

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257

oxygen for ascites tumors) stimulated the incorporation of label from glu~ose-’~C into the tumor protein. Such experiments were carried out with various transplantable tumors in both solid and ascites forms. A significantly higher labeling of tumor protein was observed when oxygen was introduced into the animal (Tagi-Zade and Shapot, 1971). The inability of tumors to fulfill in vivo their potential capacity for oxidative metabolism was indicated as well by the earlier experiments of Busch et al. (1957), who showed that the production of citrate by the tumor growing in the body (as a measure of the activity of the tricarboxylic cycle) was negligible under usual conditions, but reached the level characteristic of liver when the tumor tissue was incubated in the proper medium in the presence of oxygen. All the data described above favor the view that respiration of tumors in the organism is deficient because of local hypoxia. One can add that in the experiments of Gullino et al. (1967) with tumors grown in vivo as “tissue-isolated preparations,” whcre the blood supply was ensured by ovarian or renal vessels, the 0, consumption, thus proceeding under relatively favorable conditions, was lower than that reported for slices of the same tumor. A low level of respiration in vivo of both transplantable and spontaneous animal tumors, as well as human melanomas, was observed by Rampan (1967). Thus, it appears that in vivo anaerobic glycolysis is the main source of energy for the cancer cell. If so, one might anticipate that this process must be provided with sufficient amounts of the substrate. However, as will be shown below, the concentration of glucose in the tumor growing in the host turned out to be f a r from optimal for maximal glycolysis.

0

30

E 0

20

f

0 EI

.-E

0

f

.a Q

10

0 .c 0

min.

FIQ.1. Effect of oxygen administration in vivo on the lactic acid and glucose content of ascitic fluid (Ehrlich carcinoma). Curve 1, lactic acid; curve 2, glucose. Arrow indicates the moment oxygen was introduced.

258

V. S. SHAPOT

111. Glucose levels in Ascites Cancer Cells and Their Medium in Vivo

I n the ascitic fluid of Ehrlich carcinomas in vim, as mentioned above, lactic acid is continuously produced, indicating an extensive glycolysis. However, the concentration of glucose in the fluid was found to be surprisingly low. Warburg and Hiepler (1952) reported that no glucose could be detected in the ascitic fluid (cf. also Burgess and Sylven, 1962; G. Klein, 1956; Del Monte and Rossi, 1963). Kemp and Mendel (1957) using a chemical method determined the ascitic fluid glucose to be as low as 5-7 mg./100 ml., claiming that this is just the concentration which still allows a maximal rate of glycolysis in cancer cells as evidenced by their in vitro experiments. It was believed by those authors that the influx of glucose to the ascitic fluid, measured after the removal of cancer cells from the ascitic fluid, was sufficiently high to saturate the cells under aerobic conditions. It will be seen later on that this is not the case. Gorozhanskaya and Shapot (1964) tried to assess the true glucose content of the ascitic fluid of Ehrlich carcinoma under in vivo conditions using a slightly modified glucose oxidase method (Richterich and Colombo, 1962) which allowed us to measure glucose concentration as low as 0.008 mg./100 ml. No traces of glucose could be detected either in the ascitic fluid or in the cancer cells themselves, taken 4-6 days after transplantation from 20 different mice and placed in 0.02 M N a F as soon as they were withdrawn from the body. Thus, the glucose concentration in ascites fluid must be at most 1/10,00Oth that of the blood. It is noteworthy that four years later Nakamura and Hosoda (1968) , apparently not aware of our paper, confirmed the above results, reporting that glucose content of the Ehrlich carcinoma ascitic fluid was less than 0.03 mg./100 ml. (the limitation of their version of the glucose-oxidase method). These authors, however, found in the ascitic fluid about 2 mg./ 100 ml. of a reducing, unidentified oligosaccharide which could not be metabolized by the cancer cells. Presumably, a similar carbohydrate was erroneously recognized as glucose by the authors, who determined ascitic sugar by chemical methods. Thus, Warburg and Hiepler (1952) were right in concluding that no glucose was present in the cancer ascitic fluid in vivo. It was reasonable t o assume, however, that the absence of glucose is no more than apparent and that the removal of its avid consumersthe cancer cells-should result in the accumulation of glucose in the ascitic fluid. Indeed, after the ascitic fluid, previously freed from the cells, was reintroduced into the mouse abdominal cavity, glucose appeared in the fluid (Kemp and Mendel, 1957; Gorozhanskaya and Shapot,

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

259

1964). At the height of the glucose concentration plateau, we introduced 1 ml. of the Ehrlich cancer cells back into the cell-free ascitic fluid of the mouse. Immediately, the cells briskly reduced the glucose level to zero (Fig. 2 ) . Thus, it became clear that the apparent absence of glucose in the ascitic fluid is to be regarded as an extremely low dynamic concentration. On the other hand, human erythrocytes, although known as energetically glycolyzing cells, when introduced into the cell-free ascitic fluid of a mouse were unable to diminish within 55 minutes the glucose level, which before reached a plateau (Shapot and Gorozhanskaya, unpublished observation). From the highly reproducible curves 1 and 2 of Fig. 2 a t their very beginning, we have calculated both the real rate of glucose consumption in vivo (which cannot exceed the rate of influx of glucose) and the potential rate, respectively. The difference between the two rates turned out to be enormous: 16-fold. Under limited and optimal conditions, respectively, 0.11 mg. and 1.67 mg. glucose per minute was metabolized by 1 ml. of cancer cells. Tagi-Zade (1971) repeated the above experiments with ascites rat ovarian carcinoma, which grows somewhat more slowly than the Ehrlich tumor. He found that in these cells the potential rate exceeded by 7 times the real rate of glucose consumption. The data mentioned above imply that in vivo tumors realize their potential capacity of metabolizing glucose only partially, sometimes to 3

E 0

0

\

-p

70

c

40 20 30 40 50 60 70 80 90 (00

min.

n o . 2. Effect of the presence of Ehrlich cancer cells on the glucose concentration in the ascitic fluid in vivo. Curves 1, glucose in the cell-free ascitic fluid; curve 2, glucose after the administration of cancer cells.

260

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an insignificant extent, and their true capacities in glucose are never met. It will be shown below what consequences such a situation entails for the tumor-bearing organism. As to the effusions obtained from 28 patients with ovarian carcinoma and 14 patients with lung cancer, the concentration of glucose in the ascitic and pleural fluids, in our experience, was determined not to be significantly lower than that of the blood (Gorozhanskaya et al., 1964). This fact does not contradict the above observations concerning the animal tumors because the glucose levels depend upon the amount of viable, metabolizing cancer cells in the ascitic fluid. With the transplantable tumors the volume ratio of cancer cells to the whole ascitic fluid usually ranges from 1 : 3 to 1:1, whereas, in the human cancer effusions this ratio is negligible-about 1:1.000, as a rule. When cancer cells lost their ability to grow as a result of an effective chemotherapy, the concentration of glucose in the effusion fluid rose, reaching the blood level, and a concomitant fall of the previously elevated concentration of lactic acid was observed. I n this connection one case is noteworthy (Gorozhanskaya e t al., 1964). I n the effusion fluid of a patient with ovarian carcinoma a t her admission to hospital, no glucose was found and lactic acid was determined to be 37 mg./100 ml. Chemotherapy had a favorable effect on the patient and glucose in the ascitic fluid rose to 73 mg./100 ml. She was operated on and released from the hospital with glucose in the effusion as high as 120 mg./100 ml. and lactic acid as low as 12 mg./100 ml. The cultivation of ascites cancer cells in vitro revealed the absence of growth. Two months later the patient was readmitted to the hospital in very poor condition. Analysis of the effusion fluid, which was rich in malignant cells that rapidly grew in culture, displayed a complete absence of glucose and a very high (about 80 mg./100 ml.) level of lactic acid on the day before death. My associate (Tagi-Zade, 1971) analyzed the fluid obtained from secreting mammary cysts in 44 women patients. In 39 cases the lesions were benign and the glucose levels in the fluid ranged from 10 to 20 mg./100 ml. The cysts of 5 patients were recognized to be malignant, and no glucose could be detected in their cystic fluid. It is to be emphasized that a similar situation holds for the inner medium of solid tumors as well. According to Gullino et al. (1964), glucose in the interstitial fluid of normal tissue was present a t levels only slightly lower than in the aortic serum. However, when the growing neoplastic tissues (Walker carcinoma 256, Fibrosarcoma 4956, Novikoff hepatoma, Hepatoma 5123) encircled the diffusion chamber, inserted sub-

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

261

cutaneously into the body, glucose disappeared from the interstitial fluid. The fact that an imperceptibly low dynamic concentration of glucose is maintained both in the cancer cells themselves (observed in 8 various transplantable tumors, Shapot and Tagi-Zade, 1972) and their surrounding media poses a question as to what may be the reason for their ability to metabolize glucose under such unfavorable conditions-the riddle which baffled Warburg and Hiepler as early as 1952. Later on this surprising ability was noted by Burk et al. (1967), who showed that half of the maximal rate of glucose uptake by cancer cells was attained a t the concentration of glucose or only 1 mg./100 ml. whereas 500-750 mg./100 ml. of glucose in the medium was required to reach the same results with normal cells. In this connection, a paper by Hatanaka et aZ. (1969) is noteworthy. Mouse cells transformed by RNAmurine sarcoma virus (MSV) in vitro revealed an extremely low K , for glucose uptake, differing from both uninfected and DNA-virus transformed mouse cells. Since the latter when transplanted into animals, unlike the cells transformed by MSV, grow as benign tumors, it seems that a sharp decrease in K,,, for glucose uptake has some bearing on the malignancy, presumably, on the capacity for destructive growth (see pp. 263, 264). A possibility must be considered that the plasma membrane of cancer cells, which is known to permit leakage of proteins into the medium, is readily permeable to the enzymes of glycolysis and their cofactors. If so, glucose might start degrading before it enters the cell, i.e., in the interstitial or ascitic fluid. We have checked this proposition and obtained a negative answer. The ascitic fluid of Ehrlich carcinoma was indeed found to contain a whole set of glycolytic enzymes, but glycolysis could not proceed because of the lack of essential cofactors. However, once ATP and NAD' were added to ascitic fluid containing a sufficient amount of glucose, a marked glycolysis was observed (Gorozhanskaya et al., 1969). Then we tried to find out whether the plasma membrane of cancer cells is endowed with properties bearing on the problem. Highly purified plasma membranes, isolated from Ehrlich carcinoma cells as well as from Zajdela hepatoma (rat), solid Guelstein hepatoma 22 (mouse), and the respective homologous tissues, were studied. The whole set of glycolytic enzymes including hexokinase with an extremely high affinity for glucose [reminding one of isozyme 111 of Grossbard and Schimke (1966) ] was found in the tumor plasma membrane tightly bound to its lipoprotein complexes. In contrast, the plasma membranes of normal cells were found to be unable to glycolyze. Some hexokinase

262

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activity was manifested, however, provided a very high concentration of glucose was added (60- to 100-fold higher than that used with cancer plasma membrane) .3 Such a difference in terms of hexokinase activities was confined to the plasma cell membranes only and could not be revealed when the soluble fraction of the tumor and normal cell homogenate were compared (Davidova et al., 1968). So it might be suggested that the low K , hexokinase residing in the plasma membrane of cancer cells is able to abstract glucose from the medium even if it is present in a very low concentration. Thus, the product of the hexokinase reaction, glucose 6-phosphate, would be formed in the membrane itself and could be transformed eventually to lactic acid by the remaining glycolytic enzymes, IV. Glycolysis and Destructive Tumor Growth

The mechanism underlying the capacity of malignant tumors to grow destructively, infiltrating adjacent normal tissues, remains obscure. The most popular view relating to the problem appears to be that proteolytic enzymes or toxic substances liberated by tumors into the medium specifically destroy normal cells. The involvement of proteolytic enzymes, and especially cathepsin B, in the phenomenon of tumor invasion has been energetically advocated by Sylven (1968). However, some data are not compatible with Sylven’s suggestion. Leighton (1957) reported an inverse correlation between the extracellular proteolytic activity of various tumor explants and their ability to invade the explants of normal tissues in mixed cultures. The above idea is also not supported by Goldberg et al. (1969), who recorded a reduction in proteolytic activity a t both 6.50 and 3.75 p H in segments of malignant tissues from patients with adenocarcinomas of colon, No morphological or histochemical alterations were seen in kidney tubule cells during invasion by sarcoma 180 cells grafted into the retroperitoneal space of mice. Even the tubules in direct contact with the invading tumor cells remained perfectly normal (Nol, 1964; see Vasiliev and Guelstein, 1966). Only in the tubules completely surrounded by the tumor could lysis of the nuclei be observed (as to the probable significance of the latter finding, see p. 264). Interest in the toxic substances liberated by tumors was revived of late thanks to Holmberg’s data (1965, 1968). A polypeptide that exerted a toxic effect on cells in culture was isolated by Holmberg from ( a ) as-

’The

above phenomenon is in keeping with the isoenzyme hypothesis (Shapot,

1988, 19701, which regards the peculiarities of the tumor metabolism as a consequence

of a shift in the relative content of different molecular forms of the key cell enzymes and their subcellular localization.

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

263

citic fluid of Ehrlich carcinoma and Landschuta carcinoma; (b) interstitial fluid of solid transplantable tumors; (c) blood plasma of tumorbearing mice. The point, however, is that Holmberg tested the toxic effect not on the normal cells (as could be expected), but mainly on various cancer cells: Hela, L-strain mouse fibroblasts, M B 64E malignant lymphoid cells. Hence Holmberg’s data seem to have nothing to do with the problem of destructive tumor growth. I n addition, Rovensky and Bukhman (1965) in Vasiliev’s laboratory showed that the toxic substance with the properties described by Holmberg could never be detected in the cell-free supernatant of fresh Ehrlich ascitic fluid but appeared when ascitic fluid taken from the mouse was incubated for 20 hours at 37°C. Since Holmberg subjected cancer cell-free ascitic fluid to a prolonged dialysis before the isolation and purification of the toxic substance, it seems highly probable that the polypeptide is just an artifact, a product of autolysis. The weak point of any concept ascribing the leading role in the destruction of the neighboring normal tissues to lytic enzymes or toxic substances liberated by the tumor is that it is unable to explain why the above factors act selectively with no effect on tumor cells. A quite different view seems to be better substantiated. The destruction of normal tissues isolated from the organism by the growing tumor may be a result of starvation due to successful competition of cancer cells with normal cells for essential nutrients. As described above, cancer cells in vivo maintain in the surrounding medium an imperceptibly low dynamic concentration of glucose. When the extra supply of glucose due to hyperglycemia counterbalances the rapid glucose consumption by cancer cells, i.e., “saturates” them, glucose appears in both the interstitial fluid of solid tumors (Gullino et al., 1964) and ascitic fluid of ascites carcinomas (Tagi-Zade, 1971). These observations provide another way to detect a great discrepancy between the potential rate of metabolism of glucose by tumors and the real rate which is limited by a scarce influx of glucose from the body under the usual conditions. It is evident that the magnitude of such a discrepancy must be directly correlated with the capacity of cancer cells for anaerobic glycolysis. Weber and Morris (1963), and especially Burk et al. (1967)-with various Morris hepatomas differing in growth rate, which reflects the extent of their malignancy-have shown that the growth rate of tumors is correlated with the intensity of anaerobic glycolysis. There is no doubt that some important biological meaning must be concealed in the above correlation. We believe that the extensive

264

V. S. SHAPOT

anaerobic glycolysis, which determines the behavior of cancer cells as avid consumers of glucose, is to be regarded as a powerful instrument of destructive growth. Once the cells of the growing tumor encircle a fragment of adjacent normal tissue, isolating it from the organism, glucose, an essential nutrient, becomes practically inaccessible to the normal cells captured by the tumor, and they die of starvation (cf. the experiments of Nol, p. 262). We do not think, of course, that the proposed mechanism can be considered as universally true for every kind of tumor. It is rather a first attempt to connect a general biochemical feature of the malignant cell with one of its biological properties. V. Relative Glucose Deficiency of the Tumors Growing in the Body

The fact that the potential capacity of cancer cells for metabolizing glucose greatly exceeds the actual utilization of glucose by the tumor under in vivo conditions clearly indicates that the tumor must be constantly in a state of relative “glucose hunger,” although the glucose supply from the body is sufficient to ensure tumor growth. If so, the tumor in vivo is expected to be able to consume and metabolize extra amounts of glucose on hyperglycemia. Several lines of evidence demonstrate that this is indeed the case.

EFFECT OF HYPERGLYCEMIA ON CANCER CELLS 1. Glucose Consumption Gullino et al. (1967), using ingenious “tissue isolated preparations” for the study of tumor metabolism in vivo, have shown that when a high hyperglycemia (above 600 mg./100 ml. plasma) was induced in the host the arteriovenous (A-V) difference in glucose content in tumor blood measured 10 minutes later increased 3- to 10-fold as compared with normoglycemia. Tagi-Zade (1971) examined 12 patients with cancer of the stomach. During operation, blood was taken from the brachial artery and vein as well as from the vein draining the tumor. The second time, blood was sampled from the same vessels after 1 gm. of glucose per kilogram of body weight had been injected in the vein of another arm. It was found that in normoglycemia the A-V difference of peripheral blood in glucose averaged 6 mg. per 100 ml. of plasma while the A-V difference of tumor blood ranged from 9 to 17 mg. 100 ml., averaging 13 mg. per 100 ml. of plasma. Hyperglycemia brought about’ a 3-fold increase in the A-V difference in glucose of tumor blood.

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

265

Still more spectacular results were obtained with rabbits carrying Brown-Pearce tumor (Tagi-Zade, 1971). The tumor was implanted into a testicle, where it grew locally within the first 10 days, replacing almost entirely the tissue of the organ. At this time the animals were operated under ether narcosis to separate the veins of both testicles. The blood was taken from testicular veins as well as from the mesenteral artery and auricular vein. As seen from Table I, and A-V difference in glucose of the blood of the intact twin testicle averaged 5 mg. and in the tumor blood 18 mg. per 100 ml. of plasma. Ten minutes after the introduction of 1 mg. of glucose per gram of body weight into the vein of another ear, the A-V difference of tumor blood in glucose rose to 57 mg. per 100 ml. of plasma. It is noteworthy that, provided hyperglycemia was maintained for over 6-7 hours, the elevated consumption of glucose by the tumor declined and reached a level not exceeding 140% of the normal uptake despite the large availability (Gullino e t al., 1967; Gullino, 1970). Similar observations were reported by Tagi-Zade (1971). So it seems that prolonged hyperglycemia “saturated” the capacity of cancer cells to use large amounts of glucose. The reason for this phenomenon is not clear. I t is possible that the glucose concentration gradient between afferent blood and the tumor is being leveled off, causing a diminution of glucose uptake by the tumor. TABLE I UPTAKEOF GLUCOSP (ARTERIOVENOUS DIFFERENCE) BY TUMORin Vivo Usual conditions

THE

BROWN-PEARCE

After glucose injection of 1 mg. per gram of body weight

Animal No.

Mesenteric artery

1 2 3 4 5 6 7 8 9

66 69 82 64 83 74 75 78 80

59 62 76 60 78 69 70 73 74

48 51 63 49 64 57 55 59 60

270 301 279 296 310 286 29 1 315 321

254 282 270 288 301 279 285 307 312

221 246 209 218 248 242 251 255 266

74 f 7

69 f 7

56 f 6

296 f 17

286 f 18

239 f 19

Mean

Testicular Testicular (tumor) Mesenteric vein artery vein

~~

Testicular vein

Testi cular (tumor) vein

~~

a

As milligrams per 100 ml. of plasma.

-

266

V.

S. SHAPOT

The fact that malignant cells are avid glucose consumers in vivo has been reported by many authors (Cori and Cori, 1925, 1929; Warburg, 1930; etc., see also below). Now we are able to suggest a quantitative basis for this property of the tumor. Owing to an undetectably low glucose level which is maintained in vivo in the tumor and surrounding medium, interstitial or ascitic fluid, an enormous, quite unusual gradient in glucose concentration between afferent blood and the tumor-as high as 60-80 mg. per 100 ml. of plasma to zero-is established. Such a gradient makes the tumor act as a powerful pump, constantly draining the host in terms of glucose (see also below).

2. Lactic Acid Production An increased consumption of glucose by the tumor in vivo, induced by hyperglycemia, allowed one to anticipate a stimulation of glycolysis under these conditions. As early as 1925 Cori and Cori reported a selective accumulation of lactic acid (up to 1 W 1 8 0 mg./100 ml.) in the tumor after glucose was injected into the host, whereas the content of lactic acid in the liver of the same animal remained unchanged. In 6 cases where patients developed large tumors comprising a significant proportion of the body weight, an increase in the lactic acid content in the blood could be observed (Cori and Cori, 1925). According to Norman and Smith (1956), a statistically significant rise of the lactic acid concentration in the blood was recorded 5-10 minutes after injection of glucose into the animals with transplantable hepatoma and mammary carcinoma. Tagi-Zade (1971) has shown that a 2-fold increase in the lactic acid level of ascitic fluid of ovarian rat carcinoma occurred within an hour after 180 mg. of glucose was administered into tumor-bearing rats. 3. Shift in the p H Values

It was observed long ago (Kahler and Robertson, 1943; Voegtlin et al., 1935) that the pH of transplantable tumors dropped significantly after the administration of glucose whereas the pH of normal tissue did not change. This phenomenon undoubtedly is a result of the overproduction of lactic acid by the tumor. Recently von Ardenne et al. (1969a) , using a perfected device for pH measurement in the body, recorded a drop of the tumor pH value down to 6 when hyperglycemia, up to 600 mg. per 100 ml. of plasma, was induced in the tumor-bearing animal. The phenomenon of tumor “self-acidification” was suggested as a way to raise selectively the sensibility of tumors to the action of damaging factors (von Ardenne et al., 1969b; Shapot, 1968, 1970).

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

267

4. Stimulated Tumor Glycolysis as a Source of Extra Energy

If the unlimited glucose supply to the tumor was ensured and thereby the conditions were improved for unrestricted glycolysis of cancer cells in vivo, one would expect that at least a part of the extra energy might be utilized by the cells for protein synthesis. Tagi-Zade and Shapot (1970a) used rat sarcoma M 1, carcinoma GuBrin, ascites ovarian carcinoma, mouse Crocker sarcoma, and ascites Ehrlich carcinoma to show that hyperglycemia up to about 300 mg. per 100 ml. of plasma stimulated significantly the incorporation of essential amino acids-methi~nine-~~S, l y ~ i n e - ~ ~ G - i nthe t o tumor protein within 30-180 minutes of exposure, whereas the labeling of the liver protein of the same animals remained unchanged. 5. Utilization of Glucose by the Tumor

Gullino et al. (1967) reported that solid tumors studied in vivo as “tissue-isolated preparations” converted about one-third of the glucose consumed into lactic acid under conditions of normoglycemia. Tagi-Zade (1971) thoroughly studied 31 animals carrying rat ovarian ascites carcinoma to find the proportion of glucose, administered intraperitoneally and metabolized by cancer cells, that could be recovered as lactic acid. After the influx of the endogenous glucose into cell-free ascitic fluid within the first few minutes, and the diffusion rate of lactic acid from ascitic fluid into the body was assessed, the ratio of lactic acid formed to glucose consumed was determined to be as low as 1:8. These results prompted us to look for ways other than lactic acid production by which the bulk of glucose consumed is utilized by cancer cells. Experiments carried out with rat sarcoma M 1 and Guhrin carcinoma (Tagi-Zade, 1971) showed that even under hyperglycemia induced in the host the tumor cells remained very poor in glycogen. Thus, conversion by the tumor of excess glucose into glycogen seems unlikely. According to Campbell and Halliday (1957), injection of glu~ose-’~C into tumor-bearing animals resulted in synthesis by the tumor of amino acids, mainly alanine. Kit and Griffin (1958) observed extensive synthesis of labeled alanine, glycine, and serine in cancer cells incubated with g l u c ~ s e - ~ ~exceeding C, 30-50 times that observed in several kinds of normal cells treated under similar conditions. Shapot and Tagi-Zade (1972) studied the incorporation of alanine-I4C into tumor protein when hyperglycemia was induced in the host. Crocker carcinoma, ascites Ehrlich carcinoma, ovarian ascites carcinoma, and Gubrin carcinoma were used. I n all cases a 2- to %fold decrease was found in the labeling of the tumor protein in the hyperglycemic animals as compared with

268

V. S. SHAPOT

the control normoglycemic ones. The more glucose introduced into the body, the greater was the decrease. These results can be interpreted as indicating an extensive conversion of extra glucose available to cancer cells into unlabeled alanine, which isotopically diluted alanine-"C, thereby reducing the specific radioactivity of the alanine pool. In separate experiments, as yet unpublished, we became convinced that the label from the randomly labeled glu~ose-'~Cadministered to the tumor-bearing animals was indeed transferred into tumor protein. Thus, it seems highly probable that the 3-carbon fragments of glucose resulting from its glycolytic breakdown are actively involved in synthetic processes-in particular, in forming nonessential amino acids. It appears that cancer cells are also able to transform extensively the carbon skeleton of glucose into pentose of nucleic acids (Eltzina and Engelhardt, 1958; Eltzina, 1965). It is noteworthy in this connection that, according to Tagi-Zade (1970), a prolonged hyperglycemia maintained almost constantly from the day of transplantation of cancer cells prevents the development of tumor necrosis, although slowing down tumor growth, as already reported previously by Goranson (1955) ; Ciaccio e t al. (1965) ; and others. Presumably an enhancement of synthetic processes in the tumor due to a great pool of readily available precursors is the cause of the above phenomenon. The reason why tumor growth is not favored by hyperglycemia in spite of the stimulation of enhanced syntheses is a problem that still remains to be solved. VI. Tumor as a Glucose Trap in the Body

It was mentioned above that, because of the enormous gradient in glucose content between the arterial blood supplying the tumor and the tumor itself, the tumor acquires the capacity to consume relatively large amounts of glucose. Presumably the bulk of glucose metabolized by the tumor is completely lost for the host, being utilized to build tumor proteins and nucleic acids. The rest is converted into lactic acid which may partially be transformed by the liver back to glucose, and then to glycogen, ready to meet requirements of both the tumor and the host. If the tumor in the body functioned as a trap for glucose, some predictions might be warranted in terms of the shifts brought about. in the host's balanced metabolism and the physiological systems involved in its control. I n particular, a tendency toward hyperglycemia in the tumorbearing body, on one hand, and hyperactivity of the physiological systems counterbalancing this tendency, on the other hand, are expected to arise.

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

269

A. TUMOR AS A HYPOGLYCEMIC FACTOR 1. Diabetes

As early as 1924 Braunstein reported a significant drop in the elevated levels of the blood sugar and the disappearance of glucosuria in diabetic patients after they had developed cancer. As soon as the tumor was removed, glucosuria reappeared. More recently a case was reported (Vaissman et al., 1964) where effective treatment with cyclophosphamide of a patient with lymphosarcoma resulted in a marked regression of the tumor and return of the diabetic state. Novikoff hepatoma and Walker carcinoma 256 were found to reduce the blood sugar levels when transplanted into rats with alloxan diabetes (Goranson et al., 195413; Goranson and Tilser, 1955). Similar results with Walker carcinoma were obtained by Garvie (1968). I n control alloxanized animals, the concentration of glucose was found to be 230 mg. per 100 ml. of plasma, and the value in the diabetic animals with tumors was 160 mg. per 100 ml. of plasma. Lymphatic leukemic neoplasms in diabetic mice were reported to have a pronounced ameliorative effect on their hyperglycemia (Silverstein et al., 1964). Mouse tumors decreased the blood glucose concentration of hereditary obese-hyperglycemic mice as well as of alloxan-diabetic mice (Jehl et al., 1955). Tagi-Zade and Shapot (1971) decided to reinvestigate the problem using a relatively large number of animals carrying three different tumors. Ten days after the last portion of alloxan was administered to the animals, tumor cells were transplanted into them. A group of alloxanized animals without tumors served as control. Blood was taken for analysis twice, immediately before transplantation and 17 days afterward. The blood sugar levels of the animals from the control group, elevated as it is, rose still more within 17 days (Fig. 3 ) . Quite different results were obtained with the tumor-bearing alloxanized animals; in all cases a marked, statistically significant reduction in the concentration of blood glucose was observed. It is noteworthy that the growth of all three tumors studied in the diabetic hosts was found to be inhibited 6&70% (cf. also Vangerov and McKee, 1955) ; Goranson et al., 1954a,b; Goranson and Tilser, 1955; Garvie, 1968). However, the small tumors were greatly eroded by extensive necrosis, and the life-span of the alloxanized tumor-bearing animals did not differ from that of the control alloxanized group. 2. The Problem of Hypoglycemia in Tumor-Bearing Organisms

When one examines the data reported by many authors who-in the course of investigations aiming at some other goals measured inci-

270

V. S. SHAPOT

-

240

' t z6

I '1

0

e

220

-

216

240 E 200 ci

190 n

.a t8

I70 I60 150

1 Control

2 3 4 Gurrln Sarcoma Ascltes ovarian MI carcinoma carcinoma

FIQ.3. Blood glucose levels in alloxaniced tumor-bearing rats. dentally the blood sugar levels of the animals carrying various tumorsattention is attracted by somewhat lower values than those found in the healthy animals. For instance, the average glucose content in 20 mice with fibrosarcoma was determined chemically to be 75 A 10 mg. per 100 ml. of plasma in comparison with 115 & 15 mg. per 100 ml. of plasma of 20 normal mice (Mallick et al., 1968). Similar results were obtained by Del Monte and Rossi (1963), who noted that rats with Yoshida hepatoma were hypoglycemic: the blood sugar level was as low as 50 mg. per 100 ml. of plasma while in the control animals it was 80 mg. per 100 ml. of plasma. Tagi-Zade and Shapot (1971) made an attempt to characterize glycemia in the animals carrying 6 different tumors in both solid and ascites forms. As seen from Fig. 4, with all tumors studied there is a marked tendency toward hypoglycemia in tumor-bearing animals. The decrease in the blood sugar was not large, but it was statistically significant ( P < 0.001). On autopsy of the animals from the experimental group, a direct correlation was found between the s h e of tumors and the magnitude of the decrease in the blood sugar levels, as well as an inverse correlation between this decrement and the amount of necrosis. It appeared that the extent of hypoglycemia depended on the number of viable cancer cells. A similar correlation between the magnitude of hypoglycemia and growth of lymphatic leukemic tumor in mice was observed by Silverstein et al. (1964). These findings prompted us to undertake a new series of experiments in which the dynamics of changes in the extent of hypoglycemia in the course of development of a tumor could be followed with individual

BELATIONSHIP BETWEEN THE TUMOR AND T H E HOST

2 Rats

3

4

5

6

271

7

Mice

FIQ.4. Hypoglycemia in tumor-bearing animais. 1 and 6, control groups; 2, Gukrin carcinoma; 3, sarcoma M 1 ; 4, ascites ovarian carcinoma; 6, Crocker carcinoma; 7, ascites Ehrlich carcipoma.

tumor-bearing animals (Tagi-Zade and Shapot, 1972). Brown-Pearce carcinoma was implanted into testicles of rabbits, and blood was sampled before transplantation of the tumor cells and three times afterward. Then the animals were sacrificed, and a thorough examination of the size of tumors and metastases was performed; the results were expressed quantitatively in conventional units. Twenty animals were studied, and the same regularity, with no exception, was observed. As found previously by Tagi-Zade, 10 days after inoculation into testicles of rabbits, BrownPearce carcinoma started to develop metastases, the number of which greatly increased within 20-30 days after inoculation. The blood sugar levels diminished slightly within the first 10 days of tumor development, and the most pronounced decrement was observed within the last 20-30 days, the extent of hypoglycemia largely depending upon the spread of the tumor growth (Table 11).These data are in keeping with the earlier observations of Silverstein et al. (1964), who reported a marked decrease in the blood sugar levels of mice implanted with lymphatic leukemic cells, the decrement reaching 3&60 mg./100 ml. on day 17 after inoculation. The next step in the exploration of the problem was an attempt to find out whether a tendency toward hypoglycemia could be detected with cancer patients (Tagi-Zade, 1971), using a specific glucose-oxidase method to get the true values for the blood glucose. Patients with tumors of similar localization were separated into two groups; one group was

V. S. SHAPOT

272

TABLE I1 WITH BROWN-PEARCE CARCINOMA DEVELOPMENT OF HYPOGLYCEMIA IN RABBITS

Glucose (mg./100 ml. plasma) Days after inoculation Animal

No. 1

2 3 4 5 6 7 8 9 10

Before inoculation

10

20

30

84 73 68 69 78 87 90 89 76 79

78 68 62 60 73 80 81 81 60 72

70 53 49 45 69 62 75 69 61 60

42 36 38 50 42 30 57 39 46 33

Quantitative assessment of the extent of tumor development Final (arbitrary decrement unih)

42 37 30 19 36 57 33 50 30 46

57 94 35 28 37 103 28 81 44 96

maintained on the usual diet and the second group on a diet enriched in carbohydrates for 3 to 4 days before the analyses were performed. I n case of cancer of the esophagus or stomach, with stenosis, such patients were given glucose intravenously (20 ml., 40%, twice daily). Figure 5 shows that human neoplasms cause a slight hypoglycemia similar to that developed in tumor-bearing animals. This drop in the blood sugar levels was statistically significant, but differences in the blood glucose content between the two groups of patients were but apparent. This implies that the extra carbohydrate supply is insufficient to normalize glycemia when the tumor is draining the body of glucose, but counterregulatory mechanisms are still efficient enough to compensate for increased utilization of glucose. But what happens to the patient when the tumor exceeds the average size and becomes very large? What are the consequences of such a situation for the balance of the carbohydrate metabolism in the host? About 200 cases of severe hypoglycemia (sometimes lethal) in cancer patients have been described. The tumors were both of epithelial and mesothelial origin and did not derive from endocrine organs. The blood sugar levels of such patients ranged from 50 mg. down to 15 mg. and even to 6 mg. per 100 ml. of plasma; these levels led not infrequently to hypoglycemic comas and various kinds of mental disorders (for reviews, see Conn and Seltzer, 1955; Lowbeer, 1961; Bower and Gordon, 1965; Unger, 1966; Papaioannou, 1966; McFadzean and Jeung, 1969;

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

273

641n

62

E

54 -

0

g n o)

f

.-

52

-

5048-

-

o)

46

8

44-

u)

'

42,

20

4

2

3

4

5

FIG.5. Hypoglycemia in cancer patients. 1, healthy persons; 2, stomach cancer; 3, the same with stenosis; 4, cancer of the esophagus; 5, lung cancer. Encircled figures indicate number of patients examined. 0 , usual diet; a, cnrbohydrnte diet.

Kreisberg and Pennington, 1970; Silverstein et al., 1964; Silverstein, 1969). A common denominator of these cases was the extremely large size of the tumors, which ranged from 900 gm. to 10 kg. and averaged 3 kg. A tumor of 18 kg., comprising 40% of the body weight a t the time of the patient's death, was reported by Carey et al. (1966). Wide experience supports the view that a massive tumor is to be regarded a prerequisite to the devcloprnent of pronounced hypoglycemia (McFadzean and Jeung, 1969). Hypoglycemia appears not to be dependent solely upon the size of the tumor; the total mass of viable, metabolizing cells is what counts. The onset of extensive necrosis in the tumor as a result of effective radiotherapy or chemotherapy without a measurable diminution of its size leads to prompt amelioration of hypoglycemia (McFadzean and Jeung, 1956 ; Papaioannou, 1966). The capacity to induce hypoglycemia seems to be a property of malignant tumors, since huge benign tumors are reported to grow for years (up to 47) causing no signs of hypoglycemia. Hypoglycemia appears shortly after thc tumors become malignant (Mahon et al., 1962; Sors et al., 1962). As a rule, the hypoglycemic attacks cease after the excision of the tumor until the malignant tumor recurs or metastasizes and again becomes extensive (cf. D. R. Miller et al., 1959; Lowbeer, 1961; Crocker and Veith, 1965; Scholz et nl., 1957; Boshell et ul., 1964; Folsch et al., 1964). I n a healthy 70-kg. man the output of glucose by the liver to meet

274

V. S. SHAPOT

the daily requirements of the body is 10CL300 gm. (Renold et at., 1960). To prevent hypoglycemic attacks in cancer patients much larger amounts of glucose are necessary. It was necessary to administer 825 gm. (McFadzean and Jeung, 1956), 872 gm. and 1210 gm. (Beers and Morton, 1935), 1500 gm. (Sellman et al., 1959), and even 2250 gm. (Crawford, 1931) of glucose to patients for this purpose. I n most cases no manifestations of hepatic dysfunction were observed (Crawford, 1931; McFadzean and Jeung, 1956; Marks et al., 1965). No hypoglycemia-inducing tumor has ever been found to contain immunoassayable insulin (Lipsett et al., 1965; Unger, 1966; Tranquada et al., 1962). No more than one case (fibrosarcoma) has been reported with a high seruminsulin level during the hypoglycemic phase, measured by immunoassay (Oleesky et aE., 1962). Most attempts to reveal “insulinlike” substances released by the tumors of hypoglycemic patients failed. Only two instances are known in which increased “insdinlike” activity has been demonstrated in the serum (for references, see Field et al., 1963; Papaioannou, 1966). I n general no biochemical abnormalities other than hypoglycemia were observed in cancer patients with tumors of nonendocrine origin (Kreisberg and Pennington, 1970; Jakob et al., 1967; Carey et al., 1966). A case recently observed in the animal clinic of our Institute is noteworthy in this connection. A female dog with a giant spontaneous mammary carcinoma (6.3 kg., 41 X 23 X 19 cm.), which developed within 3 months, displayed severe hypoglycemia. Three days before death the blood sugar level measured by the glucose-oxidase method was less than 6 mg. per 100 ml. of plasma. On autopsy, no metastases could be detected. Many authors have concluded that severe hypoglycemia in cancer patients must be due mainly to the excessive consumption of glucose by the large tumor (Hines, 1943; H. Klein and Klein, 1959; Sellman et al., 1959; Bonsavaros, 1960; Folsch et al.,1964; Duncan and Schless, 1961; Landau et al., 1962; Tranquada et al., 1962; Carey et al., 1966; McFadzean and Jeung, 1969). Evidently the desperate need for glucose produced by the presence of a large mass of viable cancer cells in the body greatly exceeds the potential capacity of counterregulating mechanisms to produce the amounts of glucose required; i.e., “demand outstrips supply,” according to McFadzean and Jeung (1969). Carey et al. (1966) calculated that a tumor of 18 kg. would require 1730 gm. of glucose per day, an amount double the estimated rate of glucose replacement in the patient with such a tumor. I n this connection, the possibility of an impairment of compensatory mechanisms must be considered as well, since it may be a factor en-

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

n5

hancing the discrepancy between demand for glucose and its supply (Unger, 1966). The interrelationship of the blood sugar levels and the efficiency of compensatory mechanisms is illustrated by the different rates of replacement of glucose in a patient with a large fibrosarcoma: 1.50 mg./kg. per minute when the patient was hypoglycemic and 5.23 mg./ kg. per minute when normoglycemia set in (Volpe et al., 1965). It is noteworthy that severe hypoglycemia can develop in a cancer patient even when the tumor contains a large amount of glycogen, up to 30 mg. per gram (Silvis and Simon, 1956). Thus tumor glycogen seems to be unavailable to the body economy (McFadzean and Jeung, 1956). Moreover, it is unavailable to the tumor itself, since the latter avidly consumes glucose derived from the body while keeping its glycogen reserves.

B. ON THE DEPOSITION OF GLYCOGEN BY THE LIVEROF THE HOST It has been shown by many authors (Engel, 1942; McFadzean and Jeung, 1956; Shonfeld et al., 1961; Landau et al., 1962; Grusdeva, 1966; Carey et al., 1966; Tashiro and Sakurai, 1970) that the liver of tumorbearing organisms both human and animal contain relatively small amounts of glycogen. Unambiguous interpretation of this observation, however, is not possible so far. It may be a result of an increased need for glucose, owing to the presence of the tumor. Equally probable is an impairment of the enzyme systems of the liver involved either in glycogen synthesis or its mobilization, as a result of a toxic action of the tumor. A simple experimental approach, the epinephrine test, has been chosen to elucidate the problem (Tagi-Zade and Shapot, 1970b). The results support the first idea. The experiments were conducted with 750 rats and over 400 mice, carrying 5 different tumors, both solid and ascites. Tumor-bearing animals maintained on the usual diet responded very poorly to epinephrine administration. The peak of their hyperglycemic curves reaching but 60% of the height of the control peak (Fig. 6 ) . Histochemical examination revealed a very low content of glycogen granules in the liver of rats carrying sarcoma M 1 and Gudrin carcinoma as compared with that of normal ones. Quite a different picture is seen in Fig. 7, which represents the situation in which all the animals, both experimental and control, were “saturated” with glucose given parenterally on the day of the experiment, the injections being stopped 2 hours before the epinephrine test was started. In this case the swing of the hyperglycemic curves greatly increased, and the response of tumor-bearing animals did not lag behind that of the control rats. According to a histochemical study, the extra

276

V. S. SHAPOT 430

(20

.E 60 U 0)

8 a

50

5 40 30

0 20 40 6 0 00 400420 min.

FIG.6. Epinephrine test: rats, usual diet. 1, Control group; 2, Gu6rin carcinoma; 3, sarcoma M 1; 4, ascites ovarian carcinoma.

4P

30

{

1 0 20 40 60 80 100 120 min.

FIG.7. Epinephrine test: rats, “presaturation” with glucose. 1, Control group; 2, Gu6rin carcinoma; 3, ascites ovarian carcinoma; 4, sarcoma M 1.

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

277

140 430

- 120 0

9

-2 U

0 0

5 0)

f

c .-

-

4 30

CT

E I20

0

410

B

110

- ioo

I00

E

90

80 c

c 70 .-

70

0)

0)

g 60

g 60 0

0

5 50

50 40

30

0 20 40 60 80 400120 min.

40 30

11 0 20 40 60 80 400420 min.

FIG.8 . Epinephrine test: cancer of stomach. 1, Healthy persons: (A) Before treatment: 2, usual diet; 4, carbohydrate-rich diet. After ineffective therapy: 3, usual diet; 5, carbohydrate-rich diet. (B) Before treatment: 2, usual diet; 4, carbohydrate-rich diet. After excision of the tumor: 3, usual diet; 5, carbohydraterich diet. Each curve represents no fewer than 15 persons examined. Data on two different diets correspond to the same group of patients.

supply of glucose led to an extensive deposition of glycogen by the liver of the tumor-bearing rats, approaching that observed in the liver of control animals. These results clearly indicate that the liver enzyme systems of both glycogen synthesis and its breakdown remain intact in spite of the presence of the tumor. The most probable reason for poor glycogen reserves in the liver of such animals is the increased mobilization of glycogen presumably stimulated by glucagon (cf. Sokal et al., 1964). It is quite possible that not only liver but, in some instances, muscle glycogen is being mobilized in the host under the influence of the growing tumor (cf. Nigram et al., 1962). Tagi-Zade (1969) applied the epinephrine test to patients with cancer of the stomach, esophagus, and lung, trying to determine whether the test can reveal the presence of a tumor depleting the liver of its glycogen reserves. The results of the study seemed positive. The patient’s liver appeared

278

V. S. SHAPOT

to be depleted of glycogen to a greater extent than those of animals carrying various tumors (Fig. 8 ) . Palliative operations had no effect on the character of the epinephrine test. However, once the tumor was removed completely, there was a tendency of the hyperglycemic curves toward normalization.

C. GLUCONEOGENESIS IN THE BODYAND TUMOR DEVELOPMENT The second physiological system of the tumor-bearing organism which must, according to the above predictions, function under great strain is the adrenal cortex, which is known to produce the glucocorticoids that stimulate gluconeogenesis. There have been indications in the literature that in the course of tumor growth there is hyperplasia of the adrenal cortex and hypersecretion of glucocorticoids. The final phase of tumor development was reported to be characterized by emaciation of the gland, and depletion of the glucocorticoid reserves (Josserand, 1953 ; Snydor and Sayers, 1954; Huhn and Schneppenheim, 1954; Nadel and Burstein, 1956; Allot and Skeleton, 1960; Hilf et al., 1961; Samundjan, 1966; Lichter and Sirett, 1968; Agayev, 1968; Agayev and Ibragimov, 1969; Mizukami et al., 1969; Kawai et al., 1969; Evgenieva, 1970). One can cite some other data in the literature conflicting with these. Therefore, we tried to find out whether in the body carrying a tumor gluconeogenesis does in fact proceed at an elevated rate. It is well known that there are several links in the metabolic chains which are affected by glucocorticoids to enhance gluconeogenesis (for a review, see Weber, 1968); ( a ) liberation of amino acids and free fatty acids along with glycerol as a result of the breakdown of muscle proteins and adipose tissues, respectively, and ( b ) induction of the liver enzymes involved in the conversion of the carbon skeleton of amino acids into glucose. We have determined the extent to which tyrosine-"C, le~cine-'~C, and acetic acid-I4C (as a product of fatty acid oxidation) were transformed to glucose-14C in rats with Zajdela ascites hepatoma as compared with normal animals (Shapot and Blinov, 1972). As seen from Fig. 9, a pronounced, 2- to 3-fold stimulation of gluconeogenesis from both amino acids and acetic acid in the serum of tumor-bearing animals was observed. It is noteworthy that leucine-a ketogenic amino acid-was converted into glucose like tyrosine, but the rates of the process were different; glucose derived from leucine appeared to be over 400 times slower than from tyrosine. However, the extent of the stimulation of glycogenesis in the animals with hepatoma in both cases was similar. We are aware that a phenomenon observed with only one transplantable tumor is not sufficient for any generalization, but it merits attention since it is in keeping with the substantiated anticipations and above-

RELATIONSHIP BETWEEN THE TUMOR AND THE HOST

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mentioned data as to the hypersecretion of glucocorticoids in the tumorbearing organisms. It is quite possible that an elevated level of gluconeogenesis is caused by the hypersecretion not only of glucocorticoids, but of epinephrine (Rinard et al., 1969) or glucagon as well. The latter has been found to augment hepatic gluconeogenesis in intact man (Salter et al., 1960) and in the isolated perfused rat liver (L. L. Miller, 1960). It can be added that the treatment of tumor-bearing animals with glucocorticoids stimulates metastasis development (Gasic and Gasic, 1955 ; Iversen, 1957; Ghose, 1958; Kallum and Saldeen, 1967), presumably through the reduction of the function of RES entailing the involution of the lymphatic tissue, i.e., undermining body defense mechanisms. It is also noteworthy that the capacity of a steroid to increase metastasis is correlated with its glucocorticoid activity (Albert and Zeidman, 1962). Thus, it is likely that in some instances a tumor growing in the host paves the way for its own dissemination. VII. The Problem of Cancer Cachexia

Everything about this problem is still obscure; even the definition of the phenomenon has not yet been achieved and general agreement has not been attained as to whether cachexia observed in some cancer patients and tumor-bearing animals is to be regarded as specific for malignancy. A drastic loss of weight, breakdown of tissue proteins and lipids of

280

V. S. SHAPOT

adipose tissues, a negative nitrogen balance, and dysfunction of important physiological systems are recognized as visible signs of cancer cachexia. The above syndrome, of course, in its entirety is not often seen in patients. We believe that one of the general manifestations of the effect exerted by the developing tumor on the body might be a forced utilization of tissue proteins and lipids as a source of carbon skeleton for glucose formation, i.e., a stimulated gluconeogenesis, which we succeeded to observe in the animals with Zajdela hepatoma (see above). Observations reported by Cahill (see De Wys, 1970) support the above view. Increased alanine in the blood of patients with advanced cancer was recorded and evidence was presented that alanine enters the blood at the expense of the breakdown of muscle proteins. The alanine circulates to the liver, where it is converted into glucose. Cahill stated that this catabolic loss of amino acid from muscle may be responsible for much of the weakness and cachexia of advanced cancer. According to Cahill’s group (Felig et al., 1970a,b), alanine among the other amino acids has been shown to be quantitatively the most important gluconeogenic substrate on starvation. Gold (1968) proceeding from a quite different assumption also came to the conclusion that . . . “gluconeogenesis appears to be a most vital link in the cachexic process” since, as he suggested, it is expected to deplete body energy reservoirs in terms of ATP. Gold was seeking the ways to maintain low blood glucose values as a tool for fighting cancer cachexia. As for us, we think that an exactly opposite tactic must be effective. One has to help the host so that it will not need to mobilize its compensatory mechanisms for maintaining the normal blood glucose level at the expense of the breakdown of the body proteins and lipids. Weinhouse with his group (Reichard et al., 1964) has also come to the conclusion that a constant drain on the glucose reserves might be expected to require considerable adjustment in gluconeogenesis mechanisms and thus profoundly affect the metabolic status of the tumorbearing host. So the administration of glucose, a mixture of amino acids and lipid emulsion (as readily available source of energy) is expected to exert a beneficial effect on the cancer patient, thus preventing many signs of cachexia. Dudrick (see De Wys, 1970) described several patients with cancer who were given (through infusion) protein hydrolyzate and hypertonic glucose. This therapy was found to reverse cachexia with significant weight gain and gain in muscle mass. A continuous wasting with all obvious signs of cachexia was shown to be characteristic of those cancer patients who suffered from impaired nutrition caused by stenosis of the stomach or esophagus or anorexia as

BELATIONSHIP BETWEEN THE TUMOR AND THE HOST

281

a side effect of chemo- or X-ray thcrapy (Sudjan, 1970). Such a situation may be brought about just by starvation or by any kind of acute stress reaction as well as by lesions connected with the hypersecretion of glucocorticoids. I n this sense the above syndrome cannot be considered as.strictly specific for cancer cachexia. However, one has to bear in mind that many cancer patients die with no metastases in the vital organs, their tumors being of moderate size. Two groups of patients (120 and 230) who died of cancer of the stomach have been studied (Sorokin, 1972). About 30% of them died without developing metastases. Dystrophy of inner organs was recognized as the main cause of death. It is possible that a limitation of critical amino acids channeled into the process of forced gluconeogenesis might be one of the reasons for deficient protein synthesis in some of the inner organs. There is no doubt that the growing tumor exerted a profound distant effect on the enzyme systems of body tissues. A well-known manifestation of such an effect is a drastic reduction in liver catalase activity presumably due to the arrested synthesis of the enzyme. Recently a 15- to 20-fold diminishment in the latent endonuclease activity of highly purified liver ribosomes obtained from the tumor-bearing animals has been observed by Krechetova in our laboratory. One can add that the template activity (revealed using purified E. coli RNA-polymerase) of the isolated nuclei or chromatin from the “normal” rat liver tissue not adjacent to the chemically induced hepatoma drops by 50% according to the observations made in our laboratory by Adler and Mironov this year. It implies that the genetic expression in such a vital organ as liver seems to be substantially impaired in the host, and this effect exerted by the tumor must diminish the functional capacity of the organ. Thus, cancer cachexia seems to be an extremely complex process including both nonspecific and specific features for malignancy. It is hoped that replacement therapy, by sparing the tumor-bearing body the trouble of using and exhausting its compensatory mechanisms, might serve as a powerful instrument for fighting cancer cachexia. VIII. Conclusion

The data described above leave no doubt that the tumor growing in the host acts as a powerful hypoglycemic factor, setting in motion a long chain of events. A tendency to disturb the body homeostasis in one link-carbohydrate metabolism-may result in the derangement of the protein and lipid metabolism, owing to a forced gluconeogenesis as well, thus requiring the endocrine systems, counterbalancing the effect of the tumor, to function under a constant strain and become prone to exhaus-

282

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tion. A high need of the tumor for glucose under in vivo conditions seems to be enhanced by ( a ) the extensive, uneconomical anaerobic glycolysis operating in the tumor because of local hypoxia, and ( b ) the fact that the tumor is devoid of the enzymes of gluconeogenesis; i.e., it is unable to produce glucose itself. The tumor surely is not only a trap for glucose in the body; such a view would sound too one-sided. As far back as 1948, Mider e t al. (cf. also Mider, 1951) shrew,dly pointed out that the tumor is to be regarded as a trap for nitrogen, using nitrogen compounds derived from the body for building its own cell constituents; therefore, the nitrogen consumed is lost for use by the host. Unfortunately, the mechanism underlying this phenomenon remains obscure and due attention has not been paid to it since the brilliant investigations of the LePage group (see Henderson and LePage, 1959). It is possible that the avid consumption by the tumor of essential metabolites supplied by the host is versatile and is not confined to glucose and nitrogen compounds. It will not be surprising if the tumor proves to be a trap for vitamins a8 well, thereby depriving the enzyme systems of the host’s vital organs of essential cofactors and prosthetic groups. Thus, if the tumor is also endowed with the capacity to drain the host of its vitamin reserves into the bargain, the reason for the dystrophy of the host’s inner organs caused by the development of the tumor becomes clearer.

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NUCLEAR PROTEINS AND THE CELL CYCLE1 Gary Stein and Renato Baserga Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania

I. Introduction . . . . . . . . . . 11. The Biochemistry of the Cell Cycle . . . . A. Biochemical Events in Continuously Dividing Cells B. Prereplicative Phase in Stimulated Go Cells . . C. Conclusions . . . . . . . . . 111. The Control of Cell Proliferation . . . . . . . A. Gene Activation and Cell Proliferation B. Nuclear Proteins as Gene Regulators . . . C. Acidic Nuclear Proteins and Cell Proliferation . D. Conclusions . . . . . . . . . . . . . . . . . . . References

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I. introduction

Cell division and the events associated with it have been of continuous interest to investigators for over a century (Flemming, 1878, 1880; Wilson, 1925; Wassermann, 1929; Schrader, 1944; Swann, 1957, 1958; Mazia, 1961; Baserga, 1965) ; however, only during the past 20 years has the cell cycle been studied a t a biochemical level. The cell cycle is defined as the interval between the midpoint of mitosis in the parent cell and the midpoint of the subsequent mitosis in one or both daughter cells. Howard and Pelc (1953) have divided the cell cycle into four phases: (1) G,, the period between completion of mitosis and the onset of DNA synthesis; (2) S, the period during which DNA is replicated; (3) Gz, the period between completion of DNA synthesis and the onset of mitosis; and (4) mitosis, the period during which the chromosomes condense, segregate, and are equally distributed to the two daughter cells (prophase, metaphase, anaphase, and telophase). It should also be pointed out that mammalian cells, to which we shall limit the present discussion, can be divided into three principal populations with respect to their ability to synthesize DNA and divide (Baserga, 1968). The first population consists of continuously dividing cells that keep moving through the cell cycle from one mitosis to the next. The second population 'This work was supported by grants from the U. 5. Public Health Service (CA-73, CA-l2!227, and CA-05'222) and the Damon Runyon Fund (DRG-1138). 281

288

GARY STEIN AND RENATO BASERGA

consists of cells that have permanently lost their ability to divide, and the third population consists of quiescent cells that ordinarily do not synthesize D N A or divide but can be stimulated to do so by the application of an appropriate stimulus, so-called Go cells (Patt and Qunstler, 1963). I n continuously dividing cells as well as in G, cells that are stimulated t o proliferate, a complex and interdependent series of biochemical events precede the onset of D N A synthesis and mitosis, and in this review we discuss these processes, with particular emphasis on those events related to the synthesis and turnover of nuclear proteins that may be involved in the control of cell proliferation. I n a previous review, Baserga (1968) summarized the available evidence suggesting that G, cells and G, cells could be separated on morphological and functional grounds. Further evidence that the two cell populations can be considered distinct from each other has come from two separate observations. The first, by E. Farber and Baserga (1969), indicated that Go cells are much less sensitive to the necrotizing effect of hydroxyurea than continuously dividing cells, while the second observation by Becker et al. (1971) showed that cultured hamster embryo cells from stationary phase cultures contained less than 70% of the ribosome complement of early G, phase cells, although the volumes of the two cell types were similar. Whether or not Go cells are different from GI cells, it is easier to consider separately the biochemical events of continuously dividing cells and of quiescent cells stimulated to synthesize DNA and divide. I n the present review, therefore, we discuss first the biochemistry of the cell cycle in continuously dividing cells and afterward the biochemical events that have been described in G, cells stimulated to proliferate, The second part of the review is devoted to the proteins of the mammalian nucleus and the role that they may play in the control of cellular proliferation. II. The Biochemistry of the Cell Cycle

A. BIOCHEMICAL EVENTS IN CONTINUOUSLY DIVIDING CELLS The biochemical events occurring in various phases of the cell cycle of continuously dividing cells can be studied either in asynchronous populations or in synchronized populations of cells. Since no effective method of cell synchronization has been achieved in the intact animal, most of the biochemical studies on the cell cycle of living animals have been carried out on asynchronous populations or, a t best, on parasynchronous populations. The amount of information that can be obtained from such heterogeneous populations of cells is necessarily limited, and

289

NUCLEAR PROTEINS AND THE CELL CYCLE

better results have been obtained in tissue culture by the use of synchronized cell populations. Fortunately, a number of lines of randomly growing cells in culture can be synchronized by chemical or mechanical means (Table I ) (Cameron and Padilla, 1966), and, although caution TABLE I

METHODS OF SYNCHRONIZING RANDOMLY GROWLY CEW

IN

CULTWRE

~ _ _

Method Treatment with exceas: Thymidine

Location of block Gi/S

Deoxyadenosine

S

Deoxyguanosine

S

References Xeros, 1962; Bootsma et aE., 1964; Anderson et al., 1967; L. S. Cohen and Studainski, 1967; Kasten, 1967; J. H. Kim et d.,1965; Petersen and Andersen, 1964; Pack, 1964; Galavazi et al., 1966; Steflen and Stolzmann, 1969; Firket, 1964 Xeros, 1962; Galavazi et d., 1966; Steffen and Stolzmann, 1969 Xeros, 1962; Mueller, 1963; Calavazi el al., 1966

Deoxycytidine Amenthopterin

S

Gi/S

Fluorodeoxyuridine

S

5-Aminouracil

S

Colcemid

Mitosis

Colchicine

Mitosis

Vinblastine Temperature shock Selective detachment of mitotic cells

Mitosis

Isoleucine deficiency

GI

Galavazi et al., 1966 Rueckert and Mueller, 1960; Klenow, 1962; Huennekena et d.,1963; Kajiwara and Mueller, 1964; Schindler, 1963; Steffen and Stolzmann, 1969; Adams et al., 1965 Rueckert and Mueller, 1960; Hartman and Heidelberger, 1961; Paul and Hagiwara, 1962; Reiter and Littlefield, 1964; Till et al., 1963; Littlefield, 1962; Gold and Helleiner, 1964; Erickson and Szybalski, 1963; Eidinoff and Rich, 1959; E. R. Stubblefield and Klevecz, 1965; Steffen and Stolzmann, 1969 Schindler, 1963; Brewen, 1965; Prensky and Smith, 1964; H. H. Smith et al., 1963 E. R. Stubblefield et al., 1967; R. R. Klevecz and Stubblefield, 1967; Kleinfeld and Sisken, 1966; Dewey and Miller, 1969 E. R. Stubblefield and Klevecz, 1965; P. H. Fitzgerald and Brehaut, 1970; Borisy and Taylor, 1967; E. W. Taylor, 1965; Morris, 1967

-

Pfeiffer and Tolmach, 1967 Newton and Wildy, 1959 Terasima and Tolmach, 1963; Sinclair and Morton, 1963; Robbins and Marcus, 1964; Belli, 1965; Robbins and Scharff, 1966 Tobey and Ley, 1970; Ley and Tobey, 1970

290

GARY STEIN AND RENATO BASERGA

must be exercised in interpreting results obtained from cells which have been subjected to such treatment (Cohen and Studzinski, 1967; Lambert and Studzinski, 1969; Churchill and Studzinski, 1969), these methods have allowed biochemical investigations of the various phases of the cell cycle which would be otherwise impossible in asynchronous populations. The various methods for synchronizing cells in vitro have been critically examined by Frindel and Tubiana (1971) in a recent review and are summarized in Table I. 1. GIPhase The first biochemical event in G, that could be related to the onset of DNA synthesis was the finding by Baserga et al. (1965b) that in the G, of Ehrlich ascites tumor cells there was a step sensitive to very small doses of actinomycin D (Baserga et al., 1965a,b). If this step, presumably RNA synthesis, was inhibited, the onset of DNA synthesis was prevented. A similar actinomycin D-sensitive step was identified by Baserga et al. (1966) in the G1 phase of epithelial cells lining the crypts of the mouse jejunum. Several reports since have shown that in mammalian cells synthesis of RNA occurs during the G, period (Terasima and Tolmach, 1963; Reiter and Littlefield, 1964; J. H. Kim and Perez, 1965; Scharff and Robbins, 1965; Crippa, 1966; Mueller and Kajiwara, 1966; Klevecz and Stubblefield, 1967 ; Stubblefield et al., 1967). A more specific step was described by Borun e t al. (1967), who found that 1 hour before the onset of DNA synthesis an RNA messenger for histone synthesis was made in synchronized HeLa cells. The messenger RNA for histones appeared on cytoplasmic ribosomes 1 hour before the onset of DNA synthesis, and, since inhibition of histone synthesis promptly leads to inhibition of DNA synthesis, the lack of the appropriate messenger RNA also leads to failure of cells to initiate DNA synthesis. Protein synthesis was also found to be necessary for the ordinate flow of GI cells into the S phase. The first demonstration goes back to the findings of Terasima and Yasukawa in synchronized L cells, where they found that 2-hour periods of exposure to either puromycin or cycloheximide produced a 2-hour delay in the onset of DNA synthesis (Terasima and Yasukawa, 1966). Two enzymes are missing from G, cells that are present in cells in the S phase: thymidine kinase, as demonstrated by Brent e t al. (1965) and by Stubblefield and Mueller (19651, and deoxycytidylate deaminase as shown in HeLa cells by Gelbard et al. (1969). It is interesting to note that DNA polymerase activity instead, is not decreased in continuously dividing cells during the G1 period. Other findings that ought to be mentioned in connection with the G, period are as follows: (a) Buell and

NUCLEAB PROTEINS AND THE CELL CYCLE

291

Fahey (1969) have shown that in human lymphoid cell lines the synthesis of immunoglobulins begins in late G , and continues throughout the S phase up to mitosis; (b) Steward et al. (1968) and Hodge et al. (1969) reported that polyribosomes in the cytoplasm are reformed immediately after mitosis, preexisting messenger RNA being used; (c) in mouse leukemic lymphoblasts, Jung and Rothstein (1967) showed that, a t the end of the G, period and extending into the very early part of the S phase, there is a marked decrease in potassium content of the cell accompanied by an increase in cellular sodium content; (d) the inducibility by steroids of the enByme, tyrosine aminotransferase, is absent in hepatoma cells in the early phase of the G , period (Martin et al., 1969) ; and (e) the expression of certain surface antigens in transformed cells in culture is most accentuated in the G, period and decreases considerably in the S phase and Gz (Cikes and Friberg, 1971). These results, although fragmentary, seem to indicate that during the G , period there is a series of orderly metabolic events, some of which involve gene expression and are directly related to the onset of DNA

BIOCHEMICAL EVENTS IN

TABLE I1 TEE GI PHASE OF

Biochemical event RNA synthesis

Protein synthesis Synthesis of histone meseenger RNA Disappearance of thymidine kinaae activity Absence of deoxycytidylate deaminase activity Immunoglobulin synthesis Reformation of polyribosomes Decreased K and increased Na content Absence of tyrosine aminotransferaae inducibility Expression of cell surface antigens Increased rate of phospholipid synthesis

THE

CELL CYCLE

Referencea Baserga et al., 1965a,b,1966;Mueller and Kajiwara, 1966;Scharff and Robbins, 1965;Terasima and Tolmach, 1963; J. H. Kim and Perez, 1965;Crippa, 1966;Reiter and Littlefield, 1964;E. R. Stubblefield et al., 1967;Klevecz and Stubblefield, 1967 Terasima and Yasukawa, 1966;Robbins and SchariT, 1966 Borun et al., 1967 Brent et al., 1965;Btubblefield and Mueller, 1965 Gelbard et al., 1969 Buell and Fahey, 1969 Steward et al., 1968 Jung and Rothstein, 1967 Martin et al., 1969 Cikes and Friberg, 1971 Robbins and ScharK, 1966

292

GARY STEIN AND RENATO BASERGA

synthesis. The events in the GIperiod have been discussed in a number of recent reviews (Baserga, 1968; Baserga and Wiebel, 1969; N. Bucher, 1967a,b; H. L. Cooper, 1971; Epifanova, 1971; Mueller, 1969, 1971; Petersen et al., 1969; Stein and Baserga, 1971b; Tobey et al., 1971) and are summarized in Table 11. 2. S Phase

Through the S phase the activities of the enzymes associated with DNA synthesis remain high [e.g., DNA polymerase (R. L. Adams et al., 1965; Barka, 1965b; Fausto and Van Lancker, 1965; Giudice and Novelli, 1963; Kit et al., 1966; Lieberman et al., 1963a; Younger et al., 1966), thymidine kinase (Barka, 1965b; Bollum and Potter, 1959; Kit et al., 1966; Lieberman et al., 1963a; Maley et al., 1965; Whitlock et al., 1968), thymidylate kinase (Fausto and Van Lancker, 1965; Kit et al., 1966; Pegoraro and Baserga, 1970), thymidylate synthetase (Maley et al., 1965; Pegoraro and Baserga, 1970), deoxycytidine deaminase (Holtzer et al., 1964; Maley et al., 1965), and RNA synthesis continues (Terasima and Tolmach, 1963; Pfeiffer, 1968; Pfeiffer and Tolmach, 1967, 1968; J. H. Kim and Perez, 1965; Crippa, 1966; Stubblefield et al., 1967; Klevecz and Stubblefield, 1967; Paul and Hagiwara, 1962; Killander and Zetterberg, 1965; Killander, 1965) 1. Although Prescott (1964) has demonstrated in the protozoan Euplotes that DNA cannot replicate and transcribe RNA simultaneously, this does not appear to be the situation in mammalian cells (Prescott, 1964; Klevecz and HSU,1964; Baserga, 1962b ; Showacre et al., 1967) where DNA synthesis is markedly asynchronous in individual chromosomes (J. H. Taylor, 1960b; Painter, 1961; Morishima et al., 1962; Moorhead and Defendi, 1963) as well as in various segments of each chromosome (J. H. Taylor, 1960b; Stubblefield and Mueller, 1962; Hsu, 1964). In fact, Amaldi et al. (1969) could time the duplication of the genes coding for ribosomal RNA in hamster cells between 1.5 and 3 hours from the beginning of S. Protein synthesis also continues a t an elevated rate during S phase (Mueller et al., 1962; Lieberman et al., 1963a; Bennett et al., 1964; Stone and Prescott, 1964; Baserga et al., 1965a; Estensen and Baserga, 1966; Langen and Repke, 1966; Robbins and Scharff, 1966), and the synthesis of nonhistone chromosomal proteins, which was evident during GI, continues (Stein and Baserga, 1970b; Stein and Borun, 1971; Stein et al., 1970), although the rate of turnover of nonhistone chromosomal proteins during S phase appears to be significantly lower than that observed during G , (Stein et al., 1971; Borun and Stein, 1971). The synthesis of histones has been shown to be tightly coupled to DNA synthesis, both in stimulated Go cells (Takai et al., 1968; Borun et al., 1971), and in continuously

NUCLEAR PROTEINS AND THE CELL CYCLE

293

dividing cells (Spalding et al., 1966; Robbins and Borun, 1967), and what has been provisionally designated as histone messenger RNA is present during S phase (Borun et al., 1967). A number of investigators have recently described different types of extrachromosomal DNA, most of which are found outside the nucleus and include: mitochondria1 DNA (Nass, 1966, 1969a,b), the DNA present in the centrioles (Randall and Disbrey, 1965; Seaman, 1960), and the DNA that results from gene amplification (Brown and Dawid, 1968). These DNA’s will not be discussed in this review, nor will we deal with the unscheduled repair replication which results from damage caused by radiation (Setlow, 1966) or a number of carcinogens (Lieberman et al., 1971) and alkylating agents (Roberts et al., 1968, 1971; Lieberman e t al., 1971); however, it should be pointed out that the synthesis of these DNA’s is not restricted to the S phase of the cell cycle. 3. G, Phase

During the G, phase of the cell cycle, RNA and protein synthesis continue ; however, their rates of synthesis decrease as the cell approaches mitosis (J. H. Taylor, 1960a; Baserga, 1962a; Kishimoto and Lieberman, 1964; Tobey et al., 1965, 1966a,b; Cummins et al., 1966; Robbins and Scharff, 1966; Sisken and Wilkes, 1967). Kishimoto and Lieberman (1964) were the first to demonstrate that puromycin (5 pg./ml.), when administered during G,, effectively prevents the flow of cultured cells from Gz to mitosis; however, they found that low doses of actinomycin D (0.33 pg./ml.) were ineffective in producing a G, block. The inability of low doses of actinomycin D to produce a G2 block was confirmed by Baserga et al. (1965b) ; however, subsequent studies with higher doses of actinomycin D (2 pg./ml.) (Tobey et al., 1966b), puromycin (50 pg./ml.) (Tobey et al., 1966b), and cycloheximide (2 pg./ml.) (Tobey et al., 1966a) and with mengovirus (Tobey et al., 1965) have clearly established that the synthesis of RNA and proteins during the G, phase of the cell cycle is essential for entry of cells into mitosis. Since the G, cell contains DNA which was present during G, as well as that which was replicated during S phase, the question arises whether the RNA synthesized during G, is transcribed on the “old” DNA, the “newly replicated” DNA or both. A number of studies show that with the onset of Sphase there is an increase in the rate of RNA synthesis, as measured by the incorporation of uridine-3H, formate-”C or orthoph0sphate-3~P,which persists into G,. This was demonstrated by Terasima and Tolmach (1963), Pfeiffer (1968), Pfeiffer and Tolmach (1967, 1968), and J. H. Kim and Perez (1965) in HeLa cells synchronized by mitotic selective detachment; by Crippa (1966) in randomly growing

294

GARY STEIN AND RENATO BASERGA

Chinese hamster cells; by Stubblefield et al. (1967) and Klevecz and Stubblefield ( 1967) in Chinese hamster cells synchronized by Colcemid collection of mitotic cells; by Paul and Hagiwara (1962), Reiter and Littlefield (1964), and Killander and Zetterberg (1965) in mouse L cells; and by Killander in mouse leukemia cells (1965). The increased synthesis of RNA, which coincides with the onset of DNA replication and persists into Gz,indeed suggests that both the newly synthesized DNA as well as the “old” DNA are transcribed during G?; however, the possibility cannot be dismissed that either the old or the newly synthesized DNA alone may be active in the transcription of G, RNA and the increased rate may be due to other factors. The synthesis of nonhistone chromosomal proteins continues during G, (Stein and Borun, 1971; Stein et al., 1970, 1971) ; however, their rate of turnover is significantly increased in comparison to that observed during S phase (Stein et al., 1971; Borun and Stein, 1971). Furthermore, polyacrylamide gel electrophoretic profiles of these proteins suggest that there are qualitative differences in the nonhistone chromosomal proteins synthesized in G , , S, and G, (Stein et al., 1970, 1971). These results confirm the findings of Jockusch et al. (1970) and of Kolodny and Gross (1969), who demonstrated by gel electrophoresis the presence in cultured cells of G, specific proteins. Bosmann and Winston (1970) reported that phospholipid synthesis in synchronous L 5178Y cells, as measured by choline incorporation, is almost entirely restricted to the GI phase of the cell cycle. This is in TABLE I11 BIOCHEMICAL EVENTS OF Biochemical event RNA synthesis and sensitivity to actinomycin D Specific G, proteins Protein synthesis Sensitivity to: Puromyain Cycloheximide Mergouinus Synthesis of nonhistone chromosomal proteins Increased rate of turnover of nonhistone chromosomal proteins Synthesis of phospholipids

THE

PHAsE OF THE

CELL CYCLE

References Bseerga et d.,196513; Robbins and ScharfT, 1986; Tobey et d.,1966b; Kishimoto and Lieberman, 1964 Jockuach el al., 1970 Robbins and ScharfT, 1966; Baserga, 1962a; Cumrnins el d.,1966 Tobey et al,, 1966b Tobey et d.,1966a Tobey et d.,1965 Stein and Borun (1971); Stein el al., 1970, 1971 Stein et al., 1971; Borun and Stein (1971) Bosmann and Winston, 1970

NUCLEAR PROTEINS AND THE CELL CYCLE

295

contrast, though, to the results obtained by Bergeron et al. (1970), which showed a continuous incorporation of choline into phospholipids during the entire cell cycle with a peak during Sphase. The biochemical events in GOare summarized in Table 111. 4. Mitosis I n addition to the condensation and segregation of chromosomes, a number of biochemical events also occur during the mitotic phase of the cell cycle. In 1960, J. H. Taylor (1960a) demonstrated that mammalian cells synthesize RNA in all phases of the cell cycle except during mitosis, when there is cessation of RNA synthesis; and these findings, indicating an interruption of gene expression, have been confirmed by several other investigators (Baserga, 1962b; Prescott and Bender, 1962; Feinendegen and Bond, 1963; Prescott, 1964). A decreased rate of total cellular protein synthesis has also been observed during mitosis (Baserga, 1962a; Prescott and Bender, 1962; Konrad, 1963; Robbins and Scharff, 1966) ; however, Stein and Baserga (1970a) have found that nonhistone chromosomal proteins are actively synthesized a t this time, and their rate of turnover is greater than that which occurs during Sphase (Stein et al., 1971). T. C. Johnson and Holland (1965) have reported that the in vitro template activity of mitotic chromatin is severalfold less than

TABLE IV BIOCHEMICAL EVENTSOF

THE

MITOTICPHASEOF

Decreaee in in vitro chromatin template activity Decrease in protein synthegis Disaggregation of polyribosomes Continued synthesis of nonhistone chromosomal proteins Rapid turnover of nonhistone chromosomal proteins Persistence of messenger RNA’s Membrane changes (agglutinability)

CELLCYCLE

References

Biochemical event Condensation and segregation of chromosomes Cessation of RNA synthesis

THE

Mazia, 1961

J. H. Taylor, 196Oa; Baaerga, 1962b; Prescott and Bender, 1962; Feinendegen and Bond, 1963; Prescott, 1964 Johnson and Holland, 1965; J. Farber d d.,19710 Baserga, 196211; Prescott and Bender, 1962; Konrad, 1963; Robbins and ScharE, 1966 Robbins and SchariT, 1966; ScharE and Robbins, 1966 Stein and Baserga, 1970a Stein et d.,1971 Hodge et al., 1969 Fox et al., 1971

296

GARY STEIN AND RENATO BASERGA

that observed in interphase chromatin, and Hodge et al. (1969) have observed that in spite of the disaggregation of polysomes which occurs during mitosis (Robbins and Scharff, 1966; Scharff and Robbins, 1966), the rapidly labeled polyribosome-associated RNA made prior to mitosis persists into the subsequent GI period and new RNA synthesis is not necessary for the reformation of polyribosomes. Fox et al. (1971) have shown that mitotic cells are agglutinated by wheat agglutinins, whereas interphase cells are not, indicating changes in the structure of the cell surface. The biochemical events associated with mitosis are summarized in Table IV.

B. PREREPLICATIVE PHASE IN STIMULATED G , CELLS Quiescent cells which are stimulated to proliferate (G,, cells) provide an excellent opportunity to study the complex and interdependent structural and biochemical events that lead to DNA synthesis and ultimately mitosis, and a number of in vitro (Table V) as well as in v i m (Table VI) models of stimulated cell proliferation have been developed. In most of these systems, DNA synthesis is preceded by a lag period of 12 or more hours, which has been termed the prereplicative phase, and is followed by a GcLperiod and then mitosis. The biochemical events which occur during the prereplicative phase of the cell cycle are summarized in Table VII. An early increase in RNA synthesis has been observcd in most models of stimulated DNA synthesis, as first demonstrated in 1963 by Lieberman and co-workers in primary explants of rabbit kidney cortex cells (Lieberman e t al., 1963b). These investigators also showed that low doses of actinomycin D, which did not affect DNA synthesis per se or the rate of RNA synthesis in nonproliferating kidney cells, were capable of inhibiting the increase in RNA synthesis that occurred during the prereplicative phase as well as the subsequent onset of DNA synthesis. This increased RNA synthesis and sensitivity to actinomycin D was quickly confirmed by Lieberman’s group in the regenerating liver following partial hepatectomy (Fujioka et al., 1963; Tsukada and Lieberman, 1964a,b; Chaudhuri et al., 1967; Ove et al., 1966), and subsequently in phytohemagglutinin-stimulated lymphocytes ( H . L. Cooper and Rubin, 1965; A. D. Rubin and Cooper, 1965; B. G. T. Pogo et al., 1966; Mueller and Le Mahieu, 1966; Salaman e t al., 1966), in the isoproterenol-stimulated rat (Barka, 1966, 1970) and mouse (Sasaki and Baserga, 1970), salivary gland, in the folic acid-stimulated kidney (D. M. Taylor et al., 1966; Threlfall et al., 1966, 1967; Threlfall and Taylor, 1969), in the estrogen-stimulated uterus (Ui and Mueller, 1963 ; Hamilton, 1964, 1968; Hamilton et al., 1965; Gorski, 1964; Means and Hamilton, 1966a,b; Teng and Hamilton, 1968), and in a number of other

NUCLEAR PROTEINS AND THE CELL CYCLE

297

TABLE V In Vitro MODELSOF STIMULATED DNA SYNTHESIS AND CELLDIVISION System Contacbinhibited cells in culture

Lymphocytes

Organ cultures of skin Organ cultures of mammary gland Organ cultures of lens Hen erythrocyte and other quiescent cells

Stimulus

References

Todaro et d.,1965; Bloom et al., 1966 (b) Change of medium Wiebel and Baserga, 1969 (c) Trypsin Burger, 1970 H. Rubin, 1970 (d) Peptidases (e) Oncogenic viruses Green, 1970 Kit et al., 1966; Sauer and (1) Simian virus 40 Defendi, 1966 (2) Polyoma virus Dulbecco et d.,1965; Gershon et d.,1965 E. H. Cooper et al., 1963; H. L. (a) PhytohemagCooper and Rubin, 1965; H. L. glutinin Cooper, 1969, 1971 Powell and Leon, 1970 (b) Concanavalin A Epidermal growth S. Cohen, 1965 factor (a) Epidermal growth Turkington, 1969 factor (b) Insulin Lockwood et al., 1967 Harding et al., 1962 (a) Explantation (b) Insulin Reddan and Harding, 1969 Hybridization Harris, 1967; Jacobson, 1968; R. T. Johnson and Harris, 1969; Bolund et al., 196913 Explantation Lieberman el al., 1963b (a) Dialyzed serum

Primary cultures of kidney cells Isoleucine CHO cells after isoleucine deprivation Hormones and cyclic Thymocytes AMP Conditioned medium Mouse macrophages from L cells in culture Mouse salivary glands Epithelial growth factor

Tobey and Ley, 1970; Ley and Tobey, 1970 Whitfield et d.,1970a,b,c Virolainen and Defendi, 1967; Mauel and Defendi, 197la,b Jones and Ashwood-Smith, 1970

systems. Although measurements of incorporation of radioactive precursors into RNA suggest an increase in the rate of RNA synthesis in most models of stimulated DNA synthesis, it should be pointed out that a realistic picture cannot be obtained unless fluctuations in the specific activities of the nucleotide precursor pools (N. L. Bucher and Swaffield, 1966; Ove et al., 1966; Baserga and Heffler, 1967; Malamud and Baserga, 1968b, 1969) and the activities of enzymes, such as cytidine kinase (Lucas, 1967), uridine kinase (Lucas, 1967), and uridylate kinase

298

GARY STEIN AND RENATO BASERGA

TABLE VI In Vivo MODELSOF STIMULATED DNA SYNTHESIS AND CELLDIVISION ~

Tissue ororgan

Animal

Stimulus

Liver

Rat

Partial hepatectomy

Liver

Mouse

Partial hepatectomy

Liver Liver

Rat Alloxandiabetic rat Mouse Rat

D-Gdactosamine Insulin

Kidney Kidney

Salivary gland salivary gland Epiphyseal cartilage

Folk acid (a) Folic acid

Rat

(b) Temporary ischemia (c) Metabolic acidosis (d) Mercuric chloride necrosis Isoproterenol

Mouse

Isoproterenol

HYPOPhY- Growth hormone sectomized rat Mouse Estrone

Uterine epithelium Mouse Mammary gland

References Grisham, 1962; N. Bucher, 1963; N. Bucher, 1967a,b; Fujioka el d.,1963 Church and McCarthy, 1967a,b Leach et d.,1970 Younger et d.,1966 Baserga et d.,1968 D. M. Taylor et d.,1966; Threlfall and Taylor, 1969 Stiicker, 1966 Lotspeich, 1967 Cuppage and T a b , 1967 Barka, 1965a,b Baserga, 1966 Kember, 1971

Epifanova, 1966

Skin Skin Skeletal muscle Lens epithelium Hair follicle Pancreas Spleen

Mouse Rat Mouse

(a) Estradiolprogesterone (b) Lactation Wounding Wounding Wounding

Rabbit

Wounding

Harding and Srinivasan, 1881

Sheep Rat Mouse

Bladder Skin Adrenal

Mouse Mouse Mouse

Plucking Ethionine (a) Erythropoietin (b) Growth hormone Carcinogens Crooton oil ACTH

Downes et d.,1966 P. J. Fitzgerald et al., 1966 Hodgson, 1967 Fast et al., 1970 Lawson et d.,1970 Hennings and Boutwell, 1970 Masui and Garren, 1970

Bresciani, 1965 Traurig, 1967 Block et d.,1963 Block et al., 1963 Pietach and McCollister, 1965

NUCLEAR PROTEINS AND THE CELL CYCLE

299

(Malamud and Baserga, 1968b), are taken into account. Additional evidence for increased transcriptional activity during the prereplicative phase of the cell cycle includes (1) an increased in vitro chromatin template activity for RNA synthesis, which has been observed by A. 0. Pogo et al. (1966) in the regenerating liver following partial hepatectomy, by Teng and Hamilton (1969) in the estrogen-stimulated uterus, and by J. Farber et al. (1971b) in contact-inhibited human diploid fibroblasts stimulated by a change of medium; ( 2 ) an increased binding of actinomycin D and acridine orange (Killander and Rigler, 1965, 1969; Bolund e t al., 1969a,b; Daraynkiewicz et aZ., 1969; Ringerta et al., 1969; Auer et al., 1970; Zetterberg and Auer, 1970; Baserga, 1971; Gierthy and Rothstein, 1971) ; and (3) the synthesis of new species of messenger RNA which has been reported by Church and McCarthy (1967a,b) in the regenerating mouse liver following partial hepatectomy. Although the RNA-DNA hybridization technique used by Church and McCarthy was generally accepted a t the time their experiments were carried out, the biological relevance of their results has subsequently been open to considerable criticism (Birnboim et al., 1967; Melli and Bishop, 1969; Gelderman et al., 1971) since it appears that only the RNA messages transcribed from highly redundant sequences of the genome were actually studied. An increase in protein synthesis has been observed during the prereplicative phase of the cell cycle in most models of stimulated DNA synthesis as well as in continuously dividing cells, and studies with inhibitors of protein synthesis such as puromycin and cycloheximide suggest that the synthesis of these proteins is essential for the subsequent onset of DNA synthesis. This increased protein synthesis was initially reported by Lieberman and co-workers in primary cultures of rabbit kidney cortex cells within 1 hour after explantation (Lieberman et al., 1963a,b). Subsequent observations of increased protein synthesis during the prereplicative phase of the cell cycle have been made in serumstimulated contact-inhibited cells (Todaro et al., 1965; Wiebel and Baserga, 1969), in the regenerating liver following partial hepatectomy (Majumdar et al., 1967), in the contralateral kidney after nephrectomy (H. A. Johnson and Roman, 1966), in the folic acid-stimulated kidney (Threlfall and Taylor, 19691, in the estrogen-stimulated uterus (Noteboom and Gorski, 1963; Hamilton, 1964; Toft and Gorski, 1966; Jensen et al., 1967), in phytohemagglutinin-stimulated lymphocytes, and in the isoproterenol-stimulated salivary gland (Sasaki e t al., 1969; Stein and Baserga, 1970b). I n several systems fluctuations in the size and specific activity of amino acid pools have been observed during the cell cycle (Wiebel and Baserga, 1969); therefore, as with nucleotide pools, a

300

GARY STEIN AND RENATO BASERGA

BIOCHEMICAL EVENTSIN Biochemical event Increased RNA synthesis and sensitivity to low doses of actinomycin D

Increased RNA polymerase activity Increased activity of uridine, uridylate, and cytidine kinase Synthesis of new DNA species Increased in uitro chromatin template activity for RNA synthesis Increased binding of actinomycin D and acridine orange Increased protein synthesis and sensitivity to puromycin and cycloheximide Acetylation and phosphorylation of histones and nonhistone chromosomal proteins

Synthesis of nonhistone chromosomal proteins

THE

TABLE V I I PREREPLICATNE PHASE OF

THE

CELLCYCLE

References Lieberman et al., 1963b; Fujioka et al., 1963; Tsukada and Lieberman, 1964a; Tsukada and Lieberman, 1964b; Chaudhuri et al., 1967; Ove et al., 1966; Baaerga et al., 1965a,b, 1966; H. L. Cooper and Rubin, 1965; B. G. T. Pogo et al., 1966; Mueller and Le Mahieu, 1966; Salzman et al., 1966; Rubin and Cooper, 1965; Barka, 1966, 1970; h s a k i and Baserga, 1970; Threlfall and Taylor, 1969; D. M. Taylor et al., 1966; Threlfall et al., 1966, 1967; Ui and Mueller, 1963; Hamilton et al., 1965; Hamilton, 1964, 1968; Gorski, 1964; Means and Hamilton, 1966a,b; Teng and Hamilton, 1968 Tsukada and Lieberman, 1964b; Steiner and King, 1966 Malamud and Baserga, 1968b; Lucas, 1967 Church and McCarthy, 1967a A. 0. Pogo el al., 1966; Teng and Hamilton, 1969; J. Farber et aZ., 1971b

Auer et al., 1970; Baserga, 1971; Bolund et al., 1969; Darzynkiewicz et al., 1969; Gierthy and Rothstein, 1971; Killander and Rigler, 1965, 1969; Ringertz el al., 1969; Zetterberg and Auer, 1970 Lieberman et al., 1963a,b; Todaro et al., 1965; Wiebel and Baserga, 1969; Majumdar et al., 1967; H. A. Johnson and Roman, 1966; Noteboom and Gorski, 1963; Hamilton, 1964; Toft and Gorski, 1966; Jensen et al., 1967; Threlfall and Taylor, 1969; Sasaki et al., 1969; Stein and Baserga, 1970b Kleinsmith et al., 1966a,b; Ord and Stocken, 1967; Langan and Smith, 1966, 1967; Langan, 1968, 1969a,b; Kleinsmith and Allfrey, 1969a,b; Gershey and Kleinsmith, 1969a,b; Gershey el al., 1968; Paik and Kim, 1970a, Liew et al., 1970; B. G. T. Pogo et al., 1968; Phillips, 1963; Allfrey et al., 1964; Allfrey, 1966a,b; G. G. T. Pogo et al., 1966, 1967; Nohara et al., 1966 Teng and Hamilton, 1969; Stein and Baserga, 1970b; Barnea and Gonki, 1970; Mayol and Thrtyer, 1970; J. A. Smith et al., 1970; Stellwagen and Cole, 1969b; Rovera and Baserga, 1971

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TABLE VII (Continued) Biochemical event Increased nonhistone chromosomal protein turnover Increased synthesis of glycoproteins Increased rate of phospholipid synthesis Increased synthesis of spermidine Increased ornithine decarboxylase activity Fluctuations in glycogen concentration Increased activity of enzymes associated with DNA synthesis DNA polymerase Thymidine kinase Thymidylste kinase Thymidylate synthetase Dwxy cytidylate deaminase Synthesis of templates for enzymes associated with DNA synthesis

References Rovera and Baserga, 1971; Borun et al., 1967 Galanti and Baserga (1971) Aizawa and Mueller, 1961; Fisher and Mueller, 1968; Kay, 1968 Raina et al., 1966; Dykstra and Herbst, 1965 Russell and Snyder, 1968; Fausto, 1969; Schrock et al., 1970; Stastny and Cohen, 1970 Malamud and Baserga, 1968a; Quaglino et al., 1964; Steiner and King, 1964; Fujioka et al., 1963

Barka, 1965b; Giudice and Novelli, 1963; Fausto and Van Lancker, 1965; Lieberman et al., 1963a; Kit et al., 1966; R. L. Adams et al., 1965; Younger et al., 1966 Barka, 1965b; Lieberman et d.,1963a; Maley et al., 1965; Bollum and Potter, 1959; Kit et al., 1966; Whitlock et al., 1968 Fausto and Van Lancker, 1965; Kit et al., 1966; Pegoraro and Baserga, 1970 Pegoraro and Baserga, 1970; Maley et al., 1965 Holtzer el al., 1964; Maley el al., 1965 Pegoraro and Baserga, 1970

realistic determination of protein synthesis cannot be made without compensating for these changes in the amino acid pool. Some of the proteins synthesized during the prereplicative phase have been characterized ; they include phosphoproteins (Kleinsmith et al., 1966b), glycoproteins (Galanti and Baserga, 1971), nonhistone chromosomal proteins (Teng and Hamilton, 1969; Stellwagen and Cole, 1969b; Barnea and Gorski, 1970; Mayol and Thayer, 1970; J. A. Smith e t al., 1970; Stein and Baserga, 1970b; Rovera and Baserga, 1971), spcrmidine (Raina et al., 1966; Dykstra and Herbst, 1965), and probably a number of enzymes associated with synthesis of RNA (Steiner and King, 1966; Lucas, 1967; Malamud and Baserga, 1968b) and DNA (Bollum and Potter, 1959;

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GARY STEIN AND RENATO BASERGA

Giudice and Novelli, 1963; Lieberman et al., 1963a; Holtzer et al., 1964; R. L. Adams et al., 1965; Fausto and Van Lancker, 1965; Maley e t al., 1965; Kit et al., 1966; Younger et al., 1966; Whitlock e t al., 1968; Pegoraro and Baserga, 1970), and perhaps even with protein synthesis. The enzymes related to RNA synthesis which have been most extensively studied are RNA polymerase (Tsukada and Lieberman, 1964b; Steiner and King, 1966), uridine kinase (Lucas, 1967), uridylate kinase (Malamud and Baserga, 1968b), and cytidine kinase (Lucas, 1967) ; and those enzymes related to DNA synthesis which have been studied include DNA polymerase (Giudice and Novelli, 1963; Lieberman et al., 1963a; R. L. Adams et al., 1965; Barka, 1965b; Fausto and Van Lancker, 1965; Kit et al., 1966; Younger et al., 1966), thymidine kinase (Bollum and Potter, 1959; Lieberman et al., 1963a; Barka, 1965b; Maley et al., 1965; Kit et al., 1966; Whitlock et al., 1968), thymidylate synthetase (Maley e t at., 1965; Pegoraro and Baserga, 1970), thymidylate kinase (Fausto and Van Lancker, 1965; Kit et al., 1966; Pegoraro and Baserga, 19701, and deoxycytidylate deaminase (Holtzer et al., 1964; Maley et al., 1965). Furthermore, studies in the isoproterenol-stimulated mouse salivary gland suggest that the templates for some of these enzymes are synthesized during the prereplicative phase of the cell cycle several hours in advance of the observed increases in enzyme activity (Pegoraro and Baserga, 1970). In addition to studying protein synthesis it is perhaps just as important to consider variations in the rates of protein turnover, and indeed, increases in the rates of turnover of nonhistone nuclear proteins have been observed during the prereplicative phase of the cell cycle in contactinhibited human diploid fibroblasts following serum stimulation (Rovera and Baserga, 1971) and in the mouse salivary gland after stimulation by isoproterenol (Borun e t al., 1971). Chemical modifications of preexisting proteins have also been reported during the prereplicative phase; they include the phosphorylation of histone as well as nonhistone chromosomal proteins (Kleinsmith e t al., 1966a,b; Langan and Smith, 1966, 1967; Ord and Stocken, 1967; Langan, 1968; Gershey and Kleinsmith, 1969a,b; Kleinsmith and Allfrey, 1969a,b; Langan, 1969a,b) and the acetylation (Phillips, 1963; Allfrey, 1966a,b; Allfrey et al., 1964; Nohara et al., 1966; B. G. T. Pogo et al., 1966, 1967, 1968; Gershey et al., 1968; Liew et al., 1970; Paik and Kim, 1970a) of histones and methylation (Allfrey et al., 1964; Murray, 1964a; Comb e t al., 1966; Kleinsmith et al., 1966a; Paik and Kim, 1966, 1967, 1968, 1969a,b, 1970a,b; Gutierrez and Hnilica, 1967; Langan, 1967; Ord and Stocken, 1967; Gershey et al., 1968; Tidwell et al., 1968). The meaning of histone acetylation in terms of stimulation of cell proliferation is still obscure (Monjardino and MacGillivray, 1970).

NUCLEAR PROTEINS AND THE CELL CYCLE

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Additional biochemical events which have been reported in the prereplicative phase of the cell cycle include an increase in the activity of ornithine decarboxylase immediately after partial hepatectomy (Russell and Snyder, 1968; Fausto, 1969; Schrock et al., 1970) or epithelial growth factor (Stastny and Cohen, 1970), fluctuations in glycogen concentration (Fujioka et al., 1963; Quaglino e t al., 1964; Steiner and King, 1964; Malamud and Baserga, 1968a), an increase in the rate of phospholipid synthesis (Aizawa and Mueller, 1961; Robbins and Scharff, 1966; Fisher and Mueller, 1968; Kay, 1968; Bergeron et al., 1970), and increases in the activity of adenyl cyclase and cyclic adenosine monophosphate (Malamud, 1969; Durham and Baserga, 1971).

C. CONCLUSIONS From the preceding discussion it is evident that a complex and interdependent series of biochemical events occur during the cell cycle of quiescent cells which are stimulated to proliferate as well as during the cell cycle of continuously dividing cells, and for additional biochemical details the reader is referred to several comprehensive reviews (N. Bucher, 1967a,b; Baserga, 1968; Baserga and Wiebel, 1969; Mueller, 1969, 1971; Petersen et al., 1969; Lieberman, 1970; H. L. Cooper, 1971; Epifanova, 1971; Stein and Baserga, 1971b; Tobey et al., 1971). These findings have led a number of investigators to hypothesize that when quiescent cells are stimulated to divide there is a derepression of specific genes that code for the synthesis of the macromolecules essential for DNA replication and cell division; and it appears that modifications of gene activity also occur during the cell cycle of continuously dividing cells. Some of the biochemical events that occur during the Go to S transition in the isoproterenol-stimulated mouse salivary gland and in serum stimulated WI-38 cells have been summarized in Fig. 1. Although gene

TIME

F I ~1.. Biochemical events during the Goto S transition.

304

GARY STEIN AND RENATO BASERGA

r

M E M OF STIMUUS CHANGES IN

FIQ2. Actinomycin D-insensitive steps during the early prereplicative phase.

activation is strongly suggested, it should be pointed out that during the early portion of the prereplicative phase, a number of steps in the sequence of events which lead to DNA synthesis are insensitive to actinomycin D and hence may not be dependent on the immediate transcription of RNA’s, but rather, may be due to the activation of preexisting RNA templates. Figure 2 illustrates several of the actinomycin Dinsensitive steps which occur during the early prereplicative phase in stimulated mouse salivary glands and human diploid WI-38 cells, and a similar utilization of preexisting RNA templates has been reported in several other systems (Wool and Kiruhara, 1967; Boyadjiev and Hadjiolov, 1968; Roth et al., 1968). 111. The Control of Cell Proliferation

A. GENE ACTIVATION AND CELL PROLIFERATION The first suggestion that gene activation occurs in Go cells which are stimulated to proliferate came from the above-mentioned experiments of Lieberman and co-workers (Lieberman et al., 1963b). Indeed, their findings, subsequently confirmed by numerous investigators (Fujioka et al., 1963; Ui and Mueller, 1963; Gorski, 1964; Hamilton, 1964, 1968; Tsukada and Lieberman, 1964a,b; H. L. Cooper and Rubin, 1965; Hamilton et a,?., 1965; A. D. Rubin and Cooper, 1965; Barka, 1966; Chaudhuri et al., 1967; Means and Hamilton, 1966a,b; Mueller and Le Mahieu, 1966; Ove et al., 1966; B. G. T. Pogo et al., 1966; Salzman et al., 1966; D. M. Taylor et al., 1966; Threlfall et al., 1966, 1967; Teng and Hamilton, 1968; Threlfall and Taylor, 1969; Barka, 1970; Sasaki and Baserga, 1970), pointed to an increase in the rate of transcription as a prerequisite for DNA synthesis and mitosis. Although, as previously mentioned, Church and McCarthy (1967a,b) reported the appearance of new RNA species in the prereplicative phase of the cell cycle in the

NUCLEAR PROTEINS AND THE CELL CYCLE

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regenerating mouse liver following partial hepatectomy, their DNARNA hybridization procedures have recently been the subject of criticism (Birnboim et al., 1967; Melli and Bishop, 1969; Gelderman et al., 1971), and a formal demonstration of gene activation in Go cells stimulated to proliferate is still lacking. However, additional evidence for gene activation during the cell cycle comes from findings of an increased in vitro chromatin template activity (using an exogenous RNA polymerase) that follows the stimulation of Go cells to proliferate (A. 0. Pogo et al., 1966; Teng and Hamilton, 1969; J. Farber et al., 1971b). Furthermore, an increase in the binding of actinomycin D and acridine orange (Killander and Rigler, 1965, 1969; Bolund et al., 1969a,b; Darzynkievicz et al., 1969; Ringertz et al., 1969; Auer et al., 1970; Zetterberg and Auer, 1970; Gierthy and Rothstein, 1971; Baserga, 1971) has been observed in the nuclei and chromatin of Go cells which have been stimulated to proliferate ; however, for an appreciation of the complexity involved in the interpretation of such results, one should consult the recent paper by Kleiman and Huang (1971). The supportive evidence for gene activation has been summarized in a recent review by Stein and Baserga (1971b) and can be gleaned from the biochemical events listed in Table VII. Despite the above-mentioned reservations, the evidence suggests that after the stimulation of quiescent cells to proliferate, as well as during the cell cycle of continuously dividing cells, regions of the genome which are inactive in transcribing RNA become active in the elaboration of genetic information. Hence, the question arises as to what activates and regulates the genome, and specifically that segment of the genome which controls its own replication. B. NUCLEARPROTEINS AS GENEREGULATORS Unlike the single Escherichia coli chromosome, which consists of little more than a naked strand of DNA, the mammalian genome is a complex structure consisting of DNA and chromosomal proteins, the protein component being composed of histones and acidic proteins. Furthermore, in comparison to the bacterial chromosome where most of the genetic information is continuously expressed, in the mammalian chromosome a t any given time most of the genetic information is repressed (Huang and Bonner, 1962; Allfrey et al., 1963; Paul and Gilmour, 1966, 1968), including that information which is necessary for the synthesis of the macromolecules involved in cell proliferation. Although extreme caution should be exercised in drawing conclusions about gene regulation in mammalian cells based on results obtained from prokaryotic systems, the concept which has evolved is that, in fact, gene

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GARY STEIN AND RENATO BASERGA

regulation in mammalian cells occurs by a mechanism similar to that proposed by Jacob and Monod in 1961 for the E. coli lac operon. Specifically, it is generally believed that although the processes involved are undoubtedly somewhat more complicated in eukaryotic cells, the mammalian genome is repressed or derepressed by regulatory molecules acting on DNA and allowing or inhibiting its transcription. 1. His t ones Histones have been implicated by a number of investigators as having a regulatory function upon the mammalian genome, and in recent years B considerable amount of effort has been devoted to the study of the chemistry and biology of these basic chromosomal proteins (Table VIII). A number of reviews (Johns and Butler, 1962; Bonner et al., 1968; Hnilica, 1968; Stellwagen and Cole, 1969a; E. L. Smith et al., 1970) as well as several monographs (Bonner and Tso, 1964; Busch, 1965; De Reuck and Knight, 1966) have been written on the subject. Histones are positively charged proteins, enriched in arginine and lysine residues and completely lacking tryptophan (Daly et al., 1950; Crampton et al., 1955), and they may be separated into several classes by three principal methods. The first separation procedure involves applying histones to an Amberlite CG-50 column (a weak cation-exchange resin) followed by elution with a guanidinium chloride gradient (Luck e t al., 1958; Satake et al., 1960; Rasmussen et al., 1962) ; the second involves selective precipitation by trichloroacetic acid, acetone, and ethanol (Johns, 1964; Phillips and Johns, 1965); and the third involves polyacrylamide gel electrophoresis, either by charge and molecular weight (Reisfield et al., 1962; Johns, 1967; Panyim and Chalkley, 1969) or by molecular weight alone using polyacrylamide gel containing sodium dodecyl sulfate and urea (Shapiro et al., 1967; Weber and Osborn, 1969; Elgin and Bonner, 1970; Laemmli, 1970). Other separation procedures have been described by Busch (1965). By a combination of the above-mentioned procedures, the histones may be separated into six major classes; following the nomenclature of Murray (1964a,b) they are: I a and Ib, lysine rich; IIa and IIb, slightly lysine rich; I11 and IV, arginine rich. Butler et al. (1968) have adopted the following nomenclature for histones: F, and FZb,lysine rich; FZe1and F,, arginine rich; FZaz,an intermediate type. The molecular weights of histone monomers have been determined, and they range from 8000 to 16,000 (Trautman and Crampton, 1959) ; furthermore, the complete amino acid sequences of pea seedling histone IV (De Lange et al., 1968, 1969b) and the homologous calf thymus histone fraction (De Lange et al., 1968, 1969a) have been determined;

NUCLEAR PROTEINS AND THE CELL CYCLE

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TABLE VIII CHEMICAL AND FUNCTIONAL PROPERTIES OF HISTONES ~~

-

~

Properties Chemical properties 1. Positively charged 2. Rich in arginine and lysine residues 3. Absence of tryptophan 4. Slow rate of turnover 5. Separable into 5-10 electrophoretically distinct bands 6. May be acetylated, methylated, and phosphorylated

7. No tissue or species specificity Functional properties 1. Inhibit DNA-dependent RNA synthesis in vitro 2. Inhibit DNA-dependent DNA synthesis in vitro 3. Synthesis largely restricted to S phase of cell cycle 4. Synthesis inhibited by inhibitors of DNA synthesis 5. May be intimately involved in chromosome structure 6. Similar amounts present in active and inactive tissues 7. Similar amounts present in active and inactive chromatin 8. Synthesized in the cytoplasm

References Daly et al., 1950; Crampton et al., 1955 Daly et al., 1950; Crampton et al., 1955 Daly et al., 1950; Crampton et al., 1955 Hancock, 1969 Johns, 1967; Panyim and Chalkley, 1969; Reisfeld et al., 1962; Elgin and Bonner, 1970 Gemhey et al., 1968; Pa& and Kim, 1966, 1967, 1968, 1969a,b, 1970a,b; Liew et al., 1970; B. G. T. Pogo et al., 1966, 1967, 1968; Phillips, 1963; Allfrey et al., 1964; Allfrey, 1966a,b; Nohara et al., 1966; Shepherd et al., 1971; Murray, 1964a; Comb et al., 1966; Tidwell et al., 1968; Ord and Stocken, 1967; Langan, 1967, 1968, 1969aJb;Kleinsmith et al., 1966a,b; Gutierrez and Hnilica, 1967; Langan and Smith, 1966, 1967; Kleinsmith and Allfrey, 1969a,b; Genhey and Kleinsmith, 1969a,b De Lmge et al., 1969b; Hnilica et al., 1962; Laurence and Butler, 1965; Hnilica and Busch, 1963; Palau and Butler, 1966 Huang and Bonner, 1962; Allfrey et al., 1963; Skalka et al., 1966; Spelsberg and Hnilica, 1969; Butler and Chipperfield, 1967 Billen and Hnilica, 1964; Gurley et al., 1964 Takai et al., 1968; Borun et al., 1967; Robbins and Borun, 1967; Spalding et al., 1966 Takai et al., 1968; Borun et al., 1971; Robbins and Borun, 1967; Spalding et al., 1966 Bonner and Ts’o, 1964; Busch, 1965; De Reuck and Knight, 1966 Dingman and Sporn, 1964 Frenster et al., 1963; Littau et al., 1964; Frenster, 1965 Robbins and Borun, 1967; Borun et al., 1967

308

GARY STEIN AND RENATO BASERGA

both proteins contain 102 amino acid residues and have identical sequences except a t two positions. Histones have been shown to undergo a number of chemical modifications, both in vivo and in vitro, which include acetylation (Phillips, 1963; Allfrey, 1966a,b; Allfrey et al., 1964; Nohara et al., 1966; B. G. T. Pogo et al., 1966, 1967, 1968; Gershey et al., 1968; Liew e t al., 1970; Paik and Kim, 1970a; Shepherd et al., 1971), methylation (Allfrey et al., 1964; Murray, 1964a; Comb et al., 1966; Kleinsmith et al., 1966a; Paik and Kim, 1966, 1967, 1968, 1969a,b, 1970b; Gutierrez and Hnilica, 1967; Langan, 1967; Ord and Stocken, 1967; Tidwell et al., 1968) and phosphorylation (Kleinsmith et al., 1966b; Langan and Smith, 1966, 1967; Langan, 1967, 1968, 1969a,b). These reactions occur after completion of the histone polypeptide chain, and in the case of acetylation, the acetyl group donor is acetyl coenzyme A (Allfrey, 1966a) ; in methylation the donors are methionine (Murray, 1964a) and adenosylmethionine (S. Kim and Paik, 1965); and phosphorylation is an ATP-dependent reaction (Kleinsmith et al., 1966a). These chemical modifications of histones occur independently of histone synthesis (Allfrey, 1966a,b ; B. G. T. Pogo et al., 1966), and a number of correlations have been made between acetylation, methylation, and phosphorylation of these basic proteins and the transcription of RNA from DNA; the implication being that such processes are intimately involved in altering the structure of histones so as to render them capable of regulating the structure and function of chromatin (Allfrey et al., 1964; Allfrey, 1966b). Acetylation and phosphorylation of histones have been shown to reduce the capacity of these proteins to inhibit RNA polymerase activity (Allfrey et al., 1964, 1966) ; to be more pronounced in the diffuse euchromatin fractions, which are believed to be actively engaged in RNA synthesis, than in the clumped, inactive heterochromatin fractions (Allfrey et al., 1966, 1968) ; to be more pronounced in tissues and cell types actively engaged in RNA synthesis than in quiescent tissues (Allfrey, 1966a,b; B. G. T. Pogo et al., 1967, 1968) ; to increase in the liver of adrenalectomized rats following the administration of cortisone (Allfrey et al., 1966) in correlation to the observed increases in RNA synthesis (Feigelson et al., 1962; Kenney and Kull, 1963; Yu and Feigelson, 1969) and in vitro chromatin template activity (Dahmus and Bonner, 1965) ; and to increase during the prereplicative phase of the cell cycle in phytohemagglutinin-stimulated lymphocytes. However, it should be mentioned that recent studies, in which the mitogenic agent phytohemagglutinin has been fractionated, reveal that fractions which are incapable of eliciting the proliferative response in lymphocytes are still effective in promoting histone acetylation (Monjardino and MacGillivray, 1970). A number of studies suggest

NUCLEAR PROTEINS AND THE CELL CYCLE

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that methylation of histones may produce changes in histone structure (Tidwell et al., 1968) and, specifically, condensation of chromosomes (Tidwell et al., 1968). The main argument for considering histones as gene regulators in mammalian cells comes from the demonstration that they inhibit in vitro DNA-dependent RNA synthesis (Huang and Bonner, 1962 ; Allfrey et al., 1963; Skalka et al., 1966; Butler and Chipperfield, 1967; Spelsberg and Hnilica, 1969). However, these results are of questionable biological validity since it has been shown that, a t least under in vitro conditions, histones precipitate DNA (Johns and Hoare, 1970) and thereby make RNA polymerase and other transcribing factors inaccessible to the DNA template. I n fact, Johns and Hoare (1970) have recently pointed out that the in vitro determination of DNA-dependent RNA synthesis in a DNA-histone complex may be merely an extremely sophisticated way of measuring precipitation. Furthermore, although some investigators have concluded that the arginine-rich histones are the most effective inhibitors of DNA template activity (Allfrey et al., 1963; Hindley, 1963), others have reported that lysine-rich histones are more efficient (Barr and Butler, 1963; Huang et al., 1964). Johns and Hoare (1970) have shown that these apparent discrepancies can be resolved if the ratios of histones to DNA are taken into account, and they attribute the inconsistent results to the varying ability of each histone fraction to precipitate DNA. There is no question that histones are intimately involved in chromosome structure; however, several findings make it difficult to assign these proteins a role in the fine tuning of gene regulation. I n the first place, the histones are present in similar amounts in active and inactive tissues (Dingman and Sporn, 1964) and chromatin (Frenster et al., 1963; Frenster, 1965; Littau et al., 1964); and second, they do not possess tissue or species specificity (Hnilica and Busch, 1963; Hnilica et al., 1962; Laurence and Butler, 1965; Palau and Butler, 1966; De Lange et at., 196913). Yet, histones may be effective in completely and permanently shutting down the large portion of the mammalian genome that is inactive in differentiated cells of the adult animal; and present theory (Stellwagen and Cole, 1969a) favors the hypothesis that histones are aspecific inhibitors of DNA transcription and that they simply act by repressing large segments of the genome on a more or less permanent basis. 2. Acidic Nuclear Proteins

Recently a considerable amount of attention has been focused on nonhistone chromosomal proteins as possible regulators of gene activity in mammalian cells (Hnilica and Kappler, 1965; Stellwagen and Cole,

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GARY STEIN AND RENATO BASERGA

TABLE IX PROPERTIES OF ACIDIC NUCLEAR PROTEINS Properties

References

~

1. Negatively charged 2. Rich in aspartic and glutamic acid residues 3. Contain tryptophan 4. Faster rate of turnover than histones 5. Separable into a complex electrophoretic pattern characteristic of the tissue of origin 6. May be phosphorylated 7. Restore DNA-dependent RNA synthesis inhibited by histones 8. Synthesized in the cytoplasm 9. Synthesized throughout the cell cycle 10. Located in major groove of DNA 11. Synthesis not inhibited by inhibitors of DNA synthesis

Busch, 1965 Busch, 1965 Daly el al., 1950; Crampton et al., 1955 Stein et al., 1970; Daly et al., 1952; Smellie el al., 1953; Allfrey et al., 1955; Byvoet, 1966; Hancock, 1969; Holoubek and Crocker, 1968 Teng et al., 1970; Loeb and Creuzet, 1970; MacGillivray et al., 1971 Kleinsmith et al., 1966a,b; Kleinsmith and Allfrey, 1969a; Gershey and Kleinsmith, 1969a,b Wang, 1968, 1969a; Teng and Hamilton, 1969; Spelsberg and Hnilica, 1969 Stein and Baserga, 1971a Stein and Borun, 1971; Stein et al., 1971

J . Farber el al., 1971a Zampetti-Bosseler d al., 1969; Stein et al., 1971

1969a), and some of the properties of these proteins are summarized in Table IX. These proteins, originally described by Mirsky and Pollister in 1946, have been more recently studied by Wang and co-workers (Wang et al., 1950, 1953; Wang, l961,1963,1965,1966,1967a,b;Patel and Wang, 1964, 1965a,b; Patel et al., 1968; Wang and Johns, 1968), and by Steele and Busch (1964), Dounce and Hilgartner (19641, and Munro et al. (1970). Because of their relatively high content of glutamic and aspartic acids as determined by acid hydrolysis and amino acid analysis (Busch, 1965), these proteins have often been referred to as acidic nuclear proteins (Busch, 1965) ; unlike histones, they contain tryptophan (Daly et al., 1950; Crampton et al., 1955). A variety of techniques have been employed in the extraction, fractionation, and characterization of acidic nuclear proteins, and these have been based primarily on differences in extractability, solubility, and electrical charge between the acidic nuclear proteins and histones. These range from solubilization of acidic nuclear proteins in dilute alkali after previous extraction of the histones in dilute mineral acids (Stedman and Stedman, 1944) to disassociation of nucleoprotein complexes in strong salt solutions, followed by further fraction-

NUCLEhR PROTEINS AND THE CELL CYCLE

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ation (Mirsky and Pollister, 1946; Henson and Walker, 1970; Shaw and Huang, 1970). Other extraction procedures are based on the solubility of acidic chromosomal proteins in phenol (Steele and Busch, 1963; Vinuela et al., 1967; Shelton and Allfrey, 1970). The electrophoretic mobilities of acidic nuclear proteins have been characterized by Wang (1966, 1967a), Patel et al. (1968), Benjamin and Gellhorn (1968), and Munro et al. (1970). By modified polyacrylamide electrophoretic techniques, Stein et al. (1971) and Shelton and Allfrey (1970) have improved the resolution of acidic nuclear protein separation. Acidic nuclear proteins have also been resolved by density-gradient centrifugation, selective precipitation, and chromatographic separations. Busch (1965) has calculated an average molecular weight of 80,000400,000 for the acidic nuclear proteins from a variety of tissues on the basis of the number of NHz-terminal groups; however, acidic nuclear proteins in the range of 41,000 have also been reported (Shelton and Allfrey, 1970). Results from Wang’s laboratory (Wang and Patel, 1965; Howk and Wang, 1969, 1970) indicate that the enzyme DNA polymerase is one of the constituents of acidic nuclear protein, and Stein and Baserga (1971a) have demonstrated that acidic chromosomal proteins are synthesized in the cytoplasm. Further details on the classification and composition of these proteins can be found in a recent review by Stellwagen and Cole on chromosomal proteins (1969a). The evidence supporting the role of acidic nuclear proteins in the regulation of gene activity is summarized in Table X. These proteins are actively synthesized in mammalian cells (Steele and Busch, 1964; Stein and Baserga, 1970b; Rovera and Baserga, 1971), even during mitosis (Stein and Baserga, 1970a), and they have a more rapid turnover than histones (Daly et al., 1952; Smellie et al., 1953; Allfrey et al., 1955; Byvoet, 1966; Holoubek and Crocker, 1968; Hancock, 1969; Stein et al., 1970). In a study of protein metabolism in chromosomes of nondividing kidney, pancreas, and liver cells, Allfrey et al. (1955) found that the residual proteins incorporated glycine-I5N at a faster rate than histones. Frenster and co-workers (Frenster et al., 1963; Littau et al., 1964; Frenster, 1965) reported that the types and quantities of histones were similar in active (euchromatin) and repressed (heterchromatin) calf thymus lymphocyte chromatin ; however, active chromatin was found to contain a 2-fold excess of nonhistone residual proteins. These findings are compatible with the demonstration by Hsu (1962), Klevecz and Hsu (1964), and Berlowitz (1965) that RNA synthesis in heterochromatic areas of chromosomes is less active than in euchromatin. An increase in the synthesis of nonhistone chromosomal proteins has also been found in active tissues of chicken by Dingman and Sporn (1964),

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GARY STEIN AND RENATO BAGERGA

TABLE X ROLEOF ACIDICNUCLEARPROTEINS IN THE REQULATION OF GENE ACTIVITY Role 1. Active tissues contain more acidic nuclear 2.

3.

4.

5.

6. 7.

protein than inactive tissues Active chromatin contains more acidic nuclear proteins than inactive chromatin Acidic nuclear proteins restore histoneinhibited DNA-dependent RNA synthesis in uitro Acidic nuclear proteins increase the in uilro transcription of inactive chromatin and activate repressed gene loci Acidic nuclear proteins interact with DNA in uitro and modify transcription in a manner that is characteristic of the tissues of origin Tissue-specific nonhistone chromosomal proteins bind to DNA Acidic nuclear proteins possess tissue and species specificity

8. Acidic nuclear proteins are actively

References Dingman and Sporn, 1964 Fremter et al., 1963; Frenster, 1965; Littau el al., 1964 Wang, 1968, 1969a; Teng and Hamilton, 1969; Spelsberg and Hnilica, 1969 Wang, 1969b; Kamiyama and Wang, 1971 Gilmour and Paul, 1969, 1970; Spelsberg and Hnilica, 1970 Kleinsmith et al., 1970 Teng et al., 1970; Loeb and Creuzet, 1970; MacGillivray et al., 1971; Gilmour and Paul, 1970 Stein and Baserga, 1970a

ayntheaized during mitosis

in lactating rat mammary glands by Stellwagen and Cole (1969b), in the puffs of polytene chromosomes by Beermann (1963) and Edstrom and Beermann (1962), and in the loops of lampbrush chromosomes by Gall and Callan (1962). While the histones show uniformity in composition and a regular distribution in various tissues and organs (Frenster et al., 1963; Hnilica and Busch, 1963; Dingman and Sporn, 1964; Littau et al., 1964; Frenster, 1965; Hnilica et al., 1962; Laurence and Butler, 1965; Palau and Butler, 1966; De Lange et al., 1969b), acidic nuclear proteins can be resolved into a complex electrophoretic pattern (Shelton and Allfrey, 1970; Stein et al., 1970; Stein and Baserga, 1971a,b) that is characteristic of the tissue of origin (Loeb and Creuaet, 1970; Teng e t al., 1970; MacGillivray e t al., 1971). A possible role of acidic nuclear protein in the control of gene expression is suggested by in vitro experiments in which these proteins were found to be capable of forming insoluble complexes with histones (Wang, 196713; Wang and Johns, 1968) and of modifying the DNA-histone interaction in such a way as to bring about a restoration of histoneinhibited DNA-dependent RNA synthesis (Wang, 1968, 1969a). Langan (1967) and Langan and Smith (1967) reported that when histone is

NUCLEAR PROTEINS AND T H E CELL CYCLE

313

complexed to acidic nuclear protein it exerts only a partial inhibitory effect on DNA-dependent RNA synthesis. Wang (1968) found that acidic nuclear proteins are capable of restoring histone-inhibited DNA-dependent RNA synthesis regardless of the order of addition of DNA, histone, and acidic nuclear protein, a finding compatible with the results obtained by Teng and Hamilton (1969) ; however, Spelsberg and Hnilica (1969) found that although acidic nuclear proteins are capable of restoring histone-inhibited template activity, they are ineffective in doing so after the histoneDNA complex is established. The involvement of nonhistone chromosomal proteins in the control of gene expression is also suggested by the claim from Wang's laboratory that the augmented transcription produced by the addition of such proteins to inactive chromatin represents an activation of suppressed gene loci (Wang, 1969b; Kamiyama and Wang, 1971). The nucleotide composition, nearest-neighbor frequency analysis, and extent of RNADNA hybridization of RNA's transcribed from chromatin and nonhistone-activated chromatin suggest that additional transcription sites on DNA are rendered available as a result of gene activation by the addition of nonhistone chromosomal protein. In fact, Kamiyama and Wang (1971) claimed that the RNA made by DNA in the presence of acidic nuclear proteins codes for proteins with a different lysine : leucine ratio than the RNA made under the same conditions, but in the absence of acidic nuclear proteins. Just as important are the experiments of Paul and Gilmour (1968), Gilmour and Paul (1969, 1970), and Spelsberg and Hnilica (1970), which have demonstrated that acidic chromosomal proteins may regulate the template function of chromatin, and that acidic chromosomal proteins can interact with DNA in vitro and modify transcription in a manner that is characteristic of the tissue of origin. According to these authors and also according to Teng e t al. (1970), Loeb and Creuzet (1970), and MacGillivray et al. (1971), acidic nuclear proteins are tissue and species specific. Shelton and Allfrey (1970) have recently demonstrated that during steroid hormone-induced gene activation, the synthesis of n nonhistone chromosomal protein of molecular weight 41,000 is specifically enhanced, and Teng et al. (1970) have shown that acidic nuclear proteins will selectively bind to the DNA of thc tissue of origin. Kleinsmith et al. (1970) have also found a tissue-specific nonhistone cliromosomal protein \?rhich binds to DNA. In a study of acidic nuclear proteins in tumors, Sporn and Dingman (1966) reported that the chromatin of rat hepatomas induced by Ar-hydroxy-2-acetylaminofluorene and 3'-methyl-4-dimethylaminoazobenzene contain higher amounts of nonhistone chromosomal proteins than the chromatin of normal liver. Similar observations were

314

GARY STEIN AND RENATO BASERGA

made by Grunicke et al. (1970) when they compared highly deviated hepatomas with normal tissue; however, when they compared the acidic nuclear protein contents of minimal deviation hepatomas with normal liver, no differences in the acidic nuclear protein contents were observed. Additional evidence on the possible role of acidic nuclear proteins in the regulation of gene activity and cell function has come from the investigations of Langan (1967), Kleinsmith et al. (1966a,b), Kleinsmith and Allfrey (1969a), and Gershey and Kleinsmith (1969a,b) on nonhistone phosphoproteins; the observation by Ruddon and Rainey (1970) and Buck and Schauder (1970) that phenobarbital and insulin stimulate the synthesis of acidic nuclear proteins in liver; the observation by Swaneck et al. (1970) that both the soluble nuclear and chromosomal aldosterone-binding proteins are nonhistone proteins ; the report by Malpoix (1971) that the synthesis of nonhistone chromosomal protein is stimulated in disaggregated fetal mouse liver cells by erythropoietin ; and the findings of C. E. Smith and Mora (1971) which demonstrate that chromatin isolated from minimal deviation hepatomas contains more nonhistone chromosomal protein than liver chromatin.

PROTEINS AND CELLPROLIFERATION C. ACIDICNUCLEAR 1. Acidic Nuclear Proteins i n Models of Stimulated D N A Synthesis A number of findings have recently appeared in the literature which point to the significance of acidic nuclear proteins in models of stimulated DNA synthesis. Teng and Hamilton (1969, 1970) have suggested that nonhistone chromosomal proteins may be involved in a model of stimulated DNA synthesis, namely, the estrogen-stimulated uterus of the ovariectomized rat. They have demonstrated that the synthesis of nonhistone chromosomal proteins increases early in the response of the uterus to estrogen, and they proposed that nonhistone chromatin proteins play an antagonistic role to histones, in controlling the amount of genetic material available for transcription by RNA polymerase. Recent observations by Barker (1971) suggest that 15 minutes after stimulation by estradiol there is an increase in the specific activity of an electrophoretically distinct nonhistone protein which is associated with F, histone. Barnea and Gorski (1970) have shown that a specific acidic protein is synthesized in the rat uterus after stimulation by estrogens, and Mayol and Thayer (1970) confirmed these results using doubleisotope labeling methods and polyacrylamide gel electrophoresis. Stellwagen and Cole (1969b) reported a relationship between the synthesis of acidic nuclear proteins in the mammary gland and the stimulation of DNA synthesis, and the experiments of J. A. Smith et al. (1970)

NUCLEAR PROTEINS AND THE CELL CYCLE

315

suggest that these proteins are involved in stimulated cellular proliferation in the rat uterus by progesterone. Additional and more direct demonstrations that nonhistone chromosomal proteins may be involved in models of stimulated DNA synthesis have come from the investigations of Stein and Baserga (1970b) and Rovera and Baserga (1971). Stein and Baserga (1970b) studied the synthesis of acidic nuclear proteins in the isoproterenol-stimulated salivary gland. I n this system, a single injection of isoproterenol results, after a lag period of 20 hours, in a marked stimulation of DNA synthesis in the salivary gland of rodents (Barks, 1965a,b; Baserga, 19661, followed a few hours later by a burst of mitosis (Baserga and HeHer, 1967). During the prereplicative period changes in RNA synthesis (Baserga and HefBer, 1967; Sasaki and Baserga, 1970) and the activity of several enzymes have been observed (Pegoraro and Baserga, 1970). Changes in protein synthesis also occur in the mouse salivary gland during the prereplicstive phase (Sasaki et al., 1969). Stein and Baserga (1970b) found that the specific activities of three nonhistone nuclear protein fractions increased within 30 minutes after the administration of isoproterenol and did not return to control levels until 40 hours. The acidic nuclear protein fractions reached their peak activities 12 hours after isoproterenol, when they showed a 3- to 4-fold increase above control levels; a t 26 hours after injection of isoproterenol, when the salivary gland cells are actively synthesizing DNA, the specific activities of these acidic proteins were below their 12-hour levels. I n contrast, the peak activity of the histone fraction coincided with the peak of DNA synthesis, and at that time there was a 3-fold increase above the controI level. Since the specific activity of the leucine pool did not change in stimulated salivary glands, the increased incorporation of l e ~ c i n e - ~ H into acidic nuclear proteins suggested true changes in the rates of synthesis. Treatment with actinomycin D, 30 minutes prior to isoproterenol, inhibited isoproterenol-stimulated DNA synthesis but did not inhibit the increase in the rate of acidic nuclear protein synthesis a t 2 hours after isoprotereno1. However, the increase in the rates of acidic nuclear protein synthesis that O C C M a~ t 8 and 12 hours were effectively inhibited by actinomycin D. These results suggest that the increased synthesis of acidic nuclear proteins 2 hours after isoproterenol is not dependent on previous RNA synthesis and that the initial events in the prereplicative phase may be under translational control. These results are consistent with the demonstration of Sasaki et at. (1969) that the increased amino acid incorporation activity of free ribosomes isolated from mouse salivary gland I hour after stimulation by isoproterenol was actinomycin D insensitive, and the possibility arises that the initial increase in protein synthesis following isoproterenol

316

GARY STEIN AND BENATO BASEBGA

stimulation may be due to an activation of previously existing templates rather than to the synthesis of new templates. Such a mechanism has been reported to be operating in other systems (Wool and Kiruhara, 1967;Boyadjiev and Hadjiolov, 1968; Roth et al., 1968).The fact that Stein and Baserga found the increased synthesis of nuclear acidic proteins at 8 and 12 hours following isoproterenol to be inhibited by actinomycin suggests that a t these times transcriptional activity is a prerequisite for such synthesis. Cycloheximide administered 1 hour after isoproterenol also inhibited the increase in acidic nuclear protein synthesis a t 8 and 12 hours. At this time after isoproterenol and with the used, cycloheximide inhibits isoproterenol-stimulated DNA synthesis, although it inhibits protein synthesis in the salivary glands for only 2 hours (Sasaki et al., 1969). These results mggest that a protein (or class of proteins) synthesized 1 hour after administration of isoproterenol is esmntiai for the increased synthesis of acidic nuclear proteins a t h t e r times in the pmreplicative phase and for the onset of DNA synthesis. It also appears that the template responsible for the Synthesis of the “l-hour protein” is labile and that the “1-hour protein” may not be dependent on previous RNA synthesis. In any case, it is evident that the inhibition of protein synthesis a t 8 and 12 hours cannot be attributed to a direct effect of cycloheximide. Pilocarpine, whieh stimulates salivary gland secretion without stimulating DNA synthesis, was ineffective in producing a stimulation of acidic nuclear protein synthesis. Since the patterns of acidic nuclear protein and histone synthesis do not coincide, the possibility .of functional differences between the two classes of nuclear proteins arises. Indeed, the fact that the peaks of NaC1-soluble and tenaciously bound residual acidic nuclear protein synthesis precede the peak of DNA synthesis and that this synthesis is inhibited by inhibitors of isoproterenol-stimulated DNA synithesis, suggests a role of midic nuclear proteins in the control of DNA synthesis and cell division in the isoproterenol-stimulated salivary gland. Such a role is further suggested by differences in the SDS-polyacrylamide gel electrophoretic profiles of these proteins extracted from stimulated and unstimulated glands (Stein and Baserga, 1971b). Rovera and Baserga (1971) studied the synthesis of acidic nuclear proteins in confluent mmolayers of WI-38 e l l s stimulated to proliferate by changing the medium, a manipulation that causes approximately $060% of the cells to synthesize DNA and divide. The extraction procedure employed in these studies was similar to that used by Skin and Baserga (1970b).Rovera and Baserga found that a n increase in the synthesis of three acidic nuclear protein fractions-ribonucleoproteins, NaCl-

317

NUCLEAR PROTEINS AND THE CELL CYCLE

soluble proteins and residual acidic proteins-occurred between 1 and 3 hours after stimulation. The rate of NaC1-soluble protein synthesis then returned to control levels, while the synthesis of residual and ribonucleoproteins remained elevated between 6 and 12 hours and increased even further a t 18 hours, the peak of DNA synthesis. Pulse-chase experiments indicated that the proteins synthesized during the first hour after stimulation have a turnover time of less than 4 hours, and the same fractions in nonproliferating cells were stable for a t least 12 hours. Similar results have been obtained with stimulated 3T6 cells (Tsuboi and Baserga, 1971). 2. Acidic Nuclear Proteins in Continuously Dividing Cells

Evidence has been accumulating which suggests that acidic nuclear proteins may also be important in continuously dividing cells. Stein and TABLE X I ACIDICNUCLEAR PROTEINS AND CELLPROLIFERATION

I. Models of stimulated DNA synthesis A. In vivo models Tissue or organ Animal

Stimulus

References ~~

Uterus

Rat

Estrogen

Uterus Salivary gland

Rat Mouse

Progesterone Isoproterenol

B. In vitro models cell type

Origin

Stimulus

Mammary gland

Rat

Explantation

WI-38

Human diploid fibroblasts

Serum

11. Continuously dividing cells Cell line Origin HeLa SI

Human cervical carcinoma

Minimal deviation hepatoma

Rat liver

Barnea and Gorski, 1970; Mayol and Thayer, 1970; Teng and Hamilton, 1969, 1970 J. A. Smith et al., 1970 Stein and Baserga, 1970b References Stellwagen and Cole, 1969b Rovera and Baserga, 1971 References Stein and Baserga, 1970a; Stein and Borun, 1971; Stein et al., 1971 ; Borun and Stein, 1971 C. E. Smith and Mora, 1971

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GARY STEIN AND RENATO BASERGA

Baserga (1970a) have demonstrated that acidic nuclear proteins are synthesized in all phases of the cell cycle in synchronized HeLa S, cells, including during mitosis when RNA synthesis ceases (J. H. Taylor, 1960a; Baserga, 1962b ; Prescott and Bender, 1962; Feinendegen and Bond, 1963; Prescott, 1964) and there is a 70-90% decrease in the rate of total cellular protein synthesis (Baserga, 1962a ; Prescott and Bender, 1962; Robbins and Scharff, 1966; Konrad, 1963). Further studies have shown that there is a significant increase in the specific activity of acidic proteins during GI (Stein and Borun, 1971), and experiments with inhibitors of DNA synthesis (cytosine arabinoside and hydroxyurea) clearly established that, unlike histones whose synthesis is tightly coupled with DNA replication (Borun et al., 1967), the synthesis of these acidic proteins continues independently of DNA synthesis (Stein et d.,1971; Stein and Borun, 1971). SDS-polyacrylamide gel electrophoretic profiles of acidic nuclear proteins synthesized during G,, S, G,, and mitosis suggest that there are stage-specific differences in the acidic nuclear proteins (Stein and Borun, 1971; Stein et al., 1971). And finally, a series of recent studies not only demonstrated that acidic nuclear proteins have a faster rate of turnover than histones, but that the rates of turnover of these proteins vary in different stages of the cell cycle, the highest turnover being observed during mitosis and the lowest during Sphase (Stein et al., 1971; Borun and Stein, 1971). The evidence suggesting a major role of acidic nuclear proteins in the control of cell proliferation is summarized in Table XI.

D. CONCLUSIONS From the preceding discussion it is evident that in continuously dividing cells, as well as in G,, cells which are stimulated to proliferate, a complex and interdependent series of biochemical events precedes the onset of DNA synthesis and mitosis, and a substantial amount of data supports the hypothesis that gene activation is intimately involved in the process. It is also apparent that evidence is accumulating which suggests that acidic nuclear proteins are involved in the control of gene expression and cell proliferation in both populations of cells, and one might anticipate that in the near future additional details will become available as to the specific mechanisms by which these proteins interact with the genome and initiate, modify, or augment the transcription of informational macromolecules.

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AUTHOR INDEX Numbers in italics indicate the pages on which the complete references are listed.

A

48, 146, 168, 160, 161 Abrams, R., 292, 296, 297, 299, 300, 301, 302, 304, 394, 386 Abuelo, J. G., 102, 160 Adam, A., 217, B6 Adam, G. H. M., 139, 160 Adams, J. E., 289, 292, 301, ao2, 318 Adams, R. A., 17, 60 Adam, R. L. P.,292, 296, 297, 300, 301, 302, 304, 319, 326 Adams, W. S., 182, 187 Agayev, B. A., 278, 989 Agrell, I. P. S., 107, 160 Aisenberg, A. C., 264, 289 Aicama, Y., 301, 303, 319 Akasaka, A,, 236, 948 Alberga, A., 120, 160, 169 Albert, D., 279, 888 Albert, D. M., 12, 48 Alder, A., 122, 163 Alexander, P., 9, 48 Algranati, I., 311, 389 Ali, M. Y., 69, 89 Allen, G. V., 77, 78, 90 Allen, L. N., 97, 160 Allfrey, V. G., 113, 116, 116, 118, 119, 121, 122, 124, 126, 128, 129, 130, 136, 137, 138, 139, 140, 142, 148, 160, 163, 166, 166, 169, 160, 161, 169, 292, 296, 299, 300, 301, 302, 304, 306, 307, 308, 309, 310, 311, 312, 313, 314, 319, 381, 388, 324, 326, 3R, 398, 329 Allison, A. C., 3, 7, 48 Allot, E. N., 278, 282 Aloni, Y., 38, 48 Alstein, A. D., 4, 5, 14, 49 Amaldi, F., 292, 319 Ambrose, K. R., 24, 30, 60 Aaronson, 8. A., 12, 14, 16, 33,

Ambmaioni, J. C., 85, 90 Ananieva, L. A., 126, 131, 132, 164 Andem, M., 179, 188 Anderson, D. E., 12, 69

Anderson, E. C., 289, 292, 293, 294, 3Q3, 319, 396, 368 Anderson, K. M., 120, 163 Anderson, N. G., 24, 30, 60 Anderson, W. F., 166, 187 Andreoli, A., 187, 190 Andrews, P. A. J., 83, 90 Angell, R., 204, 986 Anken, M., 23, 33, 34, 60, 61 Apoahian, H. V., 36, 63 Arcos, J. C., 236, 848 Argus, M. L., 236, 948 Ariaon, R. N., 268, 988 Asao, T., 107, 160 Ashkenazi, A., 2, 11, 23, 24, 32, 41, @, 61

Ashwood-Smith, M. J., 297, 393 Ashworth, J. M.,304, 316, 3 s Atkinson, L., 58, 69, 79, 90, 91 Auditore, J. V., 209, 923 Auer, G., 126, 162, 299, 300, 306, 319, 330 Auld, W. H. R., 274, 984 Austin, G. E., 144, 163 Axelrod, D., 35, 39, 61, 69, 66 Aehipa, Ya. I., 242, 949, 260 B Bacopoulos, C., 194, 226 Baggot, D., 275, 286 Baguley, B. C., 170, 187 Bahn, R. C., 269, 270, 271, 273, 886 Baikie, A. G., 200, 296 Bailey, I., 274, 884 Bailey, J. S., 119, 161 Baird, S., Jr., 236, 236, 861 Bajer, A,, 103, 160 Bakay, B., 198, H6 Balchum, 0. J., 209, 823 Baliga, B. S., 168, 177, 188 Baltimore, D., 101, 160 Baluda, M. C., 214, 226 Banerjee, 9. K., 270, 884 Baney, R. W., 293, 3.84 Baranaka, W., 27, 49

331

332

AUTHOR INDEX

Barbanti-Brodano, G., 13, 40, 41, 42, 49, 64 Barhnart, F. E., 205, 884 Barka, T., 292, 298, 300, 301, 302, 304, 316, 319

Barker, K., 314, 319 Barkhan, P., 297, 381 Barnea, A., 300, 301, 314, 317, 319 Barnett, I(. M. A,, 67, 75, 87, 90 Baron, S., 3, 7, 38, 48, 63, 66 Barr, G. C., 130, 161 Barr, J. B., 274, 884 Barr, J. C., 309, 319 Barron, E. S. G., 233, 236, g@ Bartley, J., 120, 141, 161 Baeerga, R., 117, 118, 161, 287, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300,301,302,303,304,305,307, 310, 311, 312, 316, 316, 317, 318, 319, 3mJ 381, 388, 386, 386, 387, 388, 329 Basilico, C., 15, 49 Bauduin, H., 319 Baulieu, E. E., 120, 160, 169 Baum, 9. G., 5, 16, 49, 64 Baute, E. K. F., 98, 99, 161, 163 Baute, F. A., 98, 99, 161, 163 Beatty, B. R., 103, 167, 386 Becker, H., 288, 319 Beckwith, J., 100, 168 Beecher, J. L., 79, 91 Beermann, W., 121, 124, 125, 161, 163, 312, 319, 381 Beers, D. N., 274, 888 Beigelman, P. M., 274, 886 Bekhor, I., 114, 116, 119, 133, 135, 161 Belli, J. A., 289, 319 Belova, S., 8, 26, 68 Ben-Bassat, H., 28, 49 Bender, A. B., 274, 886 Bender, M. A,, 295, 318, 386 Benjamin, T. L., 15, 28, 38, 49, 96, 161 Benjamin, W., 109, 113, 115, 118, 138, 161, 311, 319 Benjamin, W. B., 138, 161 Bennett, L. L., 292, 319 Benson, R., 110, 16g Bensted, J., 9, 48 Berendes, H. D., 124, 125, 136, 161 Berg, R., 11, 60 Bergeron, J. J., 295, 303, 319

Bergman, E. D., 236, 849 Bergquist, P. L., 172, 180, 188, 189 Berlowits, L., 311, 380 Berman, E., 170, 189 Berman, L. D., 16, 49 Bernardi, G., 114, 163 Bernstein, V. A., 255, 883 Bertino, J. R., 289, 323 Beutler, E., 192, 194, 203, 208, 209, 883, 884 Bilkis, D., 274, 884 Bill, A. H., 213, .?!86 Billen, L., 307, 380 Billingham, R. E., 26, 49 Birnboim, H. C., 299, 305, 380 Birnstiel, M., 106, 166 Biserte, G., 110, 111, 166, 160 Bishop, J. O., 128, 167, 299, 305, 386 Biswas, B. B., 120, 136, 167 Bjork, G. R., 169, 188 Black, P. H., 2, 3, 11, 12, 13, 14, 16, 16, 20, 21, 30, 32, 33, 36, 38, 49, 61, 68, 68, 64 Blatti, 8. P., 144, 145, 161 Blew, D., 176, 188 Blinov, V. A., 278, 886 Bloch, D. P., 107, 161 Block, D. P., 298, 880 Block, P., 298, 380 Blois, M. S., Jr., 246, 247, 849 Bloom, S., 297, 380 Bloomquist, J. C., 293, 294, 331 Bluhm, A. L., 237, 849 Bodycote, D. J., 103, 164 Boenig, H. V., 236, 849

Bdye, A., 5, 49 Boiron, M., 3, 49 Bollinger, R. E., 273, 884 Bollum, F. J., 292, 301, 302, $80 Bolton, J. R., 228, ,961 Bolund, L., 126, 161, 169, 297, 299, 300, 305, 320, 381, 387 Boman, H. G., 180, 188 Bond, V. P., 296, 318, 38.8 Bondy, 9. C., 137, 161 Bonner, J., 102, 106, 107, 109, 110, 111, 113, 114, 115, 116, 118, 119, 120, 122, 126, 127, 130, 131, 132, 133, 134, 135, 137, 140, 146, 148, 161, 168, 163, 166,

AUTHOB INDEX

167, 169, 160, 161, 305, 306, 307, 308, 309, 312, 314, 390, 391, 393, 387 Bonney, A., 172, 188 Bonsavaros, G. A., 274, 989 Bonser, G. M., 81, 90 Booth, K., 79, 90 Bootsma, D., 289, 3.80, 39.9 Borek, E., 167, 188, 169, 170, 175, 177, 178, 179, 180, 182, 186, 187, 188, 189, 190 Borg, D. C., 228, 236, 237, 242, 249, 961 Borisy, G. G., 289, 3.90 Borman, A., 278, 284 Borman, G. S., 2, 3, 60 Borun, T. 98, 116, 117, 161, 169, 161, 290, 291, 292, 293, 294, 296, 301, 302, 307, 310, 311, 312, 317, 318, 390, 3n, 328 Boshell, B. R., 273, 989 Bosmann, H. B., 294, 320 Botchan, M., 101, 129, 164 Bothan, F., 269, 283 Boublik, M., 122, 161, 161 Bourali, M.-F., 41, 49 Boutwell, R. K., 298, 393 Bowen, J., 12, 69 Bower, B. F., 272, 989 Boyadjiev, S. I., 304, 316, 320 Boyland, E., 237, 249 Boyse, E. A., 84, 91, 212, 226 Brachet, J., 164, 188 Bradbury, E. M., 116, 121, 122, 123, 161, 167 Bragg, K. U., 69, 70, 92 Branigan, J., 3, 63 Braun, A. C., 16, 45, 49 Braunstein, A. E., 269, 28$ Brawerman, G., 95, 96, 163 Brecher, G., 200, 201, 926 Brehaut, L. A., 289, 328 Breier, B., 173, 175, 188 Bremberg, S., 220, 224 Brener, C., 278, 984 Brennan, M. J., 241, 242, 249 Brent, T. P., 290, 291, 293, 320, 3.W Bresciani, F., 298, 390 Bresler, A., 97, 166 Bresler, M. A., 291, 326 Brewen, J. G., 289, 320 Breynaert, M.D., 110, 160

w.,

333

Brigge, C. D., 237, 961 Britten, R. J., 105, 128, 148, 161, 299, 305, 329

Brody, E. N., 98, 167 Bromberg, P. A., 180, 190 Brown, D. D., 143, 144, 169, 293, 390 Brown, D. F., 294, 3.83 Brown, M., 13, 33, 41, 61, 69 Brown, S. W., 124, 161 Bruce, S., 196, 296 Brues, A. M., 233, 235, 949 Brutlag, D., 134, 167 Bucher, N. L. R., 292, 297, 298,301, 303, 320, 3 f l

Buck, A. T., 296, 298, 300, 304, 328, 399 Buck, C. A., 175, 176, 177, 188, 189 Buck, M. D., 314, 390 Buckton, K. E., 200, 996 Budke, L., 289, 320 Buhler, W., 197, ,924 Buell, D. N., 290, 291, 390 Buell, P., 70, 72, 90 Bukhman, V. M., 263, $86 Bulbrook, R. D., 84, 99 Burdick, C. J., 122, 124, 166, 167 Burdon, R. H., 120, 140, 161 Burger, M. M., 28, 29, 49, 295, 296, 297, 320, 322

Burgess, E. A., 258, 282 Burgess, R. R., 98, 161, 161 Burk, D., 261, 263, 882 Burket, A. E., 11, 12, 64 Burkitt, D. P., 205, 994 Burlakova, Ye. B., 245, 246, 949 Burns, W. H., 32, 49 Burny, A., 101, 146, 160, 161 Burstein, S., 278, 284 Burtin, P., 57, 85, 91 Busch, H., 96, 113, 130, 166, 167, 161, 257, 982, 306, 307, 309, 310, 311, 312, 320, 323, 328

Bushong, S. C., 10, 66 Bustin, M., 107, 110, 161, 169 Butler, J. A. V., 117, 130, 161, 162, 235, 249, 290, 291, 306,307, 309, 312, 319, 320, 323, 324, 326

Butel, J. S., 5, 6, 10, 12, 14, 15, 19, 20, 21, 22, 35, 41, 42, 44, 46, 49, 60, 69, 63, 64

334

AUTHOB INDEX

Byvoet, P., 117, 120, 130, 162, 169, 310, 311, 390

C Cachin, Y., 57, 85, 91 Cadena, E., 239, 242, 247, 961 Caffier, H., 40, 61 Cahan, A., 204, 826 Cahill, G. E., Jr., 280, 283 Cahn, M. B., 93, 162 Cahn, R. D., 93, 162 Callahan, P. X., 107, 167 Callan, H. G., 103, 162, 312, 329 Cameron, I. L., 289, 390 Campadelli-Fiume, G., 184, 188 Campbell, A., 35, 49 Campbell, P. N.,267, 282 Cantero, A., 277, 284 Capra, J. D., 106, 188 Carbone, P. P., 200, 201, 926 Carey, R. W., 273, 274, 275, 282 Carney, P. G., 20, 62 Carol, B. M., 298, 322 Carroll, A., 136, 148, 166, 312, 313, 324 Carroll, D., 109, 114, 115, 116, 167, 310, 312, 313, 326

Carp, R. I., 13, 14, 22, 49, 60 Cartwright, G. E., 204, 226 Cashel, M., 100, 162 Casey, M. J., 5, 61 Caspersson, T., 200, 224 Cassingena, R., 34, 41, 49, 66 Chader, G . J., 326 Chalkley, G. R.,102, 106, 107, 111, 113, 117, 120, 141, 161, 162, 167, 168, 160, 308, 307, 326 Chamberlin, M., 99, 162, 164 Chambon, P., 98, 142, 143, 144, 145, 162, 164, 166, 167 Champagne, M., 113, 162 Chaney, S. Q.,178, 185, 188 Chanock, R. M., 5, 61 Chantarakul, N., 72, 90 Chaplain, R. A., 288, 286 Chapman, R. E., 298, 321 Chaudhuri, S., 296, 300, 304, 320 Chen, B., 100, 169 Ch’en, C. C., 59, 90, 91 Cherepneva, I. Ye., 244, 261 Chesterman, F. C., 3, 7, 48

Chetverikov, A. G., 245, 260 Chheda, G. B., 182, 188 Chiappino, G., 203, 226 Chiarugi, V. P., 98, 162, 177, 188 Chibisova, V., 8, 26, 62 Chibrikeu, A., 239, 244, 246, 261 Chibrikin, V. M., 239, 244, 261 Chiga, M., 292, 301, 302, 323 Childs, B., 192, 194, 224 Chipperfield, A. R., 130, 162, 307, 309, 380 Choraey, M., 98, 167 Christensson, E. G., 107, 160 Chu, C. C., 59, 91 Chu, H. M., 59, 91 Chu, L., 314, 328 Chun, D., 76, 90 Church, R.B., 95, 96, 127, 168, 160, 298, 299, 300, 304, 320 Churchill, J. P., 290, 320 Ciaccio, E. I., 268, 883 Cikes, M., 291, 320 Clark, P. R., 121, 122, 130, 162 Clark, R. J., 121, 122, 146, 162 Clark, S. H., 280, 263, 283 Clarke, D., 275, 886 Clayson, D. B., 298, 324 Clein, G. P., 200, 224 Clever, U., 121, 124, 125, 138, 162 Clifford, P., 57, 58, 80, 05, 76, 79, 83, 84, 85, 90, 91, 195, 204, 207, 208, 209, 211, 212, 218, 219, 220, 221, 224, 226, 226 Coggin, J. H., 24, 30, 60 Coghill, S. L., 15, 16, 62 Cohen, L. S., 289, 290, 321 Cohen, M. M., 137, 167 Cohen, P. P., 128, 166 Cohen, S., 297, 301, 303, 321, 328 Cohn, P., 117, 169 Cole, R. D., 106, 107, 110, 116, 119, 129, 161, 162, 166, 166, 169, 161, 300, 301, 306, 309, 310, 311, 312, 314, 317, 328 Cole, T., 240, 241, 242, 249 Colin, M., 819 Collins, Z. V.,208, 209, 283, B.4 Colo, A., 202, 203, 224 Colombo, J., 258, 286 Comb, D. G., 120, 140, 169, 302, 307, 308, 3.91

AUTHOB INDEX

Commoner, B., 233, 234, 235, 239, 240, 243, 244, 245, 2.69, 262 Conn, J. W., 272, 283 Connor, J. D., 198, 226 Cooke, R.,79, 90 Coon, H. G., 93, 162 Cooper, E. H., 297, 321 Cooper, H. L., 292, 296, 297, 300, 303,

304, 321, 387

Cooper, J. T., 237, 249 Cooper, W. G., 292, 397 Copeland, E. S., 241, 261 Coppey, J., 34, 66 Cori, C. F., 266, 283 Cori, G. T., 266, 283 Cornell, A. G., 279, 286 Court Brown, W. M., 200, 226 Courtney, A. H., 257, 264, 265, 267, 283 Couvillion, L. A., 7, 25, 64 Cowling, D. C., 301, 303, 326 Craddock, V. M., 170, 172, 184, 188 Craig, J. W., 274, 275, 284 Cramer, R., 15, 62 Crampton, C. F., 308, 307, 310, 321, 389 Crane-Robinson, C., 116, 121, 122, 123,

335

Dalldorf, G., 205, 224 Dalton-Tucker, M. F., 12, 60 Daly, M.M., 306,307, 310, 311, 319, 321 Dameshek, W., 209, 224 Daneholt, B., 121, 162 Daniel, J. C., 95, 162 Danishepky, I., 237, 260 Darlington, G.,196, 226 Darnell, J. E., 39, 62, 299, 305, 320 Darzynkiewicz, L., 299, 800, 305, 320, 821, 327

Das, M. R., 101, 146, 260, 161 Dastugue, B., 113, 162, 166 Daughaday, W. H., 321 Dautrevaux, M., 110, 160 Davidova, S. Ya., 262, 283 Davidson, E. H., 122, 148, 161, 167 Davidson, J. N., 310, 311, 387 Davidson, N., 104, 131, 168, 162 Davidson, R. G., 192, 194, 203, 224 Davies, A. J. S., 220, 224 Davies, H. G., 102, 162 Davis, J., 196, 286 Davis, J. R., 257, 282 Davis, S., 182, 189 Dawid, I., 293, 320 161, 167 Crathorn, A. R., 290, 291, 293, 320, 387 Dawson, K. M., 298, 394 Dawson, W.R., 61, 63, 92 Crawford, W. H., 274, 983 Creuzet, C., 113, 166, 310, 312, 313, 324 Dearman, H. H., 236, 2.48 Crippa, M.,143, 144, 162, 161, 290, Bl, Deaven, L. L., 103,162,289, 290, 291, 292, 294, 328 292, 293, $81 Debatlow, V. G., 122, 169 Crocker, D. W., 273, 283 Crocker, T. T., 118, 166, 310, 311, 323 DeBellis, R. H., 118, 161 Decker, K.,298, 324 Crookeff, J. E., 273, 284 DeCrombrugghe, B., 100, 169 Crosby, W. H., 209, 224 Decter, J., 170, 189 Croes, M. E., 137, 139, 162 Defendi, V., 4, 5, 13, 14, 22, 23, 24, 26, Crosswhite, L. H., 200, 226 28, 29, 30, 31, 60, 61, 64, 292, 297, Crotty, M. W., 327 326, 327, 329 Crouch, N. A., 26, 30,31,64 de Harven, E., 84, 91, 212, 226 Crumpacker, C. S., 23, 62 Deichman, G. I., 2, 4, 7, 8, 10, 11, 14, Cummins, J. E., 293, 294, 381 26, 49, 60, 62 Cuppage, F. E., 298, 321 de la Chapelle, A., 204, 926 D De Lange, R. J., 106, 110, 111, 137, 152, 160, 306, 307, 309, 312, 314, 321, 3 f l Dacie, J. V., 209, 224 Del Monte, U., 258, 270, 283 Dahmur, M., 102, 113, 161 Delmore, E. J., 9, .@ Dahmus, G. K., 119, 135, 161 Dahmus, M. E., 118, 119, 131, 148, 161, Del Villano, B., 23, 60 DeMan, R., 192, 194, 195, 824 162, 306, 308, 320, 381 Demopoulos, H.B., 246, 247, 2@ Dallam, R. D., 310, 329

336

AUTHOR INDEX

Denis, H., 95, 163 de Reuck, A. V., 306, 307, 321 Derry, D. E.,61, 90 Desai, L., 110, 163 de Schryver, A., 57, 84, 85, 90, 91, 212, 2.94

Desgranges, C., 85, 90 De Sombre, E.R., 120, 166, 299, 300, 323 de-The, G., 57, 84, 85, 90, 91, 212, 224 de Torres, R. A., 3, 11, 23, 33, 34, 60, 61, 292, 297, 301, 302, 324 Detter, J., 199, 202, 203, 224 Dettrner, C. M., 241, 244, 249 Dewey, W. C.,289, 321 De Wys, W., 280, 283 Dexter, 9. O., 235, 260 Diamond, L., 14, 60 Diarnandopoulos, G. T., 7, 9, 12, 22, 27, 36, 45, 60, 62, 64 Dick, C.,107, 117, 163 Diderholm, H., 11, 60 Diehl, V., 57, 85, 91 Digby, K. H., 79, 90 Diggle, J. H., 131, 166 di Mayorca, G., 15, 49 Dingman, C. W., 112, 163, 161, 307, 309, 311, 312, 313, 321, 328 Dintsis, H. M., 164, 188 Doenhoff, M. J., 220, 22.4 Di Re, J., 204, 226 Diringer, R., 97, 166 Disbrey, C., 293, 326 Dische, Z., 168, 189 Ditta, T., 196, 226 Dixon, C. B., 15, 16, 49, 62 Dixon, G. H., 111, 138, 139, 140, 141, 166, 167, 161 Dobrina, S. K., 245, 260 Dobson, W. C., 78, 90 Dodonova, N. N.,4, 5, 14, 49 Doi, O., 296, 300, 304, 320 Doll, R., 86, 90 DornaniewskaSobcsak, K., 78, 91 Dormanns, E. A., 77, 78, 90 Doty, P., 95, 164 Dounce, A., 310, 311, 321, 326 Downes, A. M., 298, 321 Dreesman, G. R., 7, 64 Drens, J., 273, 274, 283

Drews, J., 95, 96, 163 Driscoll, D. H., 241, 244, 249, 268 Druckrey, H., 81, 90 Dubbs, D.R., 3, 9, 11, 13, 23, 33, 34, 36, 41, 42, 60, 61, 62, 66, 170, 173, 176, 189, 292, 297, 301, 302, 324 Dubert, J. M., 170, 189 Dubost, C., 273, 286 Duchesne, J., 239, 245, 249 DUCOS, J., 204, 224 Duff, R. G., 14, 15, 21, 27, 30, 60, 63 Duke, P. S., 246, 247, 249 Dulbecco, R., 32, 33, 36, 38, 62, 64, 66, 297, 321

Durnont, J., 319 Duncan, G. C.,274, 283 Dunn, J. J., 98, 99, 161, 163 DuPraw, E. J., 102, 163 Durham, J. P., 303, 321 Dykstra, W. G.,301, 321 Dzyuka, N. M., 246, 249

E Eagle, H., 17, 60 Eddy, R. E., 2, 3, 23, 60, 61 Edelman, I., 314, 328 Edstrorn, J-E.,121, 124, 162, 163, 312, 321 Edwards, L. J., 107, 110, 163, 166 Efirnov, M. L., 255, 283 Egyhazi, E., 121, 162 Ehrlich, R., 275, 256 Eidinoff, M. L., 289, 321, 323 Eirich, F. R., 237, 260 Elgin, S. C. R., 106, 109, 111, 113, 114, 115, 116, 120, 130, 163, 306, 307, 321 Ellgaard, E. G., 121, 138, 162, 163 Elrod, L. H., 24, 30, 60 Eltzina, N. V., 254, 256, 268, 283 Emanuel, N. M., 239, 240, 242, 244, 245, 249, 261 Emmer, M., 100, 169 Enders, J. F., 7, 9, 11, 12, 22, 27, 36, 45, 60, 62, 64. 66 Engel, R., 275, 283 Engelhardt, V. A., 256, 268, 283 Enger, M. D., 116, 164 England, H., 119, 167 Ephrussi, R., 37, 39, 66 Epifanova, 0. I., 292, 298, 303, 321

AUTHOB INDEX

Epstein, M. A., 85, 90 Erickson, R. L., 289, 321 Estefan, R. M., 239, 242, 247, 261 Estensen, R. D., 290, 291, 292, 293, 294, 300, 319, 321 Estrade, S., 41, @ Evans, J., 275, 286 Evans, V. J., 170, 173, 188 Evgenieva, T. P., 278, 283 Ezdinli, E. Z., 273, 274, 275, 282 Ezra, R., 217, 226 Ezrin, J. C., 279, 286

F Fabisch, P., 9, 10, 64 Fahey, J. L., 290, 291, 330 Fairbanks, V. F., 192, 194, 223 Fambrough, D. M., 102,106,107, 110,111, 113, 119, 131, 137, 140, 161, 162, 163, 306, 307, 309, 312, 320, 321 Fang, S., 120, 163 Farbath, N., 275, 286 Farber, E., 137, 163, 168, 189, 288, 293, 321, 324 Farber, J., 295, 299, 300, 305, 310, 321 Fasman, G. D., 122, 163, 160 Fast, D. K., 298, 321 Faulhaber, I., 114, 163 Faulkner, R., 116, 137, 140, 160, 300, 302, 307, 308, 319 Faulstich, H., 143, 167 Fausto, N., 292, 301, 302, 303, 322 Favre, M. C., 85, 90 Feigelson, M., 308, 322 Feigelson, P., 308, 322, 330 Feinendegen, L. E., 295, 318, 322 Feldman, L. A., 20, 63 Feldman, M., 173, 189 Felig, P., 280, 283 Felsenfeld, G., 121, 122, 146, 162 Fenyo, E. M., 208, 220, 226 Fernandes, M., 11, 13, 41, 60 Fialkow, P. J., 195, 199, 202, 203, 204, 207, 208, 209, 211, 212, 218, 219, 220, 221, 224 Ficq, A., 164, 188 Field, J. B., 274, 283 Fievez, M., 310, 330 Firket, H., 289, 322

337

Fisher, D. B., 301, 303, 322 Fitch, R. H., 266, 286 Fitzgerald, P. H., 200, 226, 289, 322 Fitzgerald, P. J., 139, 163, 298, 322 Fitzhugh, A., 234, 235, 237, 260 Fiume, L., 184, 188 Flanigan, C. C., 254, 284 Flemans, R. J., 200, 224 Flemming, W., 287, 322 Fletcher, G. H., 58, 90 Flexner, J. M., 209, 223 Flickinger, R. A., 95, 162 Flury, R., 274, 284 Folsch, E., 273, 274, 283 Foley, G. E., 17, 60, 110, 163 Fondelli-Restelli, A., 172, 173, 190 Forbes, W. F., 237, 249, 260 Forrester, S., 113, 120, 131, 166 Foster, M. A., 244, 260 Fowler, A. V., 130, 160, 307, 309, 3 B Fox, R. R., 168, 188 Fox, T. O., 296, 296, 322 Fraley, E. E., 12, 63 Franceshini, P., 296, 300, 304, 327 Frearson, P. M., 13, 61 Fredericq, E., 114, 121, 123, 163, 164 Freeman, N., 327 Freedman, S. O., 94, 164 Freiberg, S., Jr., 57, 84, 90 Frenster, J. H., 122, 126, 146, 147, 163, 307, 309, 311, 312, 322, 324

Friberg, S., 212, 224, 291, 320 Fric, I., 122, 161 Frie, E., 111, 200, 201, 226 Friedman, H., 8, 60 Friedman, I., 58, 90 Friedman, M., 137, 163 Friend, C., 170, 189 Friesen, S. R., 273, 284 Friis, R. R., 44, 66 Frindel, E., 290, 322 Froehner, S. C., 106, 111, 120, 130, 163 Froesch, E. R., 274, 284 Fujimoto, D., 137, 166 Fujimura, F., 102, 107, 113, 162, 163 Fujinaga, K., 40, 61 Fujioka, M., 296, 298, 300, 301, 303, 304, 322 Furlan, M., 120, 163 Furth, J. J., 144, 163

338

AUTHOR INDEX

Furuno, A., 5, 14, 66 Fueaell, C. P.,289, 387

G Gabbay, E.,310, 311 Gabrio, B. W., 289, 333 Gahrton, G., 200, 114 Galanti, N.,301, 381 Galavasi, G. H.,289, 311 Gall, J. G., 103, 108, 163 Gall, T.,312, 311 Gallagher, R. E.,170, 173, 188 Gallo, R. C., 145, 163, 170, 173, 177, 188 Gallwits, D.,116, 137, 163 Gantt, R.,170, 173, 188 Garland, M.,298, 311 Garnjana-Goochorn, S.,72, 90 Garren, L. D., 298, 326 Garrett, R. A., 123, 164 Gartler, S. M., 195, 197, 199, 203, 204, 207, 210, 213, 214, 215, 217, 218, 219, 214, 116, 116

Garven, E. V., 120, 140, 161 Garvie, W. H. H.,269, 183 Gasic, G.,279, 188 Gasic, T.,279, $83 Cause, E. M., 239, 242, 247, 161 Gaylord, W. H., Jr., 13, 61 Geering, G., 84, 91, 212, 3E.5 Gefter, M. L., 106, 188 Geiduschek, E. P., 98, 167 Gelb, L. D., 36, 60 Gelbard, A. S., 290, 291, 311 Gelderman, A. H., 299, 305, 311 Gellhorn, A., 107, 113, 115, 118, 138, 161, 311, 319 Georgiev, G. P., 106, 111, 126, 129, 131, 132, 149, 164, 166 Gerber, P., 3, 32, 33, 60, 61 Gershey, E. L., 113, 137, 138, 139, 160, 164, 168, 300, 302, 307, 308, 310, 314, 319, 391

Gershon, D.,297, 311' Ghoae, T.,279, 183 Giacomoni, D.,292, 319 Giannelli, F., 204, 116 Giblett, E. R., 199, 202, 203, 204, 219, no,221, $94 Gibson, J. F., 235, 237, 160 Gierthy, J. F., 299, 300, 305, 31.9

Gilbert, J. M., 166, 187 Gilbert, L. A., 236, 149 Gilbert, L. I., 125, 166 Gilbert, W.,100, 148, 164, 311 Gilden, R. V., 13, 14, 22, 49, 60, 261, 183 Gillam, S., 176, 188 Gillan, D. J., 297, 319 Gilmour, R. S., 102, 126, 132, 133, 134, 135, 164, 168, 305, 312, 313, 311] 386 Girardi, A. J., 2, 7, 8,24, 31, 60, 61 Gieainger, F., 143, 144, 145, 164, 166 Giudice, G., 292, 301, 302, 311 Glaser, A. N.,110, 161 Glasky, A. J., 188, 189 Glen-Bott, A. M., 203, 184 Glisin, M. V., 95, 164 Glisin, V. R., 95, 164 Glitser, N. S., 268, 183 Gniazdowski, M.,143, 144, 145, 164, 166 Goff, C. G., 98, 164 Gold, J., 280, 283 Gold, M.,179, 188, 289, 311 Gold, P.,94, 164 Goldberg, D.M., 262, 183 Goldberg, M. L., 97, 169 Goldblum, N.,11, 13, 34, 41, 61 Gold4, A., 44, 61 Goldenburg, E. W.,209, 114 Goldhuber, P.,196, 126 Coldman, H.,122, 161 Goldman, M.,177, 188 Goldner, H., 7, 8,60, 61 Goldsmith, L., 122, 163 Goldstein, B., 170, 189 Goldseiher, J. W.,239, 161 Gonano, F.,177, 188 Goodman, G. C., 320 Goodman, R. M., 138, 161 Goranson, E. S., 288, 269, 183 Gorbman, A., 129, 164 Gordon, C. S., 272, 282 Gordon, J., 119, 120, 129, 166, 180, 155 Gorman, J. G., 204, 226 Gorovsky, M. A., 121, 164 Goroshanskayn, E.G., 255, 258, 259, 260, 261, 283 Gorski, J., 120, 1/30, 290, 299, 300, 301, 304, 314, 317, 319, 322, 326, 329 Gothoskar, B., 219, 220, 221, 124, 286 Gottesman, M., 100, 169

AUTHOR INDEX

Govindan, M. V., 143, 167 Grace, J. T., Jr., 170, 171, 189 Grady, F. J., 860 Grady, L., 35, 61 Graffe, L. H., 45, 64 Granger, G. A., 177, 189 Granner, D., 96, 167 Grantham, F. H., 255, 257, 260,263, 264, 265, 267, 883 Green, H., 3, 11, 12, 13, 14, 15, 16, 38, 63, 66, 297, 299, 300, 320, 389 Green, M., 40, 61, 297, 322 Greenawalt, c., 16, 64 Greenaway, P., 110, 164 Greendyke, R. M., 202, 826 Greene, M. L.,198, 226 Griffin, A. C., 177, 188, 884 Grimes, P. A., 12, 48 Grimmo, E. P., 77, 78, 91 Grisham, J. W., 298, 322 Grisolia, S., 137, 169 Gross, A., 239, 242, 247, 861 Gross, P. R., 116, 166, 104, 188, 294, 308, 322, 324 Grossbard, L., 261, 283 Grubbs, G. E., 2, 3, 60 Grumbach, M. M., 292, 326 Grunicke, H., 314, 322 Grusdeva, K. V., 275, 283 Guelstein, V. I., 262, 286 Guentzel, M. J., 21, 42, 49 Gullino, P. M., 255, 257, 260, 263, 264, 265, 267, 283

Gulyas, G., 289, 329 Gundersen, K., 275, 886 GunvBn, P., 212, 226 Gunz, F. W., 200, 226 Gurevich, B. S., 255, 260, 261, 283 Gurley, L. R., 116, 117, 118, 138, 164, 307, 322 Gurdon, J. B., 93, 126, 164 Gutierrez, R. M., 139, 164, 302, 307, 308, 322

Guttman, A. D., 180, 190 Guttormsen, S. A., 202, 226

H Habel, K., 7, 15, 23, 34, 38, 61, 63, 64, 66 Hacha, R., 114, 164 Hacker, B., 170, 189

339

Hadjiolov, A. A., 304, 316, 380 Hadorn, E., 93, 164 E y r y , P., 26, 28, 29, 30, 61 Hagiwara, A., 289, 292, 294, 386 Hahn, W. E., 129, 164 Hajiwara, K., 117, 118, 160 Hale, A. J., 297, 321 Hall, J. G., 9, @ Hall, R. H., 170, 171, 189 Halliburton, I. W., 117, 118, 164 Halliday, J. W., 267, 282 Halpern, B. C., 178, 185, 188 Halpern, R. M.,178, 185, 188 Hamilton, T. H., 118, 128, 129, 135, 136, 164, 167, 161, 296, 299, 300, 301, 304, 305, 310, 312, 313, 314, 317, 322, 386, 328 Hamkalo, B. A., 105, 161 Hancock, R., 117, 118, 164, 307, 310, 311, 382 Hancock, R. L., 168, 170, 180,188 Harbert, P., 196, 226 Hardin, J. M., 117, 138, 164, 160, 307, 308, 327 Harding, C. V., 297, 298, 322, 323, 386 Hare, J. D., 17, 61 Harman, D., 236, 260 Harnden, D. G., 200, 226 Harrington, J. S., 236, 260 Harris, H., 32, 61, 126, 161, 224, 297, 299, 300, 305, 320, 323 Hartman, K. U., 289, 323 Hartmann, R. C., 209, 223 Hartwell, L. H., 297, 321 Haslett, G. W., 137, 166, 300, 302, 307, 308, 324 Hatanaka, M., 261, 283 Hathaway, P., 219, 226 Hauschka, T. S., 200, 226 Hausen, P., 145, 161, 297, 322 Haussler, M. R., 120, 164 Hayashi, H., 110, 166 Haydon, A. J., 121, 122, 123, 164, 167 Hayhoe, F. G., 301, 303, 386 Haynes, R. L., 279, 286 Hearst, J. E., 101, 129, 164 Heckley, R. J., 233, 260 Heddle, J. A., 103, 164 Heffler, S., 297, 315, 319 Heidelberger, C., 289, 323

340

AUTHOR INDEX

Heidema, J., 136, 148, 166, 312, 313, 384 Heil, A., 98, 99, 164, 162 Heise, J. J., 233, 235, 249 Helleiner, C. W., 289, 382 Hellman, A., 177, 190 Hellman, K. B., 177, 190 Hellstrom, I., 9, 26, 31, 61, 213, 886 Hellstrom, J., 23, 64 Hellstrom, K. E., 9, 31, 61, 213, 2&5 Henderson, J. F., 198, 226, 277, 282, 283 Henle, G., 57, 84, 85, 90, 91, 212, 224, 886, 886 Henle, W., 57, 84, 85, 90, 91, 212, 284, 826, 826 Hennings, H., 298, 323 Henry, P. H., 15, 16, 23, 27, 36, 45, 62, 96, 167 Henson, P.,122, 164, 311, 383 Herbst, J., 301, 321 Herring, B., 274, 283 Hey, A. E., 107, 166 Heyden, H. W., 119, 164 Hiepler, E., 255, 258, 261, 286 Higginson, J., 79, 81, 83, 98 Hilt, R., 278, 284 Hilgartner, C., 310, 321 Hilleman, M. R., 1, 2, 7, 24, 60, 61, 64 Hilton, J., 141, 164 Hindley, J., 130, 131, 164, 309, 323 Hines, R. E., 274, 284 Hinkle, D., 99, 164 Hirt, B., 37, 61 Hnilica, L. S., 106, 107, 110, 111, 113, 120, 126, 130, 131, 132, 134, 135, 163, 164, 166, 166, 168, 160, 302, 306, 307, 308, 309, 310, 312, 313, 320, 328, 393, 328

Ho, H. C., 57, 58, 60,60, 68, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 84, 85, 87, 90, 91, 212, 224 Hoare, T. A., 130, 166, 309, 323 Hobbs, J., 217, 226 Hochella, N. J., 280, 886 Hodge, L., 291, 295, 296, 393 Hodgett, J., 9, 48 Hoggan, M. D., 20, 61 Hodgkinson, C. P., 240, 241, 249 Hodgson, G.,298, 323 Hoffman, D., 237, 968 Hohmann, P., 110, 166

Holbrook, D., 307, 322 Holland, J. F., 175, 176, 177, 182, 188, 189, 273, 274, 275, ,989 Holland, J. J., 295, 323 Holley, R. W., 173, 175, 188 Holmberg, B., 262, 284 Holmes, E. C.,213, 226 Holoubek, V., 118, 166, 310, 311, 323 Holsman, J., 184, 188 Honna, H., 278, 284 Holt, T. K. H., 125, 166 Holtzer, R. L., 292, 301, 302, 323 Horsfield, A,, 237, 261 Hosoda, S., 258, 984 Hotta, Y., 125, 166 Hourani, B. T., 246, 247, 249 Hutchinson, J. M. S., 244, 260 Howard, A., 287, 323 Howk, R., 119, 166, 168, 311, 323 Hoyer, B. H., 95, 167 Hsiung, G. D.,13, 61 Hsu, T. C.,292, 311, 383, 384 Hu, C. H., 77, 91 Hu, Y. F., 59, 91 Huang, P. C.,133, 135, 166 Huang, R. C. C., 102, 113, 114, 115, 116, 118, 119, 126, 130, 131, 133, 135, 146,

161, 166, 160, 305, 306, 307, 309, 311, 383, 324, 327 Huberman, J., 102, 113, 161 Huebner, R. J., 5, 16, 19, 20, 28, 45, 61, 64, 261, 283 Huennekens, F. M., 289, 323 Hugher, W. L., 103, 161 Huhn, A., 278, 884 Humphrey, R. M., 291, 328 Hungerford, D. A,, 200, 226 Hunt, N., 292, 296, 299, 300, 301, 302, 324 Hunter, A. R., 166, 188 Hunter, J., 261, 263, 288 Hurst, D. J., 299, 300, 323 Hurwitz, J., 97, 130, 166, 180, 179, 188, 307, 309, 3.97 Hybner, C. J., 16, 49

I Ibragimov, E. I., 278, 282 Igel, H. J., 15, 61 Imamura, A., 247, 260

AUTHOB INDEX

Inbar, M., 17, 28, 29, 49, 61 Ingles, C. J., 117, 118, 139, 144, 145, 161, 166

Ingram, D. J. E., 228, 235, 237, 260 Inoue, A., 137, 166 Ionescu-Homoriceanu, S., 45, 64 Irvin, J., 307, 322 Irlin, I. S., 26, 61 Irwin, L. E., 208, 209, 224 Isaksson, L. A., 180, 188 Isenberg, I., 235, 236, 261 Isihara, T., 200, 286 Ishikawa, K., 110, 166 Itehaki, R. F., 121, 122, 146, 166 Ivankovic, S., 81, 90 Iversen, H. G., 279, 284 Iwai, K., 110, 166 Ieawa, M., 121, 125, 166

J Jackson, C. D., 96, 166 Jackson, R. J., 105, 188 Jacob, F., 100, 148, 166, 164, 188, 306, 323 Jacob, S. T., 96, 143, 145, 166 Jacobs, P. A., 200, 226 Jacobson, C . O., 297, 323 Janchen, L., 256, 284 Jakob, A., 274, 284 Jamison, R. M., 13, 63 Janigan, D., 273, 284 Janne, J., 301, 326 Jehl, J., 269, 284 Jenkins, A., 182,189 Jensen, E. V., 120, 166, 299, 300, 323 Jensen, F. C., 4, 11, 13, 24, 31, 32, 41, 60, 61, 62

Jensen, R. H., 102, 113, 120, 151, 168 Jergil, B., 138, 139, 166 Jericijo, M., 120, 163 Jerkofsky, M., 5, 6, 63 Jeung, T. T., 272, 273, 274, 275, 284 Jockusch, B. M., 294, 323 Johns, E. W., 106, 107, 109, 111, 112, 113, 116, 117, 120, 124, 130, 131, 148, 163, 166, 169, 162, 306, 307, 309, 310, 312, 320, 323, 326, 329 Johnson, G., 107, 111, 139, 160 Johnson, H. A., 299, 300, 323

341

Johnson, J. M., 268, 286 Johnson, P., 274, 283 Johnson, R. T., 297, 323 Johnson, T. C., 295,323 Johnston, W. M., 177, 188 Joklik, W. K., 38, 61 Jones, H. W., Jr., 209, 210, 226 Jones, R. O., 297, 323 Jordan, J. J., 110, 166 Jordan, L. E., 13, 62 Josserand, A., 278, 284 Jung, C., 291, 323 Jung, P. G., 64, 91 Jungblut, P. W., 120, 166, 299, 300, 323

K Kahler, H., 268, 284, 286 Kajiwara, K., 289, 290, 391, 292, 293, 307, 323, 326, 328 Kallum, B., 279, 284 Kalmanson, A. E., 245, 260 Kalomiris, C. G., 241, 262 Kamel, D. C., 269, 286 Kamiyama, M., 135, 166, 312, 313, 3M Kaplan, P. M., 10, 66 Kappler, H. A., 110, 166, 309, 323 Karon, H., 142, 162 Kasten, F., 289, 323 Kataoka, N., 242, 260 Kato, M., 5, 66 Kate, M., 5, 15, 25, 63, 64 Kaufman, R., 292, 301, 302, 329 Kawai, A., 278, 284 Kawashima, T., 120, 166 Kay, J. E., 301, 303, 323 Kayushin, L. P., 239, 242, 243, 249, 260 Kaenacheyev, Yu. S., 239, 244, 261 Kedes, A., 106, 166 Kedes, L. W., 116, 166 Kedinger, C., 143, 144, 145, 164, 166, 167 Keen, H., 274, 283 Kelle, D. K., 198, 226 Kelley, W. N., 198, 226 Kelly, T. J., Jr., 16, 61 Kelmers, A. D., 175, 190 Kember, N. F., 298, 323 Kemp, A., 258, 284 Kenney, F. T., 308, 323 Kensler, C. J., 235, 260 Kent, M., 232, 239, 241, 245, 960

342

AUTHOR INDEX

Keppler, D.,298, 384 Kerr, S. J., 167, 168, 170, 175, 178, 179, 188, 189 Ketchan, A. S., 213, 286 Keydar, J., 101, 146, 160, 161 Khera, K . S., 23, 24, 32, 33, 36, 61, 62 Kidson, C.,96,166 Kidwai, J. R., 38, 64 Killander, D.,126, 169, 292, 294, 299, 300, 305, 319, 323 Kim, J. H., 289, 290, 291, 292, 293, 322, 323

Kim, K-H., 128, 166 Kim, S. H., 137, 140, 166, 168, 289, 300, 302, 307, 308, 323, 324, 326 Kimmel, J. R., 274, 286 King, J., 292, 298, 300, 301, 303, 328, 330 King, M. J., 200, 226 King, R. J., 300, 301, 306, 314, 317, 328 King, R. J. B., 97, 119, 120, 129, 166, 160 Kinkade, J. M.,106, 107, 110, 166 Kirby, K . S., 96, 166 Kirchner, F. R., 58, 92 Kirkham, W. R., 310, 329 Kiruhara, K., 304, 316, 330 Kirschenfield, I. I., 273, 289 Kischer, C. W., 107, 166 Kirschstein, R. L., 3, 11, 12, 32, 60, 61, 63 Kish, V. M., 113, 169 Kishimoto, S., 294, 324 Kit, S., 3, 9, 11, 13, 14, 23, 33, 34, 36, 41, 42, 49, 60, 61, 62, 66, 170, 173, 175, 189, 267, 284, 292, 297, 301, 302, 324 Kitahara, T., 5, 20, 21, 23, 61, 63 Kleiman, L., 305,324 Klein, E., 208, 220, 224, 226 Klein, F., 121, 166 Klein, G., 23, 64, 57, 84, 85, 90, 91, 195, 204, 207, 208, 209, 211, 212, 218, 219, 220, 221, 224, 226, 226, 258, 284 Klein, H., 274, 284 Klein, S. P., 274, 284 Kleinfeld, R. G.,289, 324 Kleinsmith, L. J., 113, 119, 136, 138, 139, 148, 164, 166, 169, 300, 301, 302, 307, 308, 310, 312, 313, 314, 319, 322, 324 Klenow, H.,289, 324 Klevecz, R. R., 289, 290, 291, 292, 294, 311, 324, 328

Klietmann, W., 31, 68 Klinger, H. P., 196, 226 Klochko, E. V., 239, 244, 245, 261 Kluchahva, T. E.,8, 10, 11, 26, 60, 69 Knight, J., 306, 307, 321 Knowles, B. B., 13, 33, 40, 41, 62, 64 Koch, M . A., 19, 20, 23, 32, 36, 62, 64 Kodama, M., 247, 260 Konigsberg, I. R., 93, 166 Koga, M., 296, 298, 300, 301, 303, 304, 322 Kohne, D. E., 36, 60, 105, 128, 161 Koldorsky, P., 27, 49 Kolodny, G. M., 294, 324 Kolomiitseva, I. K., 239, 243, 260 Konrad, C. G., 295, 318, 324 Koperina, Ye. V., 239, 244, 261 Koprowski, H.,5, 11, 13, 14, 17, 27, 32, 33, 40, 41, 42, 49, 60, 61, 62, 64 Koslov, Y. U.,126, 131, 132, 164, 166 Kostraba, N. C.,112, 166 Kosako, S., 278, 284 Kozlov, Yu. P., 245, 260 Kozlova, L. E., 240, 242, 249 Krakowski, A,, 217, 226 Kreisberg, R. A., 273, 274, 984 Kroeger, H., 125, 166 Krogman, W.M.,61, 91 Kruglyak, S. A,, 239, 244, 245, 261 Kruglyakova, K. Ye., 239, 240, 242, 244, 245, 249, 261 Kruh, J., 113, 162, 166 Kubai, D. F., 101, 169 Kuchino, Y., 169, 175, 189 Kudlow, J. E., 288,319 Kiintrel, H.,145, 166 Kugelman, B. H.,289, 327 Kull, F. G.,308, 323 Kung, G. M., 114, 116, 133, 135, 161 Kuno, G. O.,279, 286 Kurashina, Y., 122, 131, 166 Kurimura, T., 3, 9, 13, 36, 41, 62 Kusyya, N., 278, 284 Kwan, H. C.,85, 90

1 Labhest, A., 274, 284 LaCour, L. F., 103, 166 Laemmli, U. K., 306, 324 Lai, C. J., 187, 189 Laidlow, A. G.,279, 286

AUTHOB INDEX

Lamb, D. C., 107, 167 Lambert, B., 121, 169 Lambert, W. C., 290, 3.94 Lancet Annotations, 83 Landau, B. R., 274, 275, 884 Landgraf, W . , 246, 247, .9@ Landgraf-Leun, I., 40, 61 Landsberg, B., 180, 190 Lane, W. T., 19, 61 Langan, T. A., 113, 119, 138, 139, 166, 167, 300, 302, 307, 308, 312, 314, 384 Langen, P., 292, 3.94 La Rock, J. F., 219, ,926 Larson, E. O., 241, 261 Lanon, V. M., 7, 24, 60, 61 Laskey, R. A,, 93, 164 Latarjet, R., 15, 62 Laufer, H., 125, 166 Laurence, D. J., 307, 309, 312, 384 Lausch, R. N., 7, 8, 10, 24, 62, 64 Law, L. W., 7, 26, 27, 62, 66 Lawler, S. D., 204, 226 Lawley, M., 68, 91 Lawson, T. A., 298, 3.94 Layde, J. P., 290, 291, 292, 300, 319 Lazar, G . K., 297, 299, 300, 3.99 Lararus, H., 17, 60 Leclerc, J., 110, 111, 166 Leclercq, M., 110, 160 Lederman, M.,58, 91 Lee, C. S., 105, 161 Lee, F. K., 77, 78, 92 Lee, G. R., 204, 886 Lee, H. W., 137,168 Lee, K. H., 76, 90 Lee, P., 327 Lee, R. E., 293, 3.94 Lee, R. H., 71, 91 Lee, S. K., 77, 78, 91 Leger, L., 273, 286 Leighton, J., 262, 284 Leistner, I., 256, 884 Le Mahieu, M., 296, 300, 304, 396 Leon, M. A., 297, 3.96 Leonard, J. R., 274, 275, 284 Leong, H. K., 79, 91 LePage, G. A., 282, 283 Lepatova, L., 249 Lerman, S., 310, 311, 326 Leach, R., 298, 324

343

Leslie, I., 119, 166, 167 Leuchars, E., 220, 8.94 Levan, A., 220, 826 Levander, 0. A., 118, 161 Levin, M. J., 15, 16, 23, 27, 36, 45, 62 Levine, A. J., 27, 35, 36, 45, 62 Levine, E. M., 17, 60 Levinthal, J. D., 11, 12, 64 Levy, B., 5, 6, 15, 63 Levy, J. P., 3, 49 Lewis, A. M., Jr., 23, 62 Lewis, U. J., 306, 307, 327 Ley, K. D., 289, 297, 324, 329 Lezzi, M., 125, 130, 166 Liang, P. C., 58, 59, 91 Liao, S., 120, f63 Libby, P. R., 137, 166 Lichter, T., 278, 284 Lieberman, I., 117, 161, 292, 294, 296, 297, 298, 299, 300, 301, 302, 303, 304, 307, 320, 322, 324, 326, 326, 328, 329

Lieberman, M. W., 293, 324 Liew, C. C., 137, 166, 300, 302, 307, 308, 324

Lilly, F., 57, 91 Limade-Faria, A,, 150, 166 Lindberg, U., 39, 62 Lindell, T. J., 144, 145, 161 Linden, G., 69, 70, 92 Linder, D., 195, 197, 208, 210, 213, 214, 215, 216, 218, 226 Lindsay, D. T., 107, 116, 166, 167 Lindsten, J., 200, 224 Ling, V., 139, 141, 167 Linsell, C. A., 183, 184, 189, 205, 2.94 Lion, Y., 245, 249 Lipchina, L. P., 245, 260 Lipkin, D., 235, 260 Lippincott, B. B., 233, 235, 2.69 Lipsett, M. B., 94, 166, 274, 284 Lisker, R., 199, 202, 203, 204, 224 Littau, V. C., 122, 124, 130, 138, 142, 160, 166, 167, 169, 305, 307, 308, 309,311, 312, 319, 324

Littauer, U. Z., 173, 189 Littlefield, J. W., 289, 290, 291, 294, 324, 327

Litwack, G., 299, 300, 315, 316, 327 Livenson, A. R., 239, ,960 Loeb, L., 179, 189

344

AUTHOR INDEX

Lockwood, D. H., 297, 324 Loeb, J. E., 113, 166, 310, 312, 313, 324 Loeb, L., 168, 189 Lofberg, R. T., 242, 261 Lorenson, M. G., 292, 301, 302, 326 Losick, R., 99, 166 Lotspeich, W. D., 298, 324 Lowbeer, L., 272, 273, 284 Lowrie, R. J., 180, 189 Lucas, L. A., 196, 886 Lucas, Z.J., 297, 300, 301, 302, 324 Lucik, F., 239, $261 Luck, J. M., 106, 169, 308, 386, 326, 327 Luther, S. W., 96, 162 Luzzatto, L., 209, 826 L’Vov, K. M., 239, 243, 260 Lwoff, A., 186, 189 Lyon, M. F., 192, 296 Lyons, M. J., 235, 237, 260 Lytle, C. D., 33, 48

Macpherson, I., 18, 62 Magee, P. N., 168, 184, 188, 189 Mahon, W. A., 273, 284 Mainwaring, W. I. P., 120, 167 Maizel, E., 306, 327 Maizel, J. V., 116, 160 Majumdar, C., 299, 300, 326 Makman, R. S., 100, 167 Malamud, D., 297, 299,300,301, 302, 303, 326 Maley, F., 292, 301, 302, 326 Maley, G. F., 292, 301, 302, 326 Maling, J. E., 246, 247, 249 Malkin, L. I., 164, 188 Mallard, J. R., 232, 239, 241, 244, 245, 260 Mallick, L., 270, 284 Malmgren, R. A., 26, 62, 213, 226, 264, 984

McFarland, P., 168, 188 McFarlane, E. S., 170, 182, 189 MacGillivray, A. J., 107, 109, 114, 115,

Malpoix, P. J., 310, 314, 326, 330 Mandel, J. L., 143, 144, 164, 166 Mandel, L. R., 170, 182, 189 Mandel, P., 142, 145, 162 Mandel, R. K., 120, 136, 167 Manolov, G., 208, 220, 226 Maor, P. T., 26, 31, 64 Marcus, P. I., 38, 62, 289, 327 Margalith, E., 34, 62 Margalith, M., 11, 13, 34, 41, 62 Marin, G., 18, 62 Marini, M., 177, 188 Marks, J. F., 198, 226 Marks, V., 274, 284 Marliss, E., 280, 283 Marsh, W. H., 137, 139, 140, 163, 168 MBrtensson, L., 208, 226 Martin, D. H., 177, 190 Martin, D. W., 96, 167, 291, 326 Martin, G. B., 274, 286 Martin, G . M., 12, 66, 195, 208, 209, 211,

116, 137, 167, 302, 308, 310, 312, 313, 326 McGrath, J., 99, 162 MacGregor, H. C., 103, 162 McGuire, W. L., 129, 168 McIndoe, W. M., 310, 311, 327 McKee, R. W., 269, 284, 286 McMahon, N. J., 196, 226 MacManus, J. P., 297, 329 McMillan, V. L., 10, 66

Martin, G. S., 18, 44, 62 Martin, L., 300, 301, 306, 314, 317, 328 Martin, M. A., 36, 39, 60, 62 Martin, S. J., 119, 167 Martin, W. E., 289, 292, 301, 302, 318 Martinage, A., 110, 111, 166 Marty, Y., 204, 224 Martyn, R., 206, 294 Marushige, I