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ADVANCES IN GENOME BIOLOGY V o l u m e 3A

9 1995

GENETICS OF HUMAN NEOPLASIA

This Page Intentionally Left Blank

ADVANCES IN GENOME BIOLOGY Editor: RAM S. VERMA Division of Genetics

The Long island College HospitalSUNY Health Science Center Brooklyn, New York Volume 1.

UNFOLDING THE GENOME

Volume 2.

MORBID ANATOMY OF THE GENOME

Volume 3A. GENETICS OF HUMAN NEOPLASIA Volume 3B. GENETICS OF HUMAN NEOPLASIA

Volume 4.

GENETICS OF SEX DETERMINATION

Volume 5.

GENES AND GENOMES

Copyright 91995 by JAI PRESSINC 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESSLTD. The Courtyard 28 High Street Hampton Hill, Middlesex TWl 2 1PD England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-835-8 Manufactured h~ the United States of America

ADVANCES IN GENOME BIOLOGY GENETICS OF HUMAN N EOPLASIA

Editor: RAM S. VERMA Division of Genetics The Long Island College HospitalSUNY Health Science Center Brooklyn, New York VOLUME 3A

91995

@ Greenwich, Connecticut

JA! PRESS INC.

London, England


CONTENTS (Volume 3A) xi

LIST OF CONTRIBUTORS PREFACE

Ram S. Verma

xiii

GENETICS OF HUMAN CANCER: AN OVERVIEW

Ram S. Verma

ONCOGENES IN TUMOR PROGRESSION

Bruce P. Himelstein and Ruth J. Muschel

THE p53 TUMOR SUPPRESSOR GENE

Thierry Soussi

GENETIC ASPECTS OF TUMOR SUPPRESSOR GENES

Bernard E. Weissman and Kathleen Conway

P21 ras: FROM ONCOPROTEIN TO SIGNAL TRANSDUCER

Johannes L. Bos and Boudewijn M. Th. Burgering

CHROMOSOMAL BASIS OF HEMATOLOGIC MALIGNANCIES

Ram S. Verma

THE MOLECULAR GENETICS OF CHROMOSOMAL TRANSLOCATIONS IN LYMPHOID MALIGNANCY

Frank G. Haluska and Giandomenico Russo

vii

17

55

143

163

185

211


CONTENTS (Volume 3B) LIST OF CONTRIBUTORS

xi

PREFACE

Ram S. Verma

TRANSCRIPTION AND CANCER Phillip M. Cox

XV

233

LOSS OF CONSTITUTIONAL HETEROZYGOSITY IN HUMAN CANCER" A PRACTICAL APPROACH

Jan Zedenius, G~nther Weber, and Catharina Larsson

THE ROLE OF THE BCR/ABL ONCOGENE IN HUMAN LEUKEMIA

Peter A. Benn

ADVENTURES IN MYC-OLOGY

Paul G. Rothberg and Daniel Heruth

279

305

337

CYTOGENETIC AND MOLECULAR STUDIES OF MALE GERM-CELL TUMORS

Eduardo Rodriguez, Chandrika Sreekantaiah, and R. S. K. Chaganti

INDEX

415 429


LIST OF CONTRIBUTORS Peter A. Benn

Department of Pediatrics University of Connecticut Farmington, Connecticut

Johannes L. Bos

Laboratory for Physiological Chemistry University of Utrecht Utrecht, The Netherlands

Boudewijn M. Th. Burgering

Laboratory for Physiological University of Utrecht Utrecht, The Netherlands

Raju S. Chaganti

Cytogenetics Laboratory Memorial Sloan-Kettering Cancer Center New York, New York

Kathleen Conway

Department of Epidemiology Lineberger Comprehensive Cancer Center University of North Carolina Chapel Hill, North Carolina

Phillip M. Cox

Department of Histopathology Royal Postgraduate Medical School Hammersmith Hospital London, England

Frank G. Haluska

Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts

Daniel P. Heruth

Molecular Genetics Laboratory The Children's Mercy Hospital University of Missouri-Kansas City Kansas City, Missouri

Bruce P. Himelstein

Division of Oncology Children's Hospital of Philadelphia Philadelphia, Pennsylvania

xi

xii

LIST OF CONTRIBUTORS

Catharina Larsson

Department of Clinical Genetics Karolinska Hospital Stockholm, Sweden

Ruth S. Muschel

Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania

Eduardo Rodriguez

Cell Biology and Genetic Program Memorial Sloan-Kettering Cancer Center New York, New York

Paul G. Rothberg

Molecular Genetics Laboratory The Children's Mercy Hospital University of Missouri-Kansas City

Giandomenico Russo

Raggio-ltalgene SpA Rome, Italy

Thierry Soussi

Institut de Genetique Moleculaire INSERM Paris, France

Chandrika Sreekantaiah

Department of Pathology New York Medical College Valhalla, New York

Ram S. Verma

Division of Genetics The Long Island College HospitaI-SUNY Health Science Center Brooklyn, New York

Gunther Weber


Bernard E. Weissman

Department of Pathology Lineberger Comprehensive Cancer Center University of North Carolina Chapel Hill, North Carolina

Jan Zedenius


DEDICATION

To Donald F. Othmer and Mildred Topp Othmer with grateful appreciation for their commitment to the Long Island College Hospital, its research and cancer care activities, and for their financial support in establishing the Othmer Cancer Center.

xiii


PREFACE

The underlying idea that cancer is a genetic disease at the cellular level was postulated over 75 years ago when Boveri hypothesized that the malignant cell was one that had obtained an abnormal chromatin content. However, it has been only the last decade where enormous strides have been made toward understanding neoplastic development. Explosive growth in the discipline of cancer genetics is so rapid that any attempt to review this subject becomes rapidly outdated and continuous revisions are warranted. Conclusive evidence has been reached associating specific chromosomal abnormalities to various cancers. We have just begun to characterize the genes which are involved in these consistent chromosomal rearrangements resulting in the elucidation of the mechanisms of neoplastic transformation at a molecular level. The identification of over 50 oncogenes has led to a better understanding of the physiological process. Tumor suppressor genes, which were discovered through inheritance mechanisms, have further shed some light towards understanding the loss of heterozygosity during carcinogenesis. The message emerging with increasing clarity concerning specific pathways which regulate the fundamental process of cell division and uncontrolled growth. The advances in molecular biology have led to a major insight in establishing precise diagnosis and treatment of many cancers resulting in prevention of death. The field is expanding so rapidly that a complete account of all aspects of genetics of cancer could not be accommodated within the scope of a single volume format. Nevertheless, I have chosen a few very specific topics which readers may find of great interest in hopes that their interest may be rejuvenated concerning the

XV

xvi

PREFACE

bewildering nature of this deadly disease. The contributors to Volume 3 have provided up-to-date accounts of their fields of expertise. Although the contributors have kept their chapters brief, they include an extensive bibliography for those who wish to understand a particular topic in depth. For more than a century, cancer has been diagnosed on the enigmatic basis of morphological features. Establishing a diagnosis based on DNA, RNA, and proteins, which is done routinely now, was once inconceivable. Cloning a gene of hematopoietic origin is no longer a fantasy. The approach has shifted over the past 15 years from identification of chromosomal abnormalities toward zeroing in on cancer genes. The impact of new diagnostic technology on the management of cancer patients is enormous and I hope readers gain an overview on the progress concerning diagnosis and prevention. I owe a special debt of gratitude to the distinguished authors for having rendered valuable contributions despite their many pressing tasks. The publisher and many staff members of JAI Press deserve much credit. My special gratitude to many secretaries for typing the manuscripts of various contributors. Ram S. Verma Editor

GLNETICS OF HUMAN CANCER" AN OVERVIEW

Ram S. Verma

I~

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

II. III.

Clonality of Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Checkpoints in the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

2 3

IV.

Cancer Predisposition and Progression

. . . . . . . . . . . . . . . . . . . . . .

4

V.

Heritable Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

VI.

Loss of Constitutional Heterozygosity . . . . . . . . . . . . . . . . . . . . . . .

5

VII. VIII. IX. X. XI.

G e n o m i c Imprinting

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

Protooncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T u m o r Suppressor Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 10

13

Gene Therapy for Neoplastic Diseases

. . . . . . . . . . . . . . . . . . . . .

14

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14 14

Advances in Genome Biology Volume 3A, pages 1-16. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

2

RAM S. VERMA !.

INTRODUCTION

It has been a long-held belief that cancer arises from a single cell involving multiple genetic events. 1 In other terms, neoplasia is a genetic disease at the cellular level. 2 The recent advances in molecular techniques have implicated the role of single genes in proliferation and growth control. 3 Of course, tumorigenesis proceeds in a multistep order requiring both the activation of transforming genes and inactivation of recessive tumor suppressor genes. 4 Detection of genetic changes at the DNA level has added a fundamental understanding of the mechanisms of carcinogenesis. 5 The expression of genes in specialized organs reflects the unique production of proteins which govern signals of the cell cycle. 6 The recognition of Mendelian inheritance in families with cancer, and the identification of genetic markers in such families who are predisposed to certain cancers, has opened new avenues for investigation through so-called "predisposing genes". 7 However, there is a wide variation in genetic susceptibility. The clustering of certain cancers based on large population studies has resulted in an awareness concerning screening and management. Dramatic improvements have been made in overall patient survival, although preventative strategies have had little impact since an understanding of the role of gene(s) in cancer remains enigmatic. A detailed description concerning the genetic basis of cancer has been covered in various chapters. As an overview, I shall highlight only the salient features concerning the latest developments.

ii.

C L O N A L I T Y OF T U M O R S

While malignancy requires multiple steps, it is generally agreed that most tumors result from a single cell. 8 The clone destined to become a tumor generally escapes a number of steps for normal growth before it is metastasized. The most widely accepted hypothesis includes the modification of genes, which are responsible for cell proliferation, and inactivation of tumor suppressor genes, which are responsible for the control of tumor development. 9 The transgenetic model proposed by Adams and Cory 1~suggests that a trans-acting gene does not provoke tumor development directly, but predisposes towards following a series of genetic changes resulting in tumorigenesis. The synergistic mutation of preneoplastic cells in transgenic mice have recently attracted much attention. The unicellular uncontrolled growth of tumor cells can result either by high rates of cell division and/or a slower rate of cell death that has again been correlated with the somatic mutation theory. ~1A number of methods for determination of clonality of human tumors have been devised. The traditional presumptions of all these methods dictates that a cell population is homogeneous with respect to a particular marker being used for investigation. It is likely that tumors may have undergone various genetic changes and may not reflect the original events. Furthermore,

Genetics of Cancer

3

heterogeneous tumors may have originated from various cells 9 In such cases, one cell line may outgrow the other; 12 this makes the assessment task quite Herculean. The recent evolution in molecular technology resulted in an increasing number of methods for assessment of clonality in human tumors 9 Wainscoat and Fey ~3 categorized the various approaches into "traditional" methods, while others based them on DNA analysis. The general approach has been based upon X-chromosome inactivation, lymphocyte analysis, somatic mutation, and viral integration. The inactivation or methylation pattern of one of the X-chromosomes provides a unique opportunity for clonality assessment. The G6PD isoenzyme method, originally reported by Linder and Gartler, 14 became a routine approach for evaluating a variety of neoplasia. The fundamental basis is that a female who is heterozygous for G6PD locus will express only one type of G6PD isoenzyme as a tumor cell, while a polyclonal neoplasm Will have double clones. 15 Restriction. fragment-length polymorphic markers (RFLP) analogous to G6PD have also been used to investigate clonality. 16 These markers have been quite useful for studying the remission of patients during bone marrow transplantation. Another approach has been taken to investigate the immuno-gl0bulin light chain (k or ~,) on cells in B-cell neoplasms, 17 while TCR gene rearrangements are routinely detected in T-cell lymphoma and leukemia. 18The discovery of consistent chromosomal abnormalities in several dozen neoplasia have opened new understanding for lineage-specific clonal chromosomal abnormalities. Cases with normal karyotypes are being evaluated by a number of the minisatellite DNA probes which can detect a large number of VNTRP (variable number of tandem repeat polymorphisms) found throughout the genome. 19 Tumor-containing viruses have been 9 90 extensively used to study the clonal evolution of neoplas~a.-

III.

CHECKPOINTS IN THE CELL CYCLE

Exploration of the mechanisms of growth regulation and development has been the major approach for understanding the fundamental basis of tumorigenesis. First, we must know which gene or genes are responsible for malignancy at a checkpoint in the cell cycle. It has become clear that cancer cells have a variety of genomic disorders ranging from aneuploidy to bizarre chromosomal aberrations which may lead to abnormal cell-cycle control. The abnormal cell-cycle duration for various compartments of interphase cells (i.e., Gl, S, and G2) has been correlated with subsequent development of cancer. 21 However, a relatively large body of evidence indicates that cell proliferation is related to five phases of the cell cycle 9 Neoplastic cells are more unstable than normal cells. However, a central question remains to be answered concerning whether genomic instability precedes tumorigenesis 9The genetic basis of genomic instability is well documented, however. There are a number of human cancers

4

RAM S. VERMA

where the role of gene(s) has been established. The role ofp53 as a cell-cycle control is gaining popularity. 22-24 DNA damage can occur during transitions of cells from GI to S and G2 to M. 25 If the proper repair of damaged DNA is not accomplished during Gl, genomic instability may result. These transitory points are under strict genetic control. It is proposed that the loss of the GI to S check points allows gene amplification as the DNA damage produced during G1 can be passed to S phase, which in turn results in a variety of chromosomal abnormalities. The role of genes in cell-cycle control at the Gl phase in cell differentiation is well documented by the retino-blastoma (RB) gene, which is a prototype tumor suppressor gene. 26

IV. CANCER PREDISPOSITION A N D PROGRESSION Generally, it is believed that a fully developed malignant tumor has gone through a series of progressive events. 27 An inherited predisposition could be the first step in cancer progression. In these cases, a genetic approach has been taken seriously towards defining the mechanism of carcinogenesis. There are many forms of cancer which aggregate in families whose inheritance can be clearly defined. The availability of single-copy gene probes and associations of many RFLPs with candidate genes have identified the difference between germinal and somatic events in heritable cases. This approach is very powerful for drawing information in families which are predisposed to cancer, and to anticipate the segregation pattern in individuals which are genetically linked to the predisposing mutation. It has become quite evident that individuals who are genetically predisposed to a particular neoplasia may be destined to be at high risk of cancer. Such information is extremely valuable for other members of families who may not be predisposed at all.

V. HERITABLE CANCER The majority of cancers do not show definite inheritance patterns. However, a twoto threefold increased risk of some cancers among first-degree relatives has been reported, suggesting a multifactorial mode of inheritance. 28 Nevertheless, the familial clustering of certain cancers should not be considered as unequivocal evidence for a hereditary role. The majority of information available on heritable cancer is very limited but has provided genetic models for detection of a predisposing gene for many familial neoplasias. Even in such cancers, genetic heterogeneity has been observed. The classic example that describes the mechanism of tumorigenesis for inherited cancer is retinoblastoma where familial retinoblastoma is due to a germline mutation and a subsequent somatic mutation of a tumor suppressor gene on chromosome 13. 29 Similarly, the relationship of p53 tumor suppressor gene has been shown to play a

Genetics of Cancer

5

major role in a variety of tumors. 3~ Through variable penetrance an expressivity may be noted. 31 The role of genes in the genesis of familial cancer has been strongly indicated in multiple endocrine neoplasia type 1, Li-Fraumeni syndrome, neurofibromatosis, and familial breast cancer. 32 A germline mutation has been regarded as a first hit theory in the Knudson model, but multisteps are required for increased cellular proliferation and growth advantage since only certain tissues are affected. The variability of age at onset is another intriguing phenomenon in familial cancer.

Vi. LOSS OF CONSTITUTIONAL HETEROZYGOSITY The loss of constitutional heterozygosity in human cancer is gaining popularity. 33 The loss of function due to homozygosity, first identified in retinoblastoma tumors by anonymous DNA probes, produced fascinating results. The application of RFLP probes in a variety of tumors served as a marker for the possible location of genes of interest. The loss of heterozygosity through various mechanisms has become quite clear, while a chromosomal basis has been investigated currently by molecular techniques. Loss of heterozygosity at even the chromosomal level has also become evident (Table 1). The nature and mode of familial segregation has been attained not only for chromosomal regions but at the single gene level. Many tumor suppressor genes have been identified through constitutional loss. The mode of inheritance of some mutant genes may reflect a recessive nature, while others may have a dominant effect in the heterozygous state. 34

VIi.

G E N O M I C IMPRINTING

To have a complete set of a diploid genome, genetic material from both parents is required for normal development. 35 However, aberrations do occur when both copies of an entire chromosome or chromosomal segment can originate from one parent. 36 Expression of a gene that depends upon whether they are inherited from the mother or the father is a phenomenon termed "imprinting". 37 The imprinting phenomenon observed in many organisms has been implicated in both the inhibition of embryonic growth and rate of cell proliferation. A parallel scenario has been drawn where genomic imprinting has been associated with the development of tumors. 38 The Beckwith-Wiedemann syndrome is associated with embryonic tumors. In this syndrome, part of chromosome 11 band p15.5 is duplicated, resulting in triplicate copies of this region. It is suggested that the duplicated region on chromosome 11 is paternal. 39 Preferential germline mutation of the paternal allele in retinoblastoma (rbl) causing a loss of heterozygosity of the RB1 gene from chromosome 13q14 is well documented.4~ Compelling evidence suggests that imprinting plays a role in many other childhood cancers, including rhabdomyosar-

Table 1. Allele Loss in Tumors*

Disease Adrenocortical carcinoma

Chromosome Region llp15 (bws); 13ql2-q21(rbl); 17(tp53)

Bilateral acoustic Neurofibromatosis/neurofibromatosis 2 Acoustic neuroma 22ql 1.2-q12 Meningioma 22q Bladder carcinoma

9q; l lp; 17p13(tp53)

Brain tumors Astrocytomas Glioblastoma multiform Gliomas Rhabdoid brain tumor

9p; 10; 17pl 1-pter (tp53); 22q

Breast carcinoma

lp; lq; 3p21-p25 (vhi?q); 6q13--q21; llp15 (bws?); 13q12-q22 (rbl); 16q; 17p13.1 (tp53), 17p13.3; 17q (brcal?); 18q21-qter (dcc?); 22q

Colorectal carcinoma

lp(35-pter); 5q21--q22 (apc); 17p13 (tp53); 18q21.3qter (dcc); 22q

Esophageal cancer

5q (apc?, mcc?); 13q (rbl?); 17p (tp53)

Gastric carcinoma

1; 5q (apc?); 13q (rbl?); 17p13 (tp53?)

Gorlin syndrome

9q31

Basal cell carcinoma Ovarian fibroma

9q31

Hemangioblastoma

3p2 l-pter (vhi)

Hepatoblastoma

1lp15 (bws?)

Hepatocellular carcinoma

1; 4ql 1--q32; 5q(apc?); 10q; 13q12-q22 (rbl?) 14q32; 16q22.3-23.2; 17p13 (tp53)

Lung adenocarcinoma (non-small cell)

3p21-p25; l lp15 (bws?); 13q12--q32 (rbl); 17p13 (tp53); 18

Lung carcinoma (small cell)

3p21-p25; llp15 (bws?); 13q12-q22.2 (rbl); 17p13

(tp53) Melanoma

1p36; Ip22-p31 ; 6q; 9p21

Multiple endocrine neoplasia type 1 lnsulinoma Parathyroid adenoma

llq13 llq13

Multiple endocrine neoplasia type 2 Thyroid medullary carcinoma

I p; 11" 22q

Phenochromocytoma

lp; lq; 11; 22q (continued)

Genetics of Cancer

7

Table 1. (continued) Disease

Chromosome Region 3q 1p36; 14q 17p12-p13 (tp53)" 17q(nfl)

Nasopharyngeal carcinoma Neuroblastoma Neurofibromatosis 1 Neurofibrosarcoma Osteosarcoma Ovarian carcinoma Prostatic carcinoma Peripheral ne uroepi the lioma Renal cell carcinoma Retinoblastoma Rhabdomyosarcoma Testicular carcinoma Thyroid follicular carcinoma Uterine cervical carcinoma

Wilms' tumor Note:

13q12-q22 (rbl); 17p13 (tp53); 18q21.3 ~ qter (dcc?) 3p; 6q; llp; 17p13 (tp53?) 17q(bracal?) 8p; 10q(22); 16q22-q24 17p13 3p12-p14; 3p21.3; 3p21-pter (vhl); 5q (APC); 10q; 13q12-q22 (rbl?); 18 (dcc?) 13ql4 (rbl) 11p15.5 (bws); 17p13 (tp53) 3p21-pter; 1lp15 (bws?) 3p 3p 1lp13 (wtl); llp15 (bws)

*AfterGoddard and Solomon pll

coma, Wilms' tumor, osteosarcoma, and neuroblastoma. 41 The preferential amplification of the paternal allele of N-myc in neuroblastoma and the maternal origin of chromosome 22 while chromosome 9s have been paternal in CML is another perplexing genetic phenomenon. These finding have clearly suggested that paternal and maternal alleles differ in their activation process during genetic mutation. 42 Alternatively, there are a number of studies which have indicated that alleles at tumor suppressor loci may differ in expression after mutations have occurred; this in turn depends upon the nature of inheritance. Embryonic imprinting may also play a significant role in an increased susceptibility to cancer later in life, as the incidence of somatic mutation may be higher depending upon the parental origin of a chromosome(s). 43

VIii. PROTOONCOGENES The latest developments in molecular genetics of neoplastic transformation induced by viral and cellular genes have decreased the role of genetic mutation in carcinogenesis. 44 Generally, the mode of action of protooncogenes is dominant with a gain of function, while tumor suppressor genes act in a recessive manor with loss of 45 46 activity. -' Genetic lesions resulting in altered DNA with an abnormal configura-

Table 2. Chromosomes

Band Location

Oncogenes

p12-p13 p13 p31-p32 p32 p32 p32 p32-31 p34 p36 p36.1-p36.2 p36-p32 q22-q24 q24--q25 qZ4-q25 q32 q42-q43

rapla nras rab3b blym mycll lck jun mpl src fgr tnfr2 ski arg abl2 trk rab4

p12-p13 cen q 13 ql4-q21 q14--q21 q2 l-q31 p24.1

rel ralb rab6 Ico

p21-pter p22-p24 p22-p24.1 p25

Location of Protooncogenes in the Human Genome Chromos omes

7

aerb2 rab5 thrb rafl

p12-p14 p12-p13 p 12-q I 1.21 p 15

p15-p22 pter--q22 9

q31 q32-q36 q33-q36 q33-q36 q25 8

9

fos mycn

Band Location

10 11

ql I

q13-qter q22 q24 q24 q24

Oncogenes berbl egfr araf2 myclkl rala pks2 ttiml met epht braflp l brafl tcl mos lyn mybll pvtl myc bvl

p

nrasll abll

q 11.2

q24

ret hox11

pl 1.2-p12 p13

spil wtl

q34.1

Chromosomes

14

Band Location

Oncogenes

q24.3 q32.1-qter q32.3 q32.33

fos elk2 tcll aktl

15

q25-qter q26.1

yes fps

16

q22-23

maf

17

18

19

p13.1 qll.2-q12 ql 1.2-q12 q21--q22 q22 q25 9

tp53 erbb2 thral ngl bcl5 erba21 neu

q21 q21 q21.3 q21.3 q22-qter

ssavl bcl3 bcl2 yesl ervl

p13.1 p13.1 p13.2 p13.2

mel melll junb

ju,ut

mil mhr kir raflpl grol gro2 gro3

pkasl kraslp pim 1 1lrasl3 trfa tnfb sYr fytl

rosl

re12 sr2 mracrl hras sea in12

fgfjl fd4 bell st3 etsl kras2 rtfrl inrl erbb3 gli sas

Pr rbl rap2a

vav bc13 bras erbal2 hck Src

sis pdgb nrasI2 arafl elk1

10

RAM S. VERMA Table 3. Mechanisms of Activation of Protooncogenes*

Mechanism

Genetic and Biochemical Consequences

Examples

Transduction

Insertion of exons of a protooncogene into a retrovirus genome, v-src usually with truncations or internal mutations of coding sequences, causing efficient production of an abnormal protein

Point mutation

Altered sequence and biochemical function of protein product

Insertion mutation Augmented production of mRNA and protein, via promoter or enhancer in LTR; sometimes accompanied by truncation, fusion or point mutation of coding sequences

c-Ha-ras c-myc, int- 1

Amplification

Augmented production of mRNA and via increased gene dosage N-myc

Chromosomal translocation

Altered regulation of expression sometimes with creation of hybrid proteins

c-myc, abl-bcr

Protein-protein interaction

Stabilization and altered biochemical function

pp60 c-src and middle TAg

Note: *AfterVarmusISll

tion has rejuvenated the field of cancer genetics. 47 It has become apparent that the role of oncogenes is directly connected to the molecular events leading to oncogenesis. Through the use of molecular techniques, we have begun to understand the molecular mechanism of carcinogenesis. Enormous strides that molecular biologists have made in identifying several dozen oncogenes have opened new avenues in the genesis of cancer (Table 2). The deletion of protooncogenes and subsequent loss of heterozygosity is an ultimate theory of tumor suppressor genes. 48 Contrarily, the amplification of DNA domains which include protooncogenes may be related to over-expression of cellular components. 49To date, about 50 oncogenes have been identified and their precise location is being mapped on the human genome (Table 2). The protein products of protooncogenes play major roles in biochemical pathways that control phenotypic expression of cells. There are a number of pathways by which protooncogenes encode nuclear proteins. 5~The activation of protooncogenes are caused by a number of mechanisms (Table 3).

IX. TUMOR SUPPRESSOR GENES The discovery of oncogenes concerning tumorigenic mutations has aroused soaring interest in understanding other factors which control the growth of cancer cells. 52 Mutations of tumor suppressor genes, which in turn release the cells from the constrains governing normal cells, have attracted much attention in recent dec-

Genetics of Cancer

11

ades. 53 The loss of controlled cell growth can happen by an oncogene and/or tumor suppressor gene. 54 The inactivation of a variety of tumor suppressor genes has been linked to a variety of human neoplasia (Table 4). There are a number of tumor suppressor genes which are responsible for cell cycle control, angiogenesis, signal transduction, and development. The genetic mechanism of tumor suppression within and between cells is just beginning to be unraveled. 55 The function of tumor suppressor genes is altered through genomic changes including point mutation, chromosomal translocation, amplification, and mitotic nondisjunction. Chromosomal instability may be caused by tumor suppressor genes which regulate DNA repair. 56 Terminally differentiated cells lose the ability to divide due to factors such as regulation of tumor suppressor genes involving growth factor receptors. The cloning of a number of tumor suppressor genes have opened new avenues for illustrating the mechanisms of these genes. However, the biochemical mechanism by which the products of tumor suppressor genes regulate cell proliferation and differentiation remain unknown. Also unknown is the inactivation of tumor suppressor genes and activation of oncogenes in a variety of hereditary tumors which have elucidated underlying pathways for the genetic basis of human cancer. 57 It is known that certain tumor suppressor genes are involved in a variety of neoplasia, while others are restricted to a single type of malignancy. Also, loss of function of a single gene in certain cancers with requirements of multiple candidate genes for progression to the complete malignant state

Table 4. Some Known or Candidate Tumor Suppressor Genes* Gene

Cancer Types

Product Location

Mode of Action

ape

Colon carcinoma

Cytoplasm?

dcc

Colon carcinoma

Membrane

nfl

Neurofibromas

Cytoplasm

nf2

Links membrane cytoskeleton? Transcription factor

vhl

Schwannomas Inner membrane? meningiomas Colon cancer; many Nucleus others Retinoblastoma Nucleus Thyroid carcinoma; Membrane phenochromocytoma Kidney carcinoma Membrane

wt-1

Nephroblastoma

Transcription factor

p53 rb ret

Note: *AfterMarxI551

Nucleus

? Cell adhesion molecule GTPase-activator

Transcription factor Receptor tyrosine kinase ?

Hereditary Syndrome Familial adenomatous polyposis

Neurofibromatosis type 1 Neurofibromatosis type 2 Li-Fraumeni syndrome Retinoblastoma Multiple endorine neoplasia type 2 von Hippel-Lindau disease Wilms tumor

Table 5. Protooncogene Amplification in Human Tumors Gene

Tumor Type

Amplification %

erbB family c-erbB1/EGFR

Breast cancers Gastric and esophageal carcinoma Head and neck squamous cell carcinoma Lung carcinoma Renal cell carcinoma Glioblastomas Medulloblastomas

1-4 4-8 10; 19 9 5 17; 38-50 5

c-erbB2/neu/HER-2

Breast cancers Gastric and esophageal carcinomas Ovarian cancers Lung carcinomas Colon carcinomas

9-12; 16-33 5-13 20-33 2 3;4

ras family c-Ki-ras2

Breast cancers Gastric carcinomas Ovarian cancers Lung carcinomas Bladder carcinomas

1; 3 10 4-8 3; 4 5

N-ras

Breast cancers Lung carcinomas Head and neck squamous cell carcinoma

1 1.5 30

myc family c-myc

Breast cancers Gastric carcinomas Head and neck squamous cell carcinoma Ovarian cancers Carcinomas of the uterine cervix Colon carcinomas Squamous cell carcinomas of the anus Myelomas Gliomas Squamous cell lung carcinomas

1-11 ; 15-23; 27-42 4; 10 9; 17 12-20;38 8; 9;48 3--6 30 8 3 12-25

N-myc

Neuroblastomas Retinoblastomas Rhabdomyosarcomas

10-31 20 31

c-myc or N-myc

Primitive neuroectodermal tumors

10

c-myc or L-myc

Lung adenocarcinomas Large cell lung carcinomas

2-11 7; 8

(continued) 12

Genetics of Cancer

13 Table 5.

(continued)

Gene

Tumor T3pe

c-myc, N-myc, or L-myc 1lq13 locus Small cell lung carcinomas Breast cancers Gastric carcinomas

Amplification % 11-23 4-9; 13-18 6

Esophageal carcinomas Head and neck squamous cell carcinomas

28-52 7; 25-48

Ovarian cancers

6

Squamous cell lung carcinomas Bladder carcinomas

13 6-7; 21

Melanomas

8

mdm2/sas

Sarcomas

37; 38

c-myb

Breast cancers

3; 4

c-met

Gastric and esophageal carcinomas

7

gli

Gliomas

2

c-etsl

Breast cancers

1

Other

Note: *AfterBrison [631

have caused an enigma for understanding the inheritance of cancer. 58 To identify the entire array of tumor suppressor genes remains an arduous task.

X. GENE AMPLIFICATION Gene amplification was first identified cytogenetically as small double minute chromosomes (DMs) and homogeneously staining regions (HSRs). 59 DMs are seen in a number of tumors including gliomas, neuroblastoma, and medulloblastoma. 6~ In neuroblastoma with DMs, the N-myc gene is amplified. Amplification of the epidermal growth factor receptor C(EGFR) gene has been reported in glioblastoma due to EGFR gene alteration. 61'62 The DNA of amplified genes varies from tumor to tumor. Numerous reports have indicated that DNA sequence amplification is frequently observed in drug-resistant cells. There are genes which are amplified in various human tumors that are shown to be of the oncogene class. 63 A number of oncogenes which are amplified in various tumors are summarized in Table 5. The genes which are amplified in those tumors were found to overexpress the protein. However, it is suggested that amplification is a later event in tumor progression. 63 The mechanism concerning the exact stage(s) at which the amplification of cellular protooncogenes occur(s) remain to be seen. 64'65

14

RAM S. VERMA

XI. GENE THERAPY FOR NEOPLASTIC DISEASES Curing disease through gene therapy is no longer a scientific fantasy. It is soon going to be an accepted practice, but presently is limited to only a few diseases. Exhaustive literature is available concerning human gene therapy. 65-69 The viral vector based delivery system for genes has made a significant contribution in gene transfer technology for a variety of diseases including cancer. 7~ The general philosophy of gene therapy for cancer is to control the overexpression of dominant oncogenes or to activate tumor suppressor genes. 72 It is imperative that we develop vectors that will deliver the gene to specific cell types since certain neoplasias are tissue-specific. The cloning and mapping of cancer genes is increasing at a rapid pace and gene therapy will have a major impact in those neoplasia which are familial in nature.

ACKNOWLEDGMENTS I acknowledge the typing assistance of Sonia Jordan-Williams. The manuscript was proofread by Michael J. Macera and Robert A. Conte, and to them I owe a debt of gratitude.

REFERENCES 1. Stubblefield, E. The genetic changes in cancer. Molec. Carcinogen 1991, 4, 257-260. 2. Rowley, J. D. Cancer is a genetic disease. Adv. Oncol. 1989, 5, 3-8. 3. Croce, C. M. Genetic approaches to the study of the molecular basis of human cancer. Cancer Res. 1991, 5015-5018. 4. Sager, R. Genetic suppression of tumor formation: A new frontier in cancer research. Cancer Res. 1986, 46, 1573-1580. 5. Nowell, P. C. Biology of disease: Cancer, chromosomes and genes. Lab. hivest. 1992, 66, 407-419. 6. Weinberg, R. A. Negative growth controls and carcinogenesis. MoL Carchloma. 1990, 3, 3-4. 7. Ponder, B. A. J. Inherited predisposition to cancer. Trends Genet. 1990, 6, 213-218. 8. Nowell, P. C. The clonal evolution of tumor cell populations. Science 1976, 194, 23-28. 9. Pines, J. Cell proliferation and control. Curr. Opin. Cell Biol. 1992, 4, 144-148. 10. Adams, J. M.; Cory, S. Transgenic models of tumor development. Science 1991, 254, 1161-1166. 11. Farber, E.; Rubin, H. Cellular adaption and development of cancer. Cancer Res. 1991, 51, 2751-2761. 12. Alexander, P. Do cancers arise from a single transformed cell or is monoclonality of tumors a late event in carcinogenesis. Br. J. Cancer 1985, 51, 453-457. 13. Wainscoat, J. S.; Fey, M.E Assessment ofclonality in human tumors: Areview. CancerRes. 1990, 50, 1355-1360. 14. Linder, D.; Gartler, S. M. Glucose-6-phosphate dehydrogenase mosaicism: utilization as a cell marker in the study of leiomyomas. Science 1965, 150, 67-69. 15. Beutler, E.; Collins, Z.; Irwin, L. E. Value of genetic variants of glucose-6-phosphate dehydrogenase in tracing the origin of malignant tumors. N. EngL J. Med. 1967, 276, 389-391. 16. Vogelstein, B.; Fearon, E. R.; Hamilton, S. R.; Feinberg, A.P. Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors. Science 1984, 227, 642-645.

Genetics of Cancer

15

17. Arnold, A.; Cossman, J.; Baskshi, A.; Jaffe, E. S.; Waldmann, T. A.; Korsmeyer, S. J. Immunoglobulin gene rearrangements as unique clonal markers in human lymphoid neoplasms. N. Engl. J. Med. 1983, 309, 1593-1599. 18. Minden, M. D." Toyonaga, B" Ha, K.; Yanagi, Y.; Chin, B.; Gelford, E." Mak, T. Somatic rearrangement of T-cell antigen receptor gene in human T-cell malignancies. Proc. Natl. Acad. Sci. 1985, 82, 1224-1227. 19. Jeffreys, A. J.; Wilson, V.; Thein, S. L. Hypervariable "minisatellite" regions in human DNA. Nat,re 1985, 314, 67-73. 20. Edman, C.; Gray, P.; Valenzuela, P.; Rall, L. B.; Rutter, W. J. Integration of hepatitis B Virus sequences and their expression in a human hepatoma cell. Nature 1980, 286, 535-537. 21. Reid, B. J.; Blount, P. L.; Rubin, C. E.; Levine, D. S.; Haggitt, R. C.; Robinovitch, P. S. Flow-Cytometric and histological progression to malignancy in Barrett's esophagus. Gastroenterology 1992, 102, 1212-1219. 22. Kastan, M. B.; Zhan, Q.; EL-Deiry, W. S.; Carrier, E; Jacks, T.; Walsh, W. V.; Plunkett, B. S.; Vogelstein, B.; Fornace, A. J. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD 45 is defective in ataxia-telangiectasia. Cell 1992, 71,587-597. 23. Livingstone, L. R.; White, A.; Sprouse, J.; Livanos, E.; Jacks, T.; Tlsty, T. D. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 1992, 70, 923-936. 24. Yin, Y.; Tainsky, M. A.; Bischoff, E Z.; Strong, L.; Wahl, G. M. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 1992, 70, 937-948. 25. O'Connor, P. M.; Ferris, D. K.; White, G. A.; Pines, J.; Hunter, T.; Longo, D. L.; Kohn, K. W. Relationship between cdc2 kinase, DNA cross-linking and cell cycle perturbations induced by nitrogen mustard. Cell Growth Diff. 1992, 3, 43-52. 26. Wiman, K. G. The retinoblastoma gene: role in cell cycle control and cell differentiation. FASEB J. 1993, 7, 841-845. 27. Cavenee, W. K.; Scrable, H. J.; James, C. D. Molecular genetics of human cancer predisposition and progression. Mut. Res. 1991, 247, 199-202. 28. Easton, D.; Peto, J. The contribution of inherited predisposition to cancer incidence. Cancer Survey 1990, 395-415. 29. Cossman, J. (Ed). Molecular Genetics in Cancer Diagnosis. Elsevier, New York, 1990. 30. Cavenee, W. K.; Pondert, B.; Solomon, E. (Eds). Genetics and Cancer(Part III). Oxford University Press, Oxford. 31. Mulligan, L. M.; Gardner, E.; Smith, B. A.; Mathew, C. G. P.; Ponder, B. A. J. Genetic events in tumor initiation and progression in multiple endocrine neoplasia. Genes, Chrom. Cancer 1993, 6, 166-177. 32. Fried, S. H. Cancer risks for germ line mutations in tumor suppressor gene. In: Tumor Suppressor Genes (Livingston, D.M.; Mihich, E., Eds.) Edigraf, Trento, Italy. 33. Lasko, D.; Cavennee, W.; Nordenskjold, M. Loss of constitutional heterozygosity in human cancer. Ann. Rev. Genet. 1991, 25, 281-314. 34. Finlay, C. A.; Hinds, P. W.; Levine, A. J. The p53 protooncogene can act as a suppressor of transformation. Cell 1989, 57, 1083-1093. 35. Surani, M. A. H.; Barton, S. C. Development of gynogenetic eggs in the mouse: implications for parthenogenetic embryos. Science 1983, 22, 1034-1036. 36. Solter, D. Inertia of the embryonic genome in mammals. Trend Genet. 1987, 3, 23-27. 37. Solter, D. Differential imprinting and expression of maternal and paternal genomes. Am~ Rev. Genet. 1988, 22, 127-146. 38. Wilkins, R. J. Genomic imprinting and carcinogenesis. Lancet 1988, I, 329-331. 39. Henry, I.; Jeanpierre, M.; Coullin, P.; Barichard, F.; Serre, J.-L.; Journel, H.; Lamouroux, A.; Turleau, C.; Grouchy, J. de; Junien, C. Molecular definition of the 11p15.5 region involved in Beckwith-Wiedemann syndrome and predisposition to adenocortical carcinoma. Hum. Genet. 1989, 81,273-277.

16

RAM S. VERMA

40. Zhu, X.; Dunn, J. M.; Phillips, R. A.; Goddard, A. D.; Paton, K. E.; Becker, A.; Gallie, B. L. Preferential germline mutation of the paternal allele in retinoblastoma. Nature 1989, 340, 312-313. 41. Scrable, H.; Cavanee, W.; Ghavimi, F.; Lovell, M.; Morgan, K.; Sapienza, C. A model for embryonal rhabdomyosarcoma tumorigenesis that involves genome imprinting. Proc. Natl. Acad. Sci. 1989, 86, 7480-7484. 42. Reik, W. Genomic imprinting and genetic disorders in man. Trends Genet. 1989, 5, 331-336. 43. Ponder, B. J. Inherited predisposition to cancer. Trends Genet. 1990, 6, 213-218. 44. Bishop, J. M. Molecular themes in oncogenesis. Cell 1991, 64, 235-248. 45. Varmus, H. An historical overview of oncogenes. In: Oncogenes and the Molecular Origins of Cancer (Weinberg, R. A., Ed.). Cold Spring Harbor Laboratory Press, New York, 1989, pp. 3--44. 46. Sager, R. Tumor suppressor genes: The puzzle and the promise. Science 1989, 246, 1406-1412. 47. Meuth, M. The structure of mutation in mammalian cells. Biochem. Biophys. Acta 1990,1032, 1-17. 48. Scrable, H. J.; Sapienza, G.; Cavenne, W. K. Genetic and epigenetic losses of heterozygosity in cancer predisposition. Adv. Cancer Res. 1990, 54, 25-62. 49. Stark, G. R.; Debatisse, M.; Giulotto, E.; Wahl, G. M. Recent progress in understanding mechanisms of mammalian DNA amplification. Cell 1989, 57, 901-908. 50. Cantley, L. C.; Auger, K.; Carpenter, C.; Duckworth, B.; Graziani, A.; Kapeller, R.; Soltoff, S. Oncogenes and signal transduction. Cell 1991, 64, 281-302. 51. Varmus, H. An historical overview of oncogenes. In: Oncogenes and the Molecular Origins of Cancer. Cold Spring Harbor Laboratory Press, New York, 1989, p. 35. 52. Weinberg, R. A. Tumor suppressor genes. Science 1991, 254, 1138-1146. 53. Hollingsworth, R. E.; Lee, W-H. Tumor suppressor genes: New prospects for cancer research. JNC11991, 83, 91-95. 54. Klein, G. The approaching era of the tumor suppressor genes. Science 1987, 238, 1539-1545. 55. Marx, J. Learning how to suppress cancer. Science 1993, 261, 1385-1387. 56. Collins, V. P.; James, C. D. Gene and chromosomal alterations associated with the development of human gliomas. FASEB J. 1993, 7, 926-930. 57. Eng, C.; Ponder, B. A. J. The role of gene mutations in the genesis of familial cancers. FASEB J. 1993, 7, 910-919. 58. Klein, G. Genes that can antagonize tumor development. FASEB J. 1993, 7, 821-825. 59. Cox, D.; Yuncken, C.; Spriggs, A. I. Minute chromatin bodies in malignant tumors of childhood. Lancet 1965, H, 55-58. 60. Schwab, M. Amplification of N-myc in human neuroblastoma. Trends Genet. 1985, 1, 271-275. 61. Sugawa, N.; Ekstrand, A. J.; James, C. D.; Collins, V. P. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastoma Proc. NatL Acad. Sci. 1990, 87, 8602-8606. 62. Collins, V. P. Amplified genes in human gliomas. Semin. Cancer Biol. 1993, 4, 27-32. 63. Brison, O. Gene amplification and tumor progression. Biochim. Biophys. Acta 1993,1155, 25--41. 64. Schwab, M.; Amler, L. C. Amplification of cellular oncogenes: A predictor of clinical outcome in human cancer. Gene, Chrom. Cancer 1990, 1, 181-193. 65. Zelhnbauer, B. A.; Small, D.; Brodeur, G. M.; Seeger, R.; Vogelstein, B. Characterization of NMYC amplification units in human neuroblastoma cells. Mol. Cell Biol. 1988, 8, 522-530. 66. Morgan, R. A.; Anderson, W. E Human gene therapy. Ann Rev. Biochem. 1993, 62, 191-217. 67. Weissman, S. M. Gene therapy. Proc. Natl. Acad. Sci. 1992, 89, 1111-1112. 68. Anderson, W. F. Human gene therapy. Science 1992, 256, 808-813. 69. Weatherall, D. J. Gene therapy in perspective. Nat, re 1991, 349, 255-276. 70. Gutierrez, A. A.; Lemonie, N. R.; Sikora, K. Gene therapy for cancer. Lancet 1992, 339, 715-721. 71. Rowley, J. D.; Aster, J. C.; SEar, J. The impact of new DNA diagnostic technology on the management of cancer patients. Arch. Path. Lab. Med. 1993, 117, 1104-1109. 72. Goddard, A. D.; Solomon, E. Genetic aspects of cancer. Adv. Hum. Genet. 1993, 21, 321-376.

ONCOGENES IN TUMOR PROGRESSION

Bruce P. Himelstein and Ruth J. Muschel

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Metastasis . . . . . . . , .................... Oncogenes and Cell Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Immune Surveillance . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes and Radiation Resistance . . . . . . . . . . . . . . . . . . . . . . Oncogenes as Predictors of Outcome . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 18 19 22 24 31 32 33 33 35 38 41 41

I. I N T R O D U C T I O N W h i l e for m a n y y e a r s it w a s h y p o t h e s i z e d that c a n c e r w a s a d i s e a s e c h a r a c t e r i z e d b y an a c c u m u l a t i o n o f s o m a t i c m u t a t i o n s , it has n o w b e e n c o n c l u s i v e l y d e m o n -

Advances in Genome Biology Volume 3A, pages 17-53. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8 17

18

BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

strated that indeed mutations can be identified in cancer cells. Many of these mutations inactivate tumor suppressor genes while others activate oncogenes. Most of these oncogenes have been identified through their ability to transform cells in tissue culture or to induce tumorigenicity, yet malignant cells have additional properties which make the therapy of cancer particularly difficult. Malignant tumors are characterized by local invasion and distant metastasis. Tumor progression is a collective term used to describe the processes by which cancer cells become increasingly aggressive and "malignant" in their behavior. The problem of determining how the presumably causal mutations in oncogenes and in suppressor genes lead to tumor progression is only beginning to be addressed. Nonetheless, it can be demonstrated that oncogenes affect the growth of cells as tumors, both through effects on growth factors and their receptors as well as on shifting the balance between apoptosis and proliferation. They have also been shown to induce angiogenesis, the ingrowth of new blood vessels to supply the nutritional and metabolic demands of a growing tumor, metastasis, and protease expression. Furthermore, there is evidence that activation of oncogenes can alter immune recognition of tumor cells. They can also result in enhanced drug and radiation resistance, even in the absence of selection. These effects of oncogene activation would be predicted to influence prognosis and indeed data is now accumulating which correlates oncogene activation with outcome in some tumors. In this review, we have described some of the experiments which link oncogene activation with these features of tumor progression. Because of the vast literature now available on this subject, we have certainly not cited all possible literature but have selected a variety of examples to illustrate the effects that oncogenes can have on the phenotypic changes associated with tumor progression.

II.

ONCOGENES

Protooncogenes are normal components of the genome which are involved in cell growth and differentiation. Oncogenes are activated homologues of protooncogenes. More liberally defined, any gene which is significantly associated with a tumor may be thought of as an oncogene. Oncogenes may be activated by a number of mechanisms. Retroviruses may transduce a neighboring protooncogene, and carry it to other cells as an oncogenic virus. ~ Transcriptional activation can also occur as a result of the insertion of viral enhancer or promoter sequences near growth-related genes; in fact, there is some evidence to suggest that enhanced expression of normal, unmutated protooncogenes may be sufficient for oncogenic function. 2 Chromosome translocations can bring together growth-related genes with transcriptional enhancers, such as seen in the Philadelphia chromosome translocation involving the c-abl and bcr genes, typical of chronic myelogenous leukemia. 3 Genes may be activated by amplification, whereby increased gene expression results from markedly increased copy number of the gene; the N-myc

Oncogenes in Tumor Progression

19

amplification typical of aggressive neuroblastoma is a well-described example. 4 Activation by point mutation is typical, for example, in the ras gene family, where several nucleotides appear to be hot spots for mutation in human cancers. 5'6 Shiu and co-workers 7 have recently suggested that the increased half-life of c-myc messenger RNA, demonstrated in the MDA-MB-231 breast cancer cell line, may be an alternative mechanism for gene overexpression. Finally, genes may be activated by the loss of other genes, known as tumor suppressor genes or antioncogenes, which normally suppress their function. An example of such a gene is the retinoblastoma gene. 8 Activation of oncogenes and loss of tumor suppressor genes are critical components of the process of tumor progression. 9-11

i11. ONCOGENES A N D CELL G R O W T H Tumor cells have a growth advantage when compared to normal cells. The focus of research in this area has been on the ability of transforming or activated oncogenes to stimulate tumor cell proliferation. For example, overexpression of c-myc or of c-myb plays a central role in deregulation of the cell cycle. 12,13Recently, however, an alternative mechanism mediating this growth advantage has come to light following the description of the transcriptional deregulation of the bcl-2 gene in the t(14;18) chromosomal translocation typical of non-Hodgkin's follicular B-cell lymphomas. 14 The bcl-2 oncogene does not function by direct deregulation of cell growth, but instead overexpression of bcl-2 blocks programmed cell death or apoptosis. Apoptosis is an energy-requiting, active form of cell death characterized by degradation of cellular DNA by endogenous endonucleases, first described by Kerr in 1972.15 This process is responsible for maintaining the normal balance of rapid proliferation of new cells with cell loss characteristic, for example, of bone marrow. 16 Loss of such a balance, through oncogene-mediated inhibition of apoptosis, may also lead to neoplastic growth. In normal human B cells 17 and in Burkitts lymphoma cell lines, 18the presence of Epstein-Barr virus (EBV)protected cells from apoptotic death; a latent membrane protein of EBV was shown to upregulate bcl-2, suggesting that EBV infection and expression of this oncogene may be linked in cancer associated with viruses. The bcl-2 oncogene may also play a role in the multistep progression towards the malignant phenotype through coordinated function with other oncogenes. Bissonnette et al. 19 have proposed a "two signal" model for tumor cell progression through the cell cycle mediated by coexpression of c-myc and bcl-2. They demonstrated that bcl-2 expression in c-myc-transfected Chinese hamster ovary cells blocked c-myc-induced apoptosis, allowing neoplastic growth of the transformed cells. These results suggested that the signal provided by overexpression of c-myc led to either transformation or to apoptosis; and that bcl-2 provided the second signal which selects for transformation by inhibiting apoptosis, resulting in uncontrolled proliferation. However, transgenic mice beating a minigene construct which

20


mimics t(14;18) and which leads to overexpression of bcl-2 developed indolent follicular hyperplasia which later progressed to malignant large-cell lymphoma, suggesting, rather, that bcl-2 may participate in very early events in tumorigenesis. 2~ Another aspect of tumor progression is the ability of tumor cells to proliferate independently from normally required growth factors, which in vitro corresponds to growth in low-serum or serum-free conditions. This independence may involve oncogenes in one of two ways; first, an oncogene may code for a growth factor-like substance, or second, such a gene may code for a growth factor receptor. Several examples of the role of such oncogene products will be described below. Nakagawara et al. 21'22 explored the expression of the trk protooncogene, which codes for part of the nerve growth factor (NGF) receptor, in neuroblastomas, tumors characterized by their failure to undergo terminal differentiation in response to NGE They found that N-myc amplification, a poor prognostic marker in neuroblastoma, 4'23 was associated with decreased or absent levels of trk expression, suggesting that the loss of expression of part of the growth factor regulation of neuroblastoma cell differentiation could, in part, be responsible for poor prognosis in these tumors. Similarly, the c-met/hepatocyte growth factor (HGF or scatter factor) receptor (see Ref. 24 for review) was found to be overexpressed (but the gene was not amplified) in a large proportion of follicular thyroid carcinomas, and was associated with both poor prognosis and locally invasive and/or metastatic disease. The authors speculated that the overexpression of this receptor led to neoplastic stimulation of thyroid follicular cells by HGF normally secreted by thyroid parafollicular cells. 25 In colon cancer cell lines, c-met may also function in the control of cell motility. HGF is a motility stimulating factor in vitro for these cell lines; however, HGF also inhibits growth of the same cell lines, raising the question as to the relative contribution of these divergent responses to HGF action in vivo and in the phenotype of malignant cells. 26 A great deal of interest has been generated in the erbB-2 (HER-2) gene in human cancer (the neu gene is homologous in rats) (see Ref. 27 for review). This gene encodes a member of the transmembrane tyrosine kinase receptor family, and bears similarity to the epidermal growth factor (EGF) receptor. In vitro, a tumorigenic phenotype could be conferred on rat ventral prostate epithelial cells by transfection with activated neu. 28 Neither EGF nor transforming growth factor-~ (TGF-~), ligands for the EGF receptor, bind to this receptor. One ligand for this receptor, heregulin, 29 which is distributed widely in normal tissue, was shown to increase tyrosine phosphorylation of p 185 erbB-2 but not of the EGF receptor, and stimulated the proliferation of breast cancer cells in culture. These results led to speculation that erbB-2 may also be involved in paracrine stimulation of breast cancer cell growth early in their neoplastic development. The clinical finding of increased expression in ductal carcinoma in situ, which may be a precursor lesion to invasive ductal cancer, also supports this hypothesis. Monoclonal antibodies to this receptor


21

induced differentiation of breast cancer cells; this finding correlated with tumor inhibition, as well as with changes in cellular morphology, secretion of milk components, and receptor translocation to cytoplasmic and perinuclear sites, consistent with reversion of the transformed phenotype. 3~ These results suggest that overexpression of c-erbB-2 may result in tumorigenesis due to abnormalities in signal transduction along normal differentiation pathways. Data derived from c-erbB-2-expressing transgenic mice support the hypothesis that this oncogene is intimately involved in differentiation pathways; these mice bearing activated c-erbB-2 experience a shortened life span due to preneoplastic proliferation, especially in the lungs and kidneys, resulting in end organ failure. 31 Growth factor production can be under the control of activated oncogenes, a further mechanism through which activation of oncogenes can lead to tumor progression (see Ref. 32 for review). These growth factors may stimulate the tumor cells themselves or they may stimulate neighboring cells to proliferate or to secrete substances which are advantageous to tumor progression. 33 Media from Moloney murine sarcoma virus-transformed cells (known to express v-mos), for example, were found to contain two transforming growth factors, TGF-t~ and TGF-]~. 34 Increased TGF-t~ expression has been seen in NIH3T3 fibroblasts transformed by ras. Transfection of NIH3T3 cells with other oncogenesmraf, Ki-ras, mos, src,fms, fes, met, and trk, but not with the neu/erbB-2 oncogene or with SV-4035--was also associated with increased TGF-o~ expression. The expression of TGF-ct could be separated from transformation since a phenotypically revertent cell line which still expressed Ki-ras maintained upregulated TGF-t~ expression. Spontaneously immortalized normal human mammary epithelial cells (MCF-10A) transformed with c-H-ras demonstrated increased production of TGF-ct, decreased expression of EGF receptors, a receptor to which TGF-~ binds, and diminished responsiveness to exogenous EGF or TGF-ot. In addition, antibodies to TGF-ct or to the EGF receptor partially blocked colony formation in soft agar. Transformed cells with the neu oncogene did not demonstrate these changes. Most interestingly, expression of recombinant TGF-ct in the parent cell line reproduced the transformed phenotype, including decreased responsiveness to exogenous TGF-ct or EGF, suggesting that TGF-o~ may be a necessary intermediary in the ras-mediated transformation of this cell line. 36 NIH3T3 cells which overexpressed the normal EGF receptor under a retroviral promoter were also phenotypically transformed in the presence of exogenous EGE Cotransfection with an expression vector for TGF-ct induced EGF-independent cell transformation, 37 again supporting a role for transforming growth factors in development of the malignant phenotype. Similar findings have been described for TGF-[3, whereby ras and mos transformation leads to increased secretion and decreased surface receptor numbers. Also, raf, Ki-ras, mos, src,fes, met, and trk, but not fms, transformation led to an increase in TGF-[3. 35 Inducible ras expression resulted in similar increases in expression of this growth factor. The promoter for TGF-[3 contains a ras-responsive element; transcription can be increased in ras-transformed cells or in cells cotransfected with

22


ras. The v-src oncogene can also stimulate TGF-~ promoter activity. 38 Coordinate overexpression of several different growth factors was seen in some of these transformed cells, suggesting that different oncogenes may stimulate a final common pathway shared by these secreted transforming growth factors. In summary, oncogene activation has been found to affect cell growth both through effects on growth factors and their receptors as well as through shifting the balance between cell death and proliferation.

IV. ONCOGENES A N D ANGIOGENESIS Experimental evidence and clinical observations support the hypothesis that progression of solid tumors depends upon development of a stable vascular supply. 39 Induction of this angiogenic phenotype may result directly from the activity of the tumor cell itself; for example by secretion of angiogenic peptides (see Ref. 40 for review) or indirectly from recruited host elements which then induce angiogenesis. This switch, like other elements of tumor progression, is not a single event but rather a complex and coordinated process similar to the metastatic cascade which requires degradation of basement membranes, endothelial cell attachment, and endothelial cell migration. 41 Although angiogenesis does not always correlate with clinical tumor aggressiveness, tumor angiogenesis was found to be an independent predictor of metastatic disease in axillary lymph nodes or in distant sites in invasive breast carci noma. 42'43 Oncogene transfection studies have shown an association ofoncogene expression with the development of neovascularization. For example, activated ras expression in murine prostate results in dysplasia and new blood vessel formation. 44 Similarly, Kallinowski and co-workers45 demonstrated the early onset of neovascularization in tumors derived from ras-transformed Rat l cell lines when compared to the spontaneously tumorigenic parent line. The c-etsl protooncogene, the cellular counterpart of the v-ets oncogene, is a nuclear transcription factor whose expression was noted to be prominent in endothelial cells during blood vessel formation in chick embryos. 46 Expression of c-etsl has been found to be associated with tumor vascutarization, particularly in Kaposi's sarcoma spindle cells, the malignant endothelial cells surrounding the rudimentary vascular spaces characteristic of this tumor. In vitro modeling of this phenomenon with human umbilical vein endothelial cells demonstrated upregulation of c-etsl expression in response to angiogenic factors such as TNF-o~ and phorbol ester. Promoters of several degradative enzymes which participate in the angiogenic cascade have been shown to contain ets-binding consensus motifs, including urokinase-type plasminogen activator, 47 interstitial collagenase, stromelysin 1,48-50 and most recently the 92-kDa type IV collagenase, 5~ suggesting that ets might induce protease gene expression during new vessel formation.


23

Montenaso et al. 52 also demonstrated the need for a balance of proteolysis and proteolytic behavior in angiogenesis by examining the effects of transformation by the polyoma middle T oncogene in normal endothelial cells. In vitro, these transformed cells grew as large hemangioma-like structures, and overexpressed urokinase-type plasminogen activator. Reversal of the proteolytic phenotype with exogenous protease inhibitors resulted in normalization of vascular morphogenesis. The protein product of the oncogene, int-2, is a member of the fibroblast growth factor (FGF) family of epithelial growth factors. 53 Members of the FGF family are important compounds in morphogenesis, tissue regeneration, and angiogenesis (see Refs. 54,55 for review). Transfection of int-2 into NIH3T3 cells is transforming. 56 Because Kaposi's sarcoma is thought to be a proliferation of vascular cells, Huang et al. 57 attempted to determine whether members of the FGF family could also be implicated in the generation of Kaposi's sarcoma. Expression of int-2 was found by the reverse-transcriptase polymerase chain reaction (PCR) in over half of the Kaposi's lesions studied, but not in normal adult skin. By immunohistochemistry, the protein was localized to the perivascular cells surrounding the dysplastic vascular spaces characteristic of this tumor. The gene was not amplified or rearranged; the mechanism of overexpression is unclear. Expression of the FGF receptors fig and bek was also found in these lesions. It is not known whether the int-2 protein product is a ligand for these receptors, although these receptors do bind several different members of the FGF family. It is attractive to speculate that an autocrine or paracrine growth stimulatory pathway is implicit in the genesis of this neoplasm, but the exact mechanism linking int-2 overexpression to tumor growth is unclear. Further evidence implicating members of the FGF family in tumor progression associated with angiogenesis comes from transgenic mice carrying the genome of bovine papillomavirus type 1.58 These mice provide a model for the multistage development of dermal fibrosarcomas. In the progression of premalignant mild fibromatosis to more aggressive fibromatosis and finally to fibrosarcomas, basic fibroblast growth factor (bFGF) changed from an intracellular to an extracellular location, suggesting that release of bFGF may be associated with development of more invasive neoplasms. Similarly, Coulier et al. 59 demonstrated that the signal peptide of another transforming member of the FGF family, FGF6, is necessary for transformation and for commitment to the secretory pathway and consequent glycosylation. An alternative FGF6 peptide lacking this leader sequence lost the ability to transform NIH3T3 cells. In this case, of course, FGF must play a role independently of any effect on angiogenesis. The loss of a tumor suppressor gene has been linked to the development of angiogenic activity in the immortalized hamster cell line, BHK21/cll3. 6~ The presence of this suppressor gene was found to be required for the production of a secreted angiogenic inhibitor, shown to be a truncated form of thrombospondin (TSP), an adhesive glycoprotein. 61 These findings led to the hypothesis that loss of angiogenic inhibitor function due to loss of a suppressor gene could result in

24


increased tumor growth and angiogenesis. Subsequent studies together suggest that the role of TSP in tumor progression is much more complex, and may depend upon a balance between its anti-angiogenic and its growth stimulatory activity. For example, in a human squamous cell carcinoma cell line with high levels of TSP production, antisense-mediated reduction in TSP expression actually reversed the malignant phenotype and produced tumors which were less hemorrhagic and more differentiated, 62 while overexpression of TSP1 in NIH3T3 cells resulted in serumand anchorage-dependent growth, but not in tumorigenicity. 63 As shown above, much of the evidence implicating oncogene activation and loss of suppressor gene function in angiogenesis is indirect, and the mechanisms of stimulation of vessel formation are still poorly understood. 64

V. ONCOGENES A N D METASTASIS There are many steps which are required for successful tumor metastasis, including tumor cell detachment from the primary tumor, invasion of local tissue by proteolysis of basement membrane and extracellular matrix, entrance into the circulation, escape from immune surveillance, adhesion to vascular endothelium, extravasation into the target site, and proliferation in this site. 4~ The activity of oncogenes has been implicated in each of these steps. Two laboratory assays are commonly used to monitor metastatic potential: (1) the spontaneous metastasis assay, in which metastases are measured following subcutaneous tumor cell inoculation, tests the full metastatic capability of a tumor cell; and (2) the experimental metastasis assay, in which metastases are counted following intravenous injection of tumor cells, tests the later steps in the metastatic pathway, not including local invasion and entry into the vasculature. The two assays often provide similar results, but not consistently. 65-67 The ability of a tumor cell to metastasize is a property which is separate from its ability to form a tumor. 68 Metastatic subpopulations are theoretically present during tumor growth, and are allowed to expand under appropriate selection. Fidler 69 described increased metastatic potential of B 16 melanoma cell lines established from pulmonary metastases following intravenous inoculation, lending support to this hypothesis. Virone et al., 7~ however, have produced contradictory results. Murine tumor cells beating a variety of activated oncogenes derived from pulmonary micrometastases following subcutaneous injection did not macrometastasize when replanted subcutaneously, a finding which might argue that metastatic cells are not selected variants, since their phenotype was indistinguishable from that of the parent cell line. Therefore, whether metastatic cells are present and genetically programmed in the primary tumor at inception, or whether they acquire the metastatic phenotype through selective pressure, remains to be proven. It should be pointed out, however, that these are not mutually exclusive hypotheses. For example, a genetic alteration which is present might alter the response to selection.


25

The metastatic phenotype can be induced in vitro through transfection of a number of oncogenes, and in v i v o studies have associated overexpression of several oncogenes with metastasis. The diversity of oncogenes involved suggests that multiple intracellular and intercellular changes due to oncogene expression may result in metastasis. The ras mutation is a frequent finding in human cancer, although some cancers, such as breast carcinoma, do not appear to contain ras mutations frequently, while others, such as lung or pancreatic adenocarcinomas, often harbor mutated oncogenes. 6 Deng et al. 71 correlated c - H - r a s mutations in gastric cancers with development of metastases, and a particular codon 61 mutation in H - r a s has been associated with bone metastases in prostate cancer. 72 In vitro, ras has been firmly established as being responsible for the metastatic transformation seen in NIH3T3 cells, mouse T 1/2 fibroblasts, and normal diploid fibroblasts, and much is known about the genes which are activated by ras transformation (see Ref. 73 for review). In NIH3T3 cells, Thorgierrson et al. 74 demonstrated that cells transformed by genomic DNA containing N - r a s from a patient with myeloid leukemia were metastatic. Other groups confirmed that this phenotypic change was due to the ras oncogene, rather than to other genes present in the genomic DNA. Muschel et al. 75 found that isolated clones derived from NIH3T3 cells transformed with H - r a s were metastatic, and Egan et al. 76 confirmed that lung colonization in an animal model correlated with measured levels of p21 ras. Other groups reported similar findings, 77'78 while some have not found this correlation to be absolute. 79 Further studies with both mutationally activated ras as well as with the transcriptionally overexpressed but nonmutated ras protooncogene suggested that there was a dose response, whereby the metastatic phenotype was expressed beyond a certain level of expression of mutated or normal p2 lras. 75'76'80'81 Similar results have been reported in normal diploid rat fibroblasts, 75 '82 in tumorigenic but nonmetastatic transformed cells, 83-85 and in mouse T1/2 fibroblasts. 86 Human breast epithelial cells display increased invasiveness and chemotaxis when they are transformed by c - H - r a s . 87 The correlation between ras transfection and metastatic potential is not absolute, as shown by Baisch et al. 88 who were unable to induce metastases by ras transfection into R 1H rhabdomyosarcoma cells, and by Muschel et al. in 1985 in C127 cells. 75 Also, Gelmann et al. 89 showed that mutated H - r a s expression in MCF-7 mammary carcinoma cell lines did not confer the metastatic phenotype, although the transfectants were more invasive in vitro. Also, there is evidence to suggest that other cytogenetic changes or the development of genomic instability associated with ras transformation may actually be more critical determinants of the metastatic phenotype than the levels of ras expression. For example, Ichikawa and colleagues 9~ demonstrated that the frequency of structural chromosomal changes was associated with the development of the metastatic phenotype, rather than with ras levels in the nonmetastatic rat prostatic cancer cell line AT2.1 transfected with v - H - r a s . Schlatter and Waghorne 91 provided further compelling evidence to support the hypothesis that ras induces

26


stablesecondary genetic changes which result in the metastatic phenotype. SP1 mouse mammary adenocarcinoma cells were transfected with H-ras in a shuttle vector which required antibiotic selection to be maintained. Following transfection and demonstration of ras protein expression, cells were isolated which had lost the shuttle vector during selection. Despite loss of ras expression, these subclones were equally metastatic when compared to parent ras transfectants, again suggesting that genetic or epigenetic changes induced by ras were, in fact, responsible for the metastatic phenotype. Ichikawa et al. 92 subsequently characterized the nature of these cytogenetic changes by studying high metastatic subclones of v-H-ras transfected RCMI rat mammary cancer cell lines whose metastatic phenotype was also not correlated with ras expression. The authors speculated that if these cytogenetic abnormalities resulted in only gain of gene expression, than fusion products of metastatic subclones with nonmetastatic parental cells should retain the metastatic phenotype, while if these abnormalities resulted in loss of a suppressor gene, similar fusion products would lose the metastatic phenotype due to replacement of suppressor function by the parent cell genome. The fusion cells, despite continued expression of p21 r a s and equivalent tumorigenicity, lost the high metastatic phenotype, suggesting that ras transfection results in loss of metastasis suppressor function. Other oncogenes have also been implicated in the origins of the metastatic phenotype. Egan et al. 76 demonstrated that NIH3T3 cells transformed by a variety of protein kinase-coding oncogenes, including the cytoplasmic serine/threonine kinases v-mos and v-raf, and tyrosine kinases v-src, v-fes, and v-fms, became metastatic in both spontaneous and experimental metastasis assays. The c-myc oncogene induced experimental metastasis at low rates, while v-myc did not induce metastasis at all. Melchiori et al. 93 found that NIH3T3 cells transfected with oncogenes representing different steps of mitogenic signaling, such as v-sis, v-erbB, v-mos, mutated c-ras, and v-fos, were more invasive than the parent line, and had an enhanced chemotactic response to laminin. Muschel and Liotta 94 could not confirm these results, but lower tumor cell doses were used in the latter study, again suggesting the possibility of a dose effect for these oncogenes. Stoker and Sieweke 95 demonstrated that a small percentage of v-src induced sarcomas in chicken wing webs became rapidly metastatic. Clinically, however, levels of pp60 v-src in human colorectal cancer metastases have been found to be increased relative to levels in primary tumors and in normal colonic mucosa, suggesting an in vivo role for src in metastasis. 96 Similarly, increased expression of c-myc is associated with metastatic outcome in cervical carcinoma. 97 Cooperation between oncogenes has also been shown to be important in tumor spread. Both ras-and myc-cotransfected rat embryo cells 98 and mouse T1/2 fibroblasts 86 displayed increased metastatic behavior. Addition of a mutated form of the tumor suppressor p53 further increased this behavior in the mouse cells. Cooperation between the host and oncogene-transformed tumor cells may also play arole in the development of metastases. For example, Takiguchi et al. 99 showed


27

that tumors derived from clones of K-ras-transformed NIH3T3 cells in nude mice expressed higher levels of K-ras than the parent line, and acquired increased metastatic capacity. This effect could be replicated by coculture of G-418-resistant tumor cells with BALB/c3T3 fibroblasts; after removal of the fibroblasts by G-418 treatment, the remaining tumor cells also maintained increased ras expression and metastatic potential during several subsequent passages in tissue culture. Himelstein et al. ~~176 have demonstrated that the ability of H-ras and v-myc transfected rat embryo cells to metastasize may be correlated not only with their ability to secrete MMP-9, but also with their ability to induce MMP-9 expression in surrounding normal fibroblasts. As noted above, several oncogenes may code for proteins which are growth factors or growth factor receptors. This class of oncogenes is also involved in generation of metastases (see Ref. 101 for review). For example, transfection of the activated c-erbB-2/neu oncogene, !~176 which encodes an EGF receptor-like transmembrane glycoprotein into NIH3T3 cells, leads to increased metastatic potential; antibody-mediated downregulation of neu reduced this metastatic potential. Unfortunately, it is impossible to separate increased growth in response to a growth factor per se from other transformation-related phenomenon (e.g., increased adhesion or motility) in being responsible for metastasis in this type of experimental system, l~ Overexpression of the nonmutated neu gene in transgenic mice also led to development of metastases in tumor-bearing animals, again supporting the hypothesis, similar to that noted above in regards to TGF-~ and the EGF receptor, that tipping the balance of growth factors and their receptors may be sufficient for transformation in certain cells. 1~ Clinically, HER-2/neu amplification and overexpression is associated with early metastasis in breast cancer, 1~176 and serum levels of the translation product of c-erbB-2, p 185, correlated with the presence of metastatic disease. 1~ In other tumor types (e.g., gastric tumors) erb-2 may play a role in tumor progression, although conflicting results have been reported concerning the association of this oncogene with metastasis. 1~ Expression of the hst gene, a member of the FGF family of growth factors homologous to the K-fgf gene of Kaposi's sarcoma, has been associated with the metastatic phenotype in the pregnancy-dependent mouse mammary tumor. 112Similarly, transformation of NIH3T3 cells with K-fgf resulted in highly metastatic cells, ll3 Egan and co-workers 114 elucidated several interesting features of the balance of growth factors and receptors necessary for tumor metastasis. They transfected NIH3T3 cells with a chimeric construct linking basic fibroblast growth factor (bFGF) to an immunoglobulin leader sequence, which targeted the protein for secretion, or with bFGF alone. Chimeric transfectants were highly metastatic in both the experimental and spontaneous metastasis assay; bFGF-only transfectants were not. The bFGF-only cells were nontransformed, and accumulated high levels of bFGF intracellularly. Exogenous administration of bFGF to ras-transformed C3H-10T1/2 cells or to ras- or src-transformed NIH3T3 cells prior to intravenous

28


injection, conversely, markedly inhibited subsequent metastasis formation. The reason for the different behavior of NIH3T3 cells exposed to chimeric bFGF versus exogenous bFGF is unclear, but may be related to different regulation of the receptor in response to these two proteins or to as yet uncharacterized other genetic changes in ras- or src-transformed cells. These results are particularly interesting in light of those discussed above which correlated secretion of bFGF with the angiogenic phenotype. More recent results from Taylor et al. 115 also demonstrated that NIH3T3 cells expressing only bFGF were noninvasive. Of note, however, these cells were found to be more motile. One such line which was highly motile also formed pulmonary micrometastases, suggesting further that bFGF-induced increases in motility acting through an intracrine pathway may contribute to the metastatic phenotype. A similar association of motility with metastatic potential was shown for cells transformed with K-fgf, which contains a leader sequence targeting it for secretion. The motility of these transformants could also be blocked by suramin, which may interfere in some way with the growth factor-receptor loop. The association of overexpression of oncogene-related epithelial growth factors with the metastatic phenotype is not limited to bFGF. Similar induction of invasive capacity and increased motility has also been shown for the related acidic fibroblast growth factor (aFGF) in both secreted and nonsecreted forms in NBT-II epithelial carcinoma cells. 116 Egan et al. ll4 also explored the response of NIH3T3 cells transformed with H-ras or with v-fms, which codes for the colony stimulating factor (CSF-1) receptor, to exogenous CSF-1. The fms transfectants, which were cultured for 3.5 hours prior to 24-hour treatment with CSF-1, responded with an increase in metastasis. Similarly, cells cultured for 41 hours with a change of medium prior to 24-hour treatment with CSF-1 also responded with increased metastases. Instead, if the cells were autocrine conditioned for 41 hours without a change in medium, CSF-1 ' treatment now markedly inhibited metastasis formation. As a control, H-ras transfected NIH3T3 cells did not show any alteration in metastatic potential under similar experimental conditions. These results suggest that other serum factors may cooperate with CSF-1 in conferring the metastatic phenotype, and that receptor transmodulation may also be important in determining whether a given growth factor stimulates or suppresses metastases. Finally, novel metastasis suppressor and nucleoside diphosphate kinase genes, nm23-Hl and nm23-H2, have been isolated. Loss of nm23 expression has been associated with increased metastasis in vitro, ~7 and expression of the metastatic phenotype in H-ras-transformed cloned-rat embryo fibroblasts was associated with reduced expression ofnm23-H1.118 In vivo, the role of nm23-H1 in tumor progression is less clear; while rim23 mutations which might be expected to reduce function have been described in neuroblastomas and in colorectal cancer, some aggressive, advanced stage neuroblastomas, in fact, have increased levels of expression of nm23.119'12~ The effects of cellular heterogeneity were not considered in some of


29

these studies, as Radinsky et al. 121 demonstrated that expression of nm23 in subclones isolated from human colon or renal carcinomas did not correlate with the metastatic phenotype. More recent data suggest that the nm23 story is even more complex. Urano et al. 122 found that nm23 proteins are expressed on the surface of a wide variety of human normal and tumor cells, suggesting an additional extracellular role for this gene product. Intracellularly, Postel and colleagues 123 identified the human c-myc transcription factor PuF as nm23-H2, suggesting that nm23 may also participate in myc transcriptional regulation.

Vi. ONCOGENES AND CELL ADHESION Alterations in cell adhesiveness and deformability may be necessary for tumor progression. Detachment of cells from primary tumors may be an initiating event in metastasis, while anchorage-independent growth is an in vitro marker for transformation. Studies implicating ras in changes in adhesion compared parental cloned rat embryo fibroblast (CREF) cells with both T24 ras transfectants and with T24 transfectants whose transformed phenotype was reversed by the Kirsten ras revertent gene (K-rev 1A). The ras-transformed cells demonstrated anchorage-independent growth and an increase in both spontaneous and experimental metastasis. This phenotype was reversed by K-rev 1A. 124 Also, ras-transformed cells were found to be more easily detachable from parent CREF monolayers at all shear-stress levels tested, and were more deformable. Elevations in cytoplasmic pH, typically associated with cell spreading, adhesion, or response to cytokines, may also be an important factor in growth permissiveness. 125 The role of increased cytoplasmic pH on anchorage-independent growth and tumorigenicity was first demonstrated by Perona and Serrano 126 who induced such a phenotype in 3T3 cells by increasing cytoplasmic pH by expression of a yeast proton pump. Subsequently, loss of changes in cytoplasmic pH between attached cells and cells in suspension was shown in NIH3T3 cells transformed by v-Ki-ras, v-src, and polyoma middle T, suggesting that oncogenic transformation could substitute for spreading in raising intracellular pH, permitting anchorageindependent growth. 127 Similar loss of pH regulation was not seen in myc transfectants. 125 Rapid changes in surface protein glycosylation of NIH3T3 cells transfected with c-H-ras, which were associated with increased invasiveness prior to morphologic transformation, were shown by Bolscher et al. in 1988.128 The ras gene family has also been implicated in the control of expression of CD44, a cell surface glycoprotein with several described variant forms involved in diverse adhesive cellular functions (see Ref. 129 for review). Standard or lymphocyte-type CD44 expression may contribute to the metastatic capacity of human lymphoma cells, 13~ while

30


CD44H expression has recently been implicated in the adhesion of ovarian cancer cells to peritoneal mesothelium. TM Exon v6-containing variant isoforms of CD44 were recently found to be downregulated in human malignant epithelial tumors, particularly in metastatic cells. 132 Conversely, increased expression of a CD44 variant containing exon 6 in human colon adenocarcinomas 133and in rat carcinoma cell line 134'135 was associated with tumor progression, suggesting that CD44 variants may play different roles in tumor progression in different cell types. Transient ras expression in CREF cells resulted in expression of this metastasisassociated variant of CD44 as well as in perturbation in the normal control of CD44 splicing. Direct activation of the CD44 promoter by ras was shown using promoter constructs linked to the chloramphenicol acetyltransferase reporter; this induction could be blocked by cotransfection of adenovirus E1A, which also blocked in vivo metastasis of r a s - t r a n s f o r m e d CREF, and by point mutations in the AP- 1-binding consensus motif in the CD44 promoter. 136 Oncogenes other than ras have been implicated in altered cell adhesiveness in tumor progression. A recent example of the role of v-src in altered adhesion was published by Matsuyoshi and co-workers. 137 Normal rat 3Y 1 cells were compared to v - s r c - t r a n s f e c t e d and v-src- and v - f o s - d o u b l e - t r a n s f e c t e d 3Y1 cells. The transfectants were metastatic, and could not maintain stable cell--cell contacts in suspension or in collagen gels despite comparable expression of P cadherin, a member of a ubiquitous transmembrane glycoprotein family which mediates calcium-dependent intercellular adhesion, and of c~-catenin, a cadherin-associated protein. The authors demonstrated tyrosine phosphorylation of a 98-kDa catenin and weaker phosphorylation of cadherins in the transformed cells. Phosphorylation blocked by vanadate or by Herbimycin A mirrored the phenotypic changes described above. Albeit somewhat circumstantial, such data suggest that oncogene expression may be intimately involved in the regulation of cell-cell contact. Calcium-binding proteins may also be involved in tumor cell adhesion and the metastatic phenotype. For example, transfection of mrs- 1138in the mouse and p9Ka in the rat, 139 homologous calcium-binding proteins, confers the metastatic phenotype, perhaps by alteration of the cytoskeleton. A ras transfection of NIH3T3 cells has also been shown to increase expression of two other calcium-binding proteins, calcyclin and osteopontin, both in concert with increases in metastatic potential (see Ref. 73 for review). Finally, a novel tumor suppressor gene, the DCC gene, has been identified on chromosome 18q which is frequently lost in colorectal neoplasias. This loss was more likely to be found late in the course of tumorigenesis, suggesting that it has a role in tumor progression. 14~ The sequence of this gene predicts that it functions in cell adhesion, an interesting observation given the need for tumor cells to lose attachments to primary tumors as a necessary step in metastasis.


31

VII. ONCOGENES AND PROTEOLYSIS The ability of tumor cells to degrade extracellular matrix components and basement membrane is essential to metastatic s u c c e s s . 41 There are now many reports of the correlation between metastatic potential both in vitro and in vivo with secretion of proteases and downregulation of protease inhibitors. Transfection studies have repeatedly implicated activated oncogenes in the shift of proteolytic balance. The ras transformation of NIH3T3 cells has provided a model for study of the effects of ras on other cellular genes (see Ref. 73 for review). It appears that ras affects the balance of proteolysis and proteolytic inhibitors. For example, ras transfection results in a rise in type IV collagenolytic activity concomitant with a decrease in expression of the tissue inhibitors of metalloproteinases. The matrix metalloproteases are a family of zinc-containing enzymes important in tissue remodeling and tumor progression (see Ref. 142 for review). Bernard et al. 98 demonstrated that metastatic potential correlated with the ability of ras- or m y c transformed rat embryo cells to secrete a 92-kDa type IV collagenase (matrix metalloproteinase 9 or MMP-9). E1A expression abrogated the metastatic and collagenolytic activity of these transfectants, in agreement with previous work. 143 SV40 transformation of human lung fibroblasts results in secretion of the same enzyme. 144 Similarly, c - e r b B - 2 overexpression in NIH3T3 cells also led to increased expression of MMP-9, an effect which could be reversed by coexpression of adenovirus 5 E1A gene product. 1~ Conversely, Sreenath et al. 145 using similar cell lines were unable to document any difference in expression of MMP-9, but rather found a striking correlation between metastatic potential and stromelysin 1 and 2 expression. Zhang et al. 146 have shown that different activated ras genes conferred a different pattern of protease expression in NIH3T3 cells, such that two metastatic phenotypes could be defined and correlated with either the expression of urokinase-type plasminogen activator or of cathepsin L, a member of the cysteine protease family, but not with expression of the metalloproteases. A change in balance has similarly been noted in NIH3T3 cells for the cysteine proteases. Malignant transformation by a variety of oncogenes, including H-ras, N - r a s , K - r a s , as well as src and mos, has also been shown to increase the expression of cathepsin L, initially identified as major excreted protein ( M E P ) . 147-149 Cathepsin B activity also increased and cysteine protease inhibitor activity decreased in proportion to the metastatic phenotype in NIH3T3 cells. ~5~ Elevated cytosolic cathepsin D levels, which were correlated with increased c - m y c expression, were characteristic of node-invasive breast carcinomas. 151 A similar correlation of metastatic potential with cathepsin L expression has also been shown in H - r a s transformed murine fibroblasts. 152 These changes did not occur, however, in rat embryo fibroblasts transformed by ras. 153 Other enzymes in oncogene-transfected cells have been implicated in tumorrelated proteolysis. For example, Schwarz et al. 154 showed that the activity of heparinase, which degrades heparan sulfate, a major glycosaminoglycan in the

32


extracellular matrix, was tightly correlated with the metastatic capacity of activated H-ras-transfected mouse T1/2 cells. In NIH3T3 cells, however, transfection with activated H-ras, as well as with v-src or v-fes, which also induced the metastatic phenotype, did not result in increased heparanase activity, suggesting that different proteases may participate in the metastatic cascade derived from tumor cell lines of differing genetic backgrounds. Transcriptional activation of many proteases requires the function of protooncogene products. For example, stimulation of stromelysin and MMP-9 mRNA requires induction of c-fos and c-jun which bind to the AP- 1 consensus binding motif present in many metalloproteinase promoters. 5~'155 Transin, the rat homologue of stromelysin, is also regulated by these early gene products; however, a fos-independent pathway of activation which still requires the AP-1 site has been found. 156 Overexpression of c-fos in several systems has been associated with enhancement of the metastatic phenotype; for example, increased fos expression in B 16 melanoma cells is associated with high metastatic variants, 157 whilefos transfection into a src-transformed rat 3Y 1 cell line increases the hydrolytic activity of procathepsin L. 158 It is therefore attractive to postulate that increasedfos expression may trigger transcription of other such metastasis-related genes. As noted above, c-etsl is also intimately involved in protease expression.

VIII.

ONCOGENES A N D IMMUNE SURVEILLANCE

Another aspect of tumor progression is the ability of tumor cells to evade immune detection. Clinically, low levels of HLA class I antigen expression and associated loss of sensitivity to killing by natural killer cells have been detected in a variety of human tumors (see Refs. 159,160 for examples). Major histocompatibility complex (MHC) class I antigens are cell surface proteins required for immune recognition by cytotoxic T cells. TM Bernards and co-workers 162 first demonstrated the association of overexpression of an oncogene, N-myc, with downregulation of MHC class I expression in neuroblastoma. An inverse relationship of N-myc and MHC class I expression was seen in patient samples; downregulation of MHC class I expression resulting from N-myc transfection into B 104 neuroblastoma cells which express low endogenous levels of N-myc transcripts was also shown. To show that the association was not due to clonal selection of cells with stable, genetically programmed high N-myc and low MHC expression, revertents of this genotype were shown to be able to increase MHC expression and decrease N-myc expression. MHC modulation was also shown to be reversible by treatment with y-interferon, even in the presence of persistent high N-myc expression. This MHC response was cell-type specific; altered MHC expression was not seen in Rat l fibroblasts expressing high levels of transfected N-myc. The authors speculated that loss of MHC I expression may be related to the metastatic phenotype of N-mycamplified neuroblastomas; however, subcutaneous tumor growth rates of cells with


33

varying MHC class I expression were not markedly different in immunocompetent and immunodeficient hosts, suggesting that N-myc amplification, rather than modulation of MHC class I, may be the critical determinant of in vivo tumor progression in this system. In other models, though, enhancement of immune recognition does result in decreased malignancy. Wallich et al., 163 for example, showed that a metastatic subclone of the TI0 sarcoma cell line, which did not express the H2-K alleles of the MHC, lost its metastatic phenotype when H2-K expression was restored. Furthermore, oncogene activation may also result in decreased immunogenicity. Lu et al. 164 showed that cell lines bearing certain point mutations in v-H-ras are associated with reduced expression of MHC class I antigens as well as the ability to metastasize in immunocompetent mice. Again, this in vitro finding has been supported by findings in the clinical arena, as Solana et al. 165 demonstrated a close relationship between increasing levels of p21 ras and decreasing HLA class I antigen expression in breast carcinomas. However, some breast carcinomas expressed both high p21 r a s and HLA class I levels, suggesting that ras mutation may not be the only genetic alteration responsible for immune evasion by breast cancers.

IX. ONCOGENES AND DRUG RESISTANCE In addition to the well-described amplification of specific drug target genes, such as dhfr, the dihydrofolate reductase gene responsible for methotrexate metabolism, 166'167or mdrl, the gene which codes for P-glycoprotein, the drug effiux pump responsible for the multidrug resistant (MDR) phenotype, 168-17~oncogenes have also been implicated in both in vitro and in vivo resistance to chemotherapeutic agents. For example, Gusterson et al., 171 who found that c-erbB-2 expression correlated with disease-free survival in node-positive patients with breast cancer, also reported for the first time convincing data suggesting that tumors overexpressing c-erbB-2 were less sensitive to treatment with cyclophosphamide, fluorouracil, and methotrexate. A similar trend towards drug resistance in node-negative breast cancers expressing c-erbB-2 was reported by Allred et al. 172Chin and colleagues 173 elegantly demonstrated that ras, as well as mutant p53, were able to activate the promoter of mdrl, implicating ras in resistance to a wide range of drugs whose clinical efficacy is reduced by mdr, such as vinca alkaloids or anthracyclines (see Ref. 174 for review). The overexpression of myc was also found to be associated with the MDR phenotype. Delaporte et al. 175demonstrated, however, that reversion of the MDR phenotype in myc-transfected cells may not be correlated with loss of expression of mdrl; in fact, mdrl levels increased in certain clones with reversion of the MDR phenotype despite expression of high levels of myc, casting some doubt on the role of mdrl in this setting. Neuroblastomas with amplified N-myc are often characterized by rapid progression, development of drug resistance, and poor prognosis; 4'23 here, too, an inverse

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correlation of N-myc amplification and mdrl expression was found by one group 176 but not by another. 177 These studies provided further evidence that alterations in mdrl expression may not be the only genetic changes which may result in the MDR phenotype. Cisplatinum (CDDP) is a chemotherapeutic agent which has shown great efficacy in many human cancers. Sklar 178 demonstrated that NIH3T3 cells transfected with activated ras oncogenes (c-H-ras, N-ras, v-H-ras, and v-K-ras) were resistant to CDDP; this resistance did not depend upon the activating mutation or the copy number of the transfected genes. Overexpression of normal ras protein or expression of unrelated oncogenes (v-fms, v-mos) only marginally increased platinum resistance. Using similar procedures, however, Toffoli et al. 179 were unable to confirm CDDP resistance due to activated c-H-ras transfection. Isonishsi et al. 18~ demonstrated that induction of mutationally activated c-H-ras in NIH3T3 cells resulted in platinum resistance coincident with decreased intracellular drug accumulation and decreased platinum-DNA adduct formation, as well as an increase in metallothionein content. These experiments were carried out with an inducible mouse mammary tumor virus promoter, allowing examination of resistance without the compounding problem of variance between transfected cells and untransfected controls. Kashani-Sabet and co-workers 181obtained serial samples of peritoneal cells from a patient with colon adenocarcinoma and refractory malignant ascites during the course of the development of clinical resistance to CDDP and 5-fluorouracil. The oncogenes, c-myc, H-ras, and c-fos, were amplified 2-, 4-, and 15-fold, respectively, corresponding to development of drug resistance. Expression of dTMP synthase and DNA polymerase 13, enzymes involved in DNA synthesis and repair, respectively, were also increased. Activation of c-H-ras can activate fos, a nuclear early response gene which has been implicated in CDDP resistance. 182 Both CDDPresistant cells in culture and from patients with clinical CDDP resistance have elevated expression of c-fos. Several genes whose expression are also increased in the setting of CDDP resistance, such as dTMP synthase, topoisomerase I, and metallothionein, contain AP-1 binding domains in their promoters which mediate the response to fos. 183 Further, transfection of CDDP-resistant A2780DDP cells with a dexamethasone-inducible anti-fos ribozyme resulted in reversal of CDDP resistance, as well as decreased expression of dTMP synthase, DNA polymerase 13, and topoisomerase 1.184 The mdrl gene as well as the glutathione-S-transferase gene also have AP-1 promoter sites, lending support to the hypothesis thatfos may act as a coordinator for the drug resistance phenotype. Surprisingly, though, expression of c-fos in NIH3T3 cells by transfection did not confer CDDP resistance in experiments performed by Isonishi. 18~ Other oncogenes may be involved in CDDP resistance. The overexpression of c-myc in the peritoneal cells described above 181 also correlated with CDDP resistance in vitro. Sklar and Prochownik 185 also correlated CDDP resistance in Friend erythroleukemia cells with expressed levels of transfected c-myc, demonstrated


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return of CDDP sensitivity following removal of c-myc expression in a dexamethasone -inducible promoter, and showed return of CDDP sensitivity in overexpressing cell lines following co-expression of c-myc antisense RNA. The ability of bcl-2 to block apoptosis has also been implicated in resistance to chemotherapy. Many chemotherapeutic agents stimulate apoptosis in malignant cells. 186'187By gene transfer, bcl-2 was shown to induce a drug-resistant phenotype in T-lymphoid clones 188 as well as in a human pre-B-cell leukemia line 697.189 Unlike resistance in the setting of mdrl overexpression, resistance was seen to drugs with many different mechanisms of action, such as alkylating agents, antimetabolites, topoisomerase inhibitors, and microtubule inhibitors. In transgenic mice expressing high levels of bcl-2, immature thymocytes were highly resistant to killing by glucocorticoids, T-irradiation, and antibodies to the T-cell receptor, all agents known to cause cell death by apoptosis. 19~ It is interesting to speculate that bcl-2 expression-associated inhibition of apoptosis was responsible for the poorer prognosis in patients whose lymphomas carried t(14;18) translocations. 189 Benz et al. 193 assessed the role of HER-2/neu in therapeutic resistance in breast cancer. They transfected full-length HER-2 cDNA into estrogen receptor-containing MCF-7 breast cancer cells. Overexpressing subclones did not differ in their sensitivity to 5-fluorouracil or to adriamycin, but had acquired 2- 4-fold resistance to CDDP and were no longer sensitive to the anti-estrogenic agent, tamoxifen. Tumors were more rapidly induced by estradiol administration in athymic ovariectomized nude mice injected with HER-2 transfected tumor cells than in those mice injected with parental control cells. Tamoxifen, which rapidly stopped the proliferation of control tumors, failed to arrest the progression of tumors derived from HER-2 transfectants. The authors suggest that this hormone-dependent, tamoxifenresistant phenotype may model therapeutic resistance seen in human breast cancers which overexpress HER-2. Finally, certain chemotherapeutic agents may actually increase the sensitivity of cells to malignant transformation in cooperation with activated oncogenes. In one model of this phenomenon, increased drug sensitivity of phenotypic revertent fibroblasts bearing activated ras or myc oncogenes, but not in parent fibroblasts, to the hypomethylating chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC), both in vitro and in an animal model, was noted. 194DNA hypomethylation has been seen in patient tumor samples, 14~but the clinical relevance of increased sensitivity to 5AzadC or to related hypomethylating agents, as well as their mechanism of action, are unknown.

X. ONCOGENES AND RADIATION RESISTANCE Radiotherapy is a mainstay of the treatment of localized solid tumors. Recent attention has been focused on the genetic changes in populations of tumor cells which accompany the development of clinical radioresistance. Previous studies

36


demonstrated that clinical radioresistance appears to be a stable, heritable phenotype which can be tested in vitro. 195-199 Further, overexpression of oncogenes such as myc and r a f w a s identified in radioresistant cells. 199'2~176 Two sentinel studies first documented the involvement of the ras oncogene in radioresistance. FitzGerald et al. 2~ first suggested the role of ras in increased radioresistance of transformed NIH3T3 cells, but the role of ras was not clearly proven, and the effect was only shown at the high dose rate of 200 cGy/min. Sklar2~ examined the changes in Dom the slope of the exponential component of the X-ray survival curve-- due to expression in NIH3T3 cells of different mutation-activated forms of ras, including c-H-ras, v-H-ras, N-ras, and v-K-ras derived from either genomic DNA or from cloned DNA transfer. All of these activated oncogenes resulted in increased Do and radioresistance. Since both cloned ras genes and genomic DNA containing activated ras genes had the same effect, it appeared that other non-ras sequences were not responsible for this phenotype. Also, overexpression of the transformationally activated c-H-ras protooncogene under the control of the Moloney virus LTR or overexpression of an unrelated oncogene, v-fms, did not result in radioresistance, demonstrating that this phenotype was not a non-specific result of transformation itself. Finally, phenotypic revertents which still carried activated ras sequences maintained their radioresistance. No obvious changes in cell cycle parameters were noted. Samid et al. 2~ demonstrated that NIH3T3 cells with c-H-ras transcriptionally activated by the LTR of Harvey murine sarcoma virus were radioresistant, as were nontumorigenic revertants which still expressed ras. C-raf-1 has been implicated in radioresistance, both by gene transfer 2~176 and in radioresistant fibroblasts derived from a patient with the Li-Fraumeni syndrome. 2~ Further, sensitivity of a radiation-resistant human squamous cell carcinoma line was found to be increased by transfection with antisense complementary DNA to c-raf- 1.2~ These results are particularly interesting given what is now known about the role of r a f i n ras signal transduction. 2~176 Miller and colleagues 2~ using radioresistant osteosarcoma cells transfected with ras elegantly demonstrated the requirement for plasma membrane association of p21 ras in maintaining the radioresistant phenotype. Treatment of EJras-carrying radioresistant human osteosarcoma cells with lovastatin restored their radiosensitivity. Lovastatin blocks the posttranscriptional modification of p21 r a s by inhibiting its isoprenylation, thereby reducing the amount of p21 ras found at the plasma membrane. Specificity of this result was shown by the ability of exogenously supplied mevalonate, the by-product of HMG-CoA reductase, to rescue radioresistant cells treated with lovastatin, by the inability of other nonselective inhibitors of cholesterol metabolism to alter radiation sensitivity, and by the lack of a response to lovastatin in parental cells or in parental cells transfected with an unrelated oncogene, met. It has also been shown previously that p21 r a s bearing amino acid substitutions at the carboxy terminal sites where isoprenylation occurs was nontransforming. 21~


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Although lovastatin at doses required to alter radiation sensitivity may be toxic, 211 these studies and others have far-reaching implications for clinical oncology practice. For example, two recent reports demonstrated the ability of farnesyltransferase inhibitors which act on a different posttranscriptional modification of p21 r a s also required for membrane targeting, to block ras-dependent transformation. 212'213 Surprisingly, there did not appear to be a deleterious effect on the cells due to alterations in normal ras function. One could speculate that such agents may find clinical utility in increasing radiosensitivity in human tumors with activated ras.

However, ras does not alter radiation resistance in all cell types. For example, Grant et al. 214 were unable to demonstrate ras-mediated radioresistance in transformed human embryonal retinal cell lines, although two of three transformants with the greatest radioresistance expressed the highest levels of p21 r a s protein. Also, Alapetite et al. 215 could not detect radioresistance in a human mammary epithelial cell line transfected with activated ras. Harris et al. 216 actually demonstrated increased radiosensitivity due to ras transformation, and suggested that decreased repair of sublethal DNA damage was due to oncogene-mediated changes in the shoulder region of the radiation dose response curve. One possible explanation for the different results reported by these investigators is that there may be a threshold of ras expression above which cells become radioresistant. 2~ Other oncogenes besides ras have been implicated in the radioresistant phenotype. For example, McKenna et al. 217 demonstrated synergy between ras and myc in conferring radioresistance on transformed rat embryo cells; this synergy was particularly notable at low doses, those most widely used in clinical practice, and affected both Do as well as the shoulder region of the radiation curve. Although myc itself had little effect on radioresistance itself in McKenna's studies, it is interesting that Carmichael et al. 218 demonstrated a similar change in the shoulder region in cell lines derived from human small-cell lung cancers which only harbored amplified myc. Increases in Do as a result of myc-only transfection have also been reported, however. 219 FitzGerald et al. 22~showed increased radioresistance only after irradiation at low dose rates (5 cGy/min) in hematopoietic stem cells 32D cl 3 transfected with v-src, v-abl, or v-erbB, and NIH3T3 cells transfected with v-abl, v-fms, v-fos, or H-ras, but not with v-src. However, in a follow-up study, Santucci et al., 221 using a temperature sensitive v-src, demonstrated in the same cell line that radiation resistance was unchanged at the permissive or nonpermissive temperature, suggesting that either the temperature-sensitive phenotype was leaky, or that something other than src may be implicated in the radiation resistance seen in the original study. In Rat-1 fibroblasts, Shimm et al. 222 also reported that activation of a temperature-sensitive v-src mutant did not alter any parameter of radioresistance, in agreement with FitzGerald's data in NIH3T3 cells. Oncogene activation, in summary, does not always confer the radioresistant phenotype, and may be a cell line-specific phenotypic change.

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The mechanism underlying radioresistance in oncogene-transformed cells is unclear. Iiiakis et al. 223 demonstrated that radioresistant v-myc- and H-ras-transformed rat embryo cells did not differ from radiosensitive parental rat embryo cells immortalized with myc in the induction of double-strand breaks or in their ability to repair such DNA damage. The transformed cells, however, did display a prolonged G2 delay in the cell cycle, suggesting that reduction in the fixation of lethal DNA damage could underlie radioresistance in these oncogene-transformed cells. 223'224Similar delays in G2 have been shown in SV40 T antigen-immortalized radioresistant human diploid fibroblasts transformed by activated H-ras. 225 The molecular mechanism of radioresistance due to G2 delay is not clear; Muschel et al. 226have shown alterations in the rate of cyclin B expression in radioresistant cells during this G2 delay. The increased activity of DNA topoisomerase II has also been shown in NIH3T3 cells beating activated raf or ras oncogenes to correlate with radioresistance, although the role of this enzyme in the repair of DNA damage due to radiation is unclear. 227

Xi. ONCOGENES AS PREDICTORS OF OUTCOME In addition to the enormous interest in the role of oncogenes in the molecular mechanisms of tumor progression, oncogenes have also become the focus of intense research into their prognostic utility in the clinical arena. N-myc amplification is a uniquely accurate marker of disease stage and outcome in neuroblastoma. 4'23 The exact molecular basis for its role in neuroblastoma is still unclear; 228'229the typical amplicon is usually larger than the genetic unit of transcription for N-myc, is transposed to random sites within the genome, and occurs in a regular pattern of clustered tandem repeats. Keim et al. 23~demonstrated that levels of PCNA (proliferating cell nuclear antigen), a component of DNA polymerase 8, were correlated with levels of N-myc amplification. PCNA levels decreased in response to treatment with the differentiation-inducing agent, retinoic acid, in vitro. N-myc amplification correlated with advanced (stage III or IV) disease, as well as with poorer prognosis in lower (stage I and II) disease which otherwise would be expected to do well clinically. Stage IV-S disease, which involves multiple organs at diagnosis, typically resolves spontaneously, with the exception of the rare IV-S tumors with N-myc amplification (see Ref. 231 for review). Growth factor-like oncogenes and their receptors have also been used as prognostic markers in human cancer. Perhaps the most widely studied are the c-erbB2 (HER-2 or neu) and int-2/hst-1 genes. Oncogenes int-2 and hst-1 are two related members of the FGF growth factor family. These oncogenes are approximately 35 kilobases apart in the human genome 232 and may be coamplified with each other or with bcl-I and prad-l, which are also found on chromosome 11q 13. 233Therefore, int-2 may be a marker for amplification of any of these oncogenes. It is, of course, possible that the amplification of a gene such as prad-1, which codes for the cell


39

cycle-associated cyclin D1 protein, TM is the mechanistically important gene in tumor progression. Prognostically, int-2 amplification itself in some cases of breast cancer correlates with large tumor size and reduced disease-free and overall survival. 235 Similarly, Borg et al. 236 found that coamplification of int-2/hst-1 correlated with shorter disease free survival in node-negative patients, although there was no correlation with tumor size. Those tumors with amplification were largely estrogen receptor-positive; the few estrogen receptor-positive patients without coamplification of int-2/hst-1 had a much better prognosis. Unlike int2/hst-1 amplification in esophageal squamous cell carcinoma, 237 which was associated with eventual metastasis, amplification of these oncogenes did not appear to be associated with distant spread, suggesting that int-2/hst-1 amplification may be an earlier genetic event in breast cancer. Amplification of HER-2/neu (or c-erbB-2), conversely, may be prognostically related to later events in tumor progression, such as lymph node metastasis, as well as to diminished overall survival in breast cancer, first reported by Slamon et al. in 1987. 238 Poor survival has also been correlated with expression of this oncogene in endometrial and ovarian cancers. 239'24~Reported studies in breast cancer, however, are conflicting. 27'241Toikkanen et al. 242reported a historical study which correlated HER-2 expression with poorer prognosis in node-positive, but not node-negative disease. Gusterson et al. 171 reported similar results, with c-erbB-2 expression prognosticating poorly for node-positive more than node-negative tumors. In addition, they showed that c-erbB-2 expression also correlated with estrogen and progesterone receptor negativity and higher tumor grade. Kallioniemi 1~ reported a similar association with hormone receptor negativity. Allred et al. 172demonstrated poorer survival in HER-2/neu expressing node-negative tumors, most prominently in patients with small, estrogen receptor-positive tumors" a similar survival-reducing effect of c-erbB-2 expression in estrogen receptor positive patients was also shown by Wright et al. 243 Schroeter et al. 244 have suggested that some of these conflicting results may relate to the length of follow-up; in short follow-up, c-erbB-2 expression was correlated with metastatic outcome. For example, Narita et al. 245 correlated metastasis with increased serum levels of c-erbB-2 protein, whereas in longer follow-up, as in several other published studies (e.g., Ref. 246), the prognostic significance diminished. Several investigators have suggested that c-erbB-2 may be a better prognostic marker when combined with other genetic markers. For example, coexpression of c-erbB-2 with the EGF receptor was found to be a marker of very poor prognosis by Osaki and colleagues, 247 although the data suggests that they represent different biologic characteristics of the tumors when examined independently. Babiak et al. 248 found that the combination of amplification of c-erbB-2 and DNA aneuploidy were predictive of poorer survival in patients with node negative cancer, although the number of patients studied was small. Similar findings of coordinate amplification of c-erbB-2 and DNA aneuploidy and its association with worse prognosis have been described in gastric carcinomas. 1~ Schimmelpenning et al. 249 demon-

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strated, in fact, that the DNA ploidy was more prognostically significant in all breast cancer patients than was expression of c-erbB-2, with diploid tumors fating better prognostically than aneuploid tumors. It is interesting to note in breast cancer that amplification of HER-2, located on chromosome 17q, is also correlated with loss of heterozygosity at 17p, the location of the tumor suppressor gene p53. This suggests that HER-2 amplification may be prognostically significant because it reflects other genetic changes on 17p which may be central to breast carcinogenesis. 250 The expression of c-myc has also been used as a prognostic marker. RouxDosseto et al. TM reported that c-myc overexpression independently correlated with a high rate of early relapse in node-negative breast cancer. A similar finding in both node-negative and node-positive patients was described by Borg 252 and Pertschuk, 253 but not by Locker. 254 The correlation between c-myc expression and prognosis has also been described in early invasive cervical carcinoma. 97 In squamous cell carcinoma of the lung, expression was not correlated with clinical parameters. 255 Finally, ras mutations, which occur frequently in many human cancers, 6 have prognostic significance in a number of different neoplasms. Overall levels of p21 r a s expression are a prognostic marker of poor survival in colon cancer; 256 this association, like that of HER-2 and aneuploidy in breast and gastric carcinomas was strengthened when combined with DNA ploidy and S-phase fraction, other putative markers of cell proliferation. The ras mutations are distributed differently according to the tumor origin. For example, lung and colon adenocarcinomas are characterized by K-ras mutations, while N-ras mutations predominate in certain hematopoietic neoplasms. 257'258The poor prognostic significance of ras mutations is most clearly shown in pulmonary adenocarcinomas. In surgically resected early stage lung adenocarcinomas, a ras mutation was the strongest unfavorable prognostic sign. 259 In a more recent study of similar patients, a ras mutation did not correlate with tumor size, lymph node invasion, or metastasis, yet was still associated with a poorer overall survival. 26~Nishio et al. 261 reported similar findings in adenocarcinomas, but not in squamous cell, large cell, or small cell lung carcinomas. Rodenhuis and Slebos 258also noted diminished survival in patients with K-ras mutations in lung adenocarcinomas; it is interesting to note that codon 12 K-ras mutations were typical of such tumors in smokers. In colorectal adenocarcinoma, K-ras mutations are also frequent findings. The prevalence of mutations in K-ras codons 12 and 13 was 25% and 71% for nonrecurring and recurring Dukes B or C colon cancers, respectively. 262Mutations other than the usual GGT to TAT occurred almost exclusively in patients with recurrent disease. Thus, alterations in oncogenes, including overexpression and mutation, are beginning to be correlated with prognostic features in cancer.


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Xll. CONCLUSIONS The activation of oncogenes leads to many of the phenotypic alterations in cancer cells which are associated with tumor progression, including increased growth, angiogenesis, invasion and metastasis, altered cell adhesion, evasion of the host immune response, and the development of drug and radiation resistance. It is not surprising, therefore, that activated oncogenes may be useful prognostic markers in the clinical arena. Gains in understanding the molecular mechanisms of oncogene activation and their involvement in tumor progression has been exponential in the last several years. Only with detailed understanding of the mechanisms of the genetic changes resulting in malignant disease can new strides in the design of cancer therapies be made.

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243. Wright, C.; Nicholson, S.; Angus, B., et al. Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Br. J. Cancer 1992, 65, 118-121. 244. Schroeter, C. A.; De Potter, C. R.; Rathsmann, K.; Willighagen, R. G. J.; Greep, J. C. C-erbB-2 positive breast tumours behave more aggressively in the first years after diagnosis. Br. J. Cancer 1992, 66, 728-734. 245. Narita, T.; Funahashi, H.; Satoh, Y.; Takagi, H. C-erbB-2 protein in the sera of breast cancer patients. Breast Cancer Res. Treat. 1992, 24, 97-102. 246. Bianchi, S.; Paglierani, M.; Zampi, G., et al. Prognostic significance of c-erbB-2 expression in node negative breast cancer. Bl: J. Cancer 1993, 67, 625-629. 247. Osaki, A.; Toi, M.; Yamada, H.; Kawami, H.; Kuroi, K.; Toge, T. Prognostic significance of co-expression of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer patients. Am. J. Surg. 1992, 164, 323-326. 248. Babiak, J.; Hugh, J.; Poppema, S. Significance of c-erbB-2 amplification and DNA aneupioidy. Cancer 1992, 70, 770-776. 249. Schimmelpenning, H.; Eriksson, E. T.; Faikmer, et al. Prognostic significance of immunohistochemical c-erbB-2 protooncogene expression and nuclear DNA content in human breast cancer. Eur. J. Surg. Oncoi. 1992, 18, 530-537. 250. Knyazev, P. G.; Imyanitov, E. N.; Chernitsa, O. I.; Nikiforova, I. E Loss of heterozygosity at chromosome 17p is associated with HER-2 amplification and lack of nodal involvement in breast cancer. Int. J. Cancer 1993, 53, 11-16. 251. Roux-Dossetto, M.; Romain, S.; Dussault, N., et al. C-myc gene amplification in selected node-negative breast cancer patients correlates with high rate of early relapse. Eur. J. Cancer 1992, 28A, 1600-1604. 252. Borg, A.; Baldetorp, B.; Ferno, M.; Olsson, H.; Sigurdsson, H. C-myc amplification is an independent prognostic factor in postmenopausal breast cancer, hit. J. Cancer 1992, 51,687-691. 253. Pertschuk, L. P.; Feldman, J. G.; Kim, D. S., et al. Steroid hormone receptor immunohistochemistry and amplification of c-myc protooncogene. Relationship to disease-free survival in breast cancer. Cancer 1993, 71, 162-171. 254. Locker, A. P.; Dowle, C. S.; Ellis, I. O., et al. C-myc oncogene product expression and prognosis in operable breast cancer. Br. J. Cancer 1989, 60, 669-672. 255. Volm, M.; Efferth, T.; Mattern, J. Oncoprotein (c-myc, c-erbB 1, c-erbB2, c-fos) and suppressor gene product (p53) expression in squamous cell carcinomas of the lung. Clinical and biological correlations. Anticancer Res. 1992, 12, 11-21. 256. Sun, X.; Wingren, S.; Carstensen, J. M., et al. Ras p21 expression in relation to DNA ploidy, S-phase fraction, and prognosis in colorectal adenocarcinoma. Eur. J. Cancer 1991, 27, 16461649. 257. Rodenhuis, S. Ras and human tumors. Semin. Cancer Biol. 1992, 3, 241-247. 258. Rodenhuis, S.; Slebos, R. J. C. Clinical significance of ras oncogene activation in human lung cancer. Cancer Res. 1992, Supp152, 2665s-2669s. 259. Slebos, R. J. C.; Kibbelaar, R. E.; Dalesio, O., et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N. EngL J. Med. 1990, 323, 561-565. 260. Sugio, K.; Ishida, T.; Yokoyama, H.; lnoue, T.; Sugimachi, K.; Sasazuki, T. Ras gene mutations as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res. 1992, 52, 2903-2906. 261. Nishio, H.; Nakamura, S.; Horai, T.; Ikegami, H.; Matsuda, M. Clinical and histopathologic evaluation of the expression of Ha-ras andfes oncogene products in lung cancer. Cancer 1992, 69, 1130-1136. 262. Benhatter, J.; Losi, L.; Chuabert, P.; Givel, J.-C.; Costa, J. Prognostic significance of K-ras mutations in colorectal cancer. Gastroenterology 1993, 104, 1044-1048.


THE

p53 TUMOR SUPPRESSOR GENE

Thierry Soussi

I. II.

Introduction

...................................

56

p53 Protein and D N A T u m o r Viruses . . . . . . . . . . . . . . . . . . . . . . A~ SV40 and Polyoma Virus . . . . . . . . . . . . . . . . . . . . . . . . . .

58 58

SV40 Large-T Antigen and p53 Protein . . . . . . . . . . . . . . . . . . Adenoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenovirus El B Protein and p53 . . . . . . . . . . . . . . . . . . . . . . Papillomavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Papilloma Virus E6 Protein and p53 . . . . . . . . . . . . . . . . . . . . p53, RB, and DNA Tumor Virus Oncogene Products . . . . . . . . . . . p53 Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p53 cDNA Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. p53 Gene Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . p53 Localization in the Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p53 Distribution in Normal and Transformed Cell . . . . . . . . . . . . . B. Nuclear Localization Signal in p53 Protein . . . . . . . . . . . . . . . . The p53 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p53 Protein Organization . . . . . . . . . . . . . . . . . . . . . . . . . . B. p53 Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. p53 Interaction with SV40 Large-T Antigen . . . . . . . . . . . . . . . .

60 61 62 63 64 64 66 66 68 71 71 72 74 74 75 78

B.

III.

IV.

C. D. E. E G. The A.

Advances in Genome Biology Volume 3A, pages 55-141. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8 55

.

. .

56

VI.

VII.

VIII. IX. X.

XI.

THIERRY SOUSSI D. p53 Interaction with Adenovirus El B Protein . . . . . . . . . . . . . . . E. p53 Interaction with E6 Papilloma Virus Protein . . . . . . . . . . . . . . F. p53 Interaction with hsp70 . . . . . . . . . . . . . . . . . . . . . . . . . G. p53 Interaction with p53 . . . . . . . . . . . . . . . . . . . . . . . . . . H. p53 Interaction with Mdm-2 . . . . . . . . . . . . . . . . . . . . . . . . p53 Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p53 as a DNA Binding Protein . . . . . . . . . . . . . . . . . . . . . . . B. p53 as a Sequence-Specific DNA Binding Protein . . . . . . . . . . . . . C. p53 as a Transactivating Protein via a DNA Binding Activity . . . . . . . D. p53 as a Transactivating Protein via Protein Binding . . . . . . . . . . . E. Cellular Genes Regulated by Wild-Type p53 . . . . . . . . . . . . . . . . p53 and the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p53 Expression during the Cell Cycle . . . . . . . . . . . . . . . . . . . B. Wild-Type p53 is Antiproliferative . . . . . . . . . . . . . . . . . . . . . C. p53 Adopt Distinct Conformational States . . . . . . . . . . . . . . . . . D. Wild Type and Mutant p53 . . . . . . . . . . . . . . . . . . . . . . . . . E. p53 and the Cellular Response to DNA Damage: A Final Model for p53 Function? . . . . . . . . . . . . . . . . . . . . . . p53 in Differentiation and Embryogenesis . . . . . . . . . . . . . . . . . . . p53 as a Tumor Suppressor Gene . . . . . . . . . . . . . . . . . . . . . . . . p53 Alteration in Human Cancer . . . . . . . . . . . . . . . . . . . . . . . . . A. Frequency of the p53 Mutations in Human Cancer . . . . . . . . . . . . B. Distribution of p53 Mutations in tlae Molecule . . . . . . . . . . . . . . . C. Mutational Events, p53 Mutations, and Cancer Types . . . . . . . . . . . D. Immunohistochemical Analysis of p53 Accumulation in Tumor Cells E. Serological Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Updated Material Added in Proofs . . . . . . . . . . . . . . . . . . . . . . .

i.

79 79 80 81 81 83 83 83 84 84 85 86 86 86 90 90 92 94 95 96 96 102 103 . . 103 104 115 116 117 135

INTRODUCTION

The discovery that certain viruses are able to transform cells in vitro and to induce tumors in rodents has led to extensive efforts to identify the gene(s) involved in the transforming process. In contrast to the majority of oncogenic R N A retroviruses, which carry modified cellular genes, D N A tumor viruses (papovaviruses, papillomaviruses, adenoviruses, herpesviruses, and hepadnaviruses) carry their own specific genetic information which is responsible for their ability to transform cells. 1-3 S o m e of these viruses (papovavirus, adenovirus) have been shown to have a high oncogenic potential in animals in appropriate and often specialized experimental circumstances, but they have not been associated with human cancers, while others (papillomaviruses, herpesviruses, hepadnaviruses) have been found to be associated with human neoplasias (Table 1). Indeed, genes that encode protein products

Table 1. Viruses

DNA Tumor Virus O n c o g e n e s

Oncogene

Function

Location

In Vitro Properties

Mode of Function

Linkage to Human Cancer

SV40

Large T antigen

Nucleus

Immortalizing and transforming

Small T antigen Large T antigen

Complex p53 and p 105-RB ?

No

Polyoma

Transcription(+/-) DNA synthesis 9 Transcription (+/-) DNA synthesis

Immortalizing

Complex c-src

No

Transforming

Complex p 105-RB

No

Immortalizing

Complex p53

Middle T antigen

Adenoviruses

Papillomaviruses (HPV 16 and 18) Epstein-Barr Virus

Inner plasma membrane Cytoplasm Nucleus

Transforming

Small T antigen E 1A(26 kDa) EIA(36 kDa) E1B(55 kDa) EIB(19 kDa) E6

9 Transcription (+ ou -) mRNA transport Transcription

Nuclear

Immortalizing

Complex p53

E7 EBNA 1 and 2 ?

Transcription 9

Nuclear Nuclear

Transforming

Complex p 105-RB

LMP ?

Plasma membrane Nuclear

EBNA 5

Hepatitis B virus

Cytoplasm Nucleus

X protein ?

9

?

Cervical cancer Burkitt's lymphoma Nasopharyngeal cancer

Complex p 105-Rb and p53 Complex p53

Hepatocellular carcinoma

58

THIERRY SOUSSI

actively participating in the cell transformation process have been identified for papovaviruses, adenoviruses, and papillomaviruses. The most exciting outcome of these studies was the identification of cellular proteins which interact specifically with such viral oncoproteins. The first to be identified was the p53 protein, which binds to simian virus 40 (SV40) large-T antigen, to adenovirus E1B protein, and to human papilloma virus (HPV) E6 protein. The second to be identified was the retinoblastoma gene (rbl) product (p110-RB), which binds to SV40 large-T antigen, adenovirus E1A protein, and HPV E7 protein. Such interactions are thought to be involved in the transforming behavior of these viruses. The importance of the p53 and rbl genes and their encoded product is reinforced by the discovery that both genes can be altered in a wide variety of human neoplasias in the absence of any virus. Since the history of p53 is closely linked with that of small DNA tumor viruses, a brief description of several viruses is necessary to fully appreciate its behavior in normal and transformed cells.

Ii. p53 A N D DNA TUMOR VIRUSES A. SV40 and PolyomaVirus SV40 and polyoma are among the smallest viruses known. SV40 was originally isolated from cultures of rhesus monkey kidney, the permissive host of the virus. 1 Polyoma virus was detected as a contaminant in cell-free extracts of tissues used for the transmission of murine leukemia. Polyoma virus can be propagated in mouse cells. Both virus genomes are constituted of a double-stranded DNA molecule which is covalently closed, circular, and supercoiled (Figure 1). In their respective permissive cells, both viruses direct an ordered sequence of events leading from the early phase prior to viral DNA replication to late phases of infection in which viral progeny are produced in large numbers. The early phase of infection is centered upon subverting cellular control mechanisms to prepare the cell for viral DNA replication by stimulation of cellular transcription and induction of quiescent cells to synthesize cellular DNA (induction of S phase). SV40 encodes two early protein products, SV40 large-T antigen (90 kDa) and small-t antigen (17 kDa), whereas polyomavirus encodes three protein products, polyoma large-T antigen (90 kDa), polyoma middle-T antigen (55 kDa), and polyoma small-t antigen (17 kDa) (Figure 1 and Table 1). SV40 large-T antigen is involved in several functions, including stimulation of cellular and viral transcription, initiation of viral DNA replication, and the switch leading to the late phase, with synthesis of the three capsid proteins (VP 1, VP2, and VP3) and massive synthesis of the virus, coupled with cell death. 4-6 Cells in which the late phase of viral infection follows the early phase are said to be permissive. In such cells (monkey for SV40 and mouse for polyoma), lytic infection occurs.

The p53 Tumor Suppressor Gene

59

Figure 1. Organization of the three DNA tumor viruses. The three maps have been simplified for clarity and only relevant genes have been described.

In infected cells from many species, the induction of the S phase does not lead to viral replication, probably due to incompatibility of cellular and viral processes. In such nonpermissive cells, there is no switch to late phase and no cell death. Nevertheless, as long as the expression of the early viral gene continues, the infected cells cannot rest in GO because they are continually induced to proceed through the cell cycle. Mostly, the viral genome is lost by degradation or during cell division and the cell reverts to normal. This process is termed "abortive transformation". In some cases, the viral genome can integrate the genome of the nonpermissive cell. This process led to malignant transformation of the cell. It should be stressed that transforming or tumorigenic properties of SV40 and polyoma virus are not a natural feature. They occur only in laboratory experiments using specific conditions. SV40 large-T antigen is both necessary and sufficient for transformation of rat embryo fibroblast (REF). For polyoma virus, only the cooperation between large-T antigen and middle-T antigen is able to fully transform REF, whereas large-T antigen alone leads only to immortalization of such cells. 7 These experiments, associated with other observations concerning adenovirus or papillomaviruses proteins or cellular oncogenes, have led to the notion that transformation is a multistep mechanism necessitating several steps in order to produce a fully transformed phenotype. Two classes of viral and cellular oncogenes have been recog-

60

THIERRY SOUSSI

nized: the class of immortalizing genes which includes adenovirus E 1A, polyoma large-T, mutated p53 and myc, and the class of transforming genes which includes adenovirus E1B, polyoma middle-T, and ras. 8'9 One product from each category is required to obtain a fully transformed phenotype of rat (or mouse) embryo fibroblast. Nevertheless, it is not known whether these functional similarities reflect similar biochemical mechanisms. The only exception is SV40 large-T antigen, which is able to fully transform REF, but here again, other cellular events are almost certainly necessary for transformation. Careful genetic dissection of SV40 large-T antigen enables us to distinguish between distinct regions of the molecule which are involved in immortalization or transformation. One of these domains corresponds to the first 120 amino acids of the protein. It is sufficient to immortalize REF in culture. A second domain, localized between amino acid 272 and 625, is required for the transformation of REF cells in culture, l~ The exact contribution of these viral oncogenes to transformation is unknown, but there is now clear evidence that other events at the cellular level are also necessary.

B. SV40 Large-T Antigen and p53 Protein Studies of SV40-transformed cells show that a 55-kDa protein is coprecipitated with the large-T antigen. 12'13 This association was shown to be the result of an in vivo association between the two proteins. ~2 It was then postulated that this protein could be encoded by the cellular genome. (It should be kept in mind that no middle-T antigen was found for SV40 and that the molecular mass of this protein was similar to that of polyoma middle-T antigen). Linzer and Levine 13 found that the 54-kDa protein was overexpressed in a wide variety of murine SV40 transformed cells, but also in uninfected embryonic carcinoma cells. A partial peptide map from this 54-kDa protein was identical among the different cell lines, but was clearly different from the peptide map of SV40 large-T antigen. 13 It was then postulated that SV40 infection or transformation of mouse cells stimulates the synthesis or stability of a cellular 54-kDa protein. When monoclonal antibodies (mAb) became available, it was found that elevated levels of the 54-kDa protein were present in a wide variety of transformed and tumor cells from different species, regardless of the transforming agent. 14'15'16 The apparent molecular mass of this protein ranged between 50 and 55 kDa, depending on both the species and the gel system used in different laboratories. Several groups working on this protein agreed to use p53 as the name for this 53-kDa phosphoprotein, which binds to SV40 large-T antigen overexpressed in transformed cells. 17 p53 accumulation in transformed cells may be the result of transcriptional, posttranscriptional, translational, or posttranslational control. In a comparison of 3T3 and SV3T3 cells, Oren et al. 18 showed that levels of translatable mRNA were the same in both cell types. By pulse chase analysis, these authors showed that SV40-transformed cells contain stabilized p53 protein. In 3T3 cells, the p53


61

half-life is less than 90 min, whereas this protein is stable over a 24-hour period in SV3T3 cells. As a result of this stabilization, SV40-transformed cells contained p53 levels which are 100- to 1000-fold higher than those of normal cells. A similar observation was made in other virally transformed cells, methylcholanthrene-transformed cells, and tumor cells. 19 It is currently accepted that p53 accumulation is the result of the stabilization of the protein without any gross variation at the transcriptional level. An increase in p53 mRNA in tumor cells is generally due to the increased number of cycling cells frequently found in neoplastic tissue. 2~ Undifferentiated F9 embryonal carcinoma cells and murine erythroleukemia cells have a more unusual behavior than SV40-transformed cells. F9 cells contain high levels of p53 protein, but when they are induced toward differentiation by treatment with retinoic acid, there is a marked decrease in the p53 protein. 21 There is no alteration in turnover or stability of p53 in F9 cells relative to their differentiated progeny. However, there is a marked decrease in p53 mRNA in the differentiated cell culture, suggesting some other mechanism. Examination of the transcription rate of the p53 gene through this differentiation process indicates that it is regulated by a posttranscriptional control. 22 Similar results were obtained in murine erythroleukemia cells when they were induced to differentiate by hexamethylene bisacetamide. 23'24Careful analysis indicated that the stability of the p53 protein was not affected during differentiation, whereas the level of the protein decreased 2 hours after the input of the inducer, reaching a basal level of about 30% of the starting value. 24 This drop was parallel to a decrease in the corresponding RNA, but the rate of transcription was not affected, also suggesting posttranscriptional control for RNA expression. The main difference between these two systems was in the nature ofp53, p53 is of a wild type in F9 cells, whereas it is mutant in murine erythroleukemia cells.

C. Adenoviruses Adenoviruses have been found in a wide variety of species including human, simian, bovine, canine, murine, and avian. In humans, adenoviruses cause acute infections of the upper respiratory and intestinal tracts, but do not seem to be associated with neoplastic processes. In contrast to that ofpapovaviruses, the genome of adenoviruses is a linear duplex of DNA molecules (35-45 kb) that codes for at least 20-30 polypeptides (see Figure 1 and Table 1). Human adenoviruses have been classified according to their potential to induce tumors in hamsters: class A viruses (e.g., Adl2) are highly oncogenic, class B viruses (e.g., Ad7) are weakly oncogenic, and class C viruses (e.g., Ad2 and Ad5) are nononcogenic. Nevertheless, viruses from all three classes are able to transform primary rodent cells in tissue culture. Functions required for transformation and tumorigenicity are encoded by an early region (El), one of the viral regions expressed early in lytic infection. This region

62

THIERRY SOUSSI

is located within the left 11.5% of the viral genome and consists of two transcription units, E1A and E1B. The El A region encodes two major mRNA which are referred to their sedimentation values of 13S and 12S. These two RNAs arise from differential splicing of a common precursor. Related peptides of 289 and 243 amino acids are synthesized from the 13S and 12S mRNA species. Among the described activities of E 1A is the ability to regulate transcription from a wide variety of promoters. 25 The E1B region encodes one major (2.2 kb) and four minor transcripts. The major mRNA encodes two unrelated proteins: the small E 1B protein (Ad 12:19 kDa, Ad5: 21 kDa), and the large E1B protein (Adl 2:54 kDa, Ad5:55 kDa). The function of the small E1B protein resides in its ability to protect the cell against DNA degradation after viral infection, whereas the large E1B protein seems to be involved in the transport and accumulation of viral mRNA. Expression of the E 1A region alone can lead to immortalization of primary rodent cells. 26 However, efficient and complete morphological transformation requires expression of the E1B region as well. 27 This E1B activity could be replaced by a mutated ras oncogene, which led to the classification of E1A as a myc-like nuclear oncogene. Similarly, E1A can be replaced by the polyoma large-T antigen, by members of the myc family, or by a mutated form of the p53 gene.

D. Adenovirus EIB Protein and p53 The p53 interaction with the large EIB protein is more complicated than with SV40 large-T antigen or with the E6 protein. The first observation of a p53 interaction with the E1B protein was made by Sarnow et al. in 1982. 28 Fortunately, those authors used a cell line transformed by the weakly oncogenic adenoviruses 2 and 5. They showed, by immunoprecipitation and proteolysis peptide mapping, that the 54-kDa protein associated with SV40 antigen was identical to those associated with Ad2 E lB. Further work from Zentema et al. 29 showed that p53 did not complex with the Adl2 large E1B protein and that the adenovirus serotype determines the localization of p53. In transformed cells expressing the Adl2 large E1B, the lack of association led to nuclear accumulation of p53 in the nucleus of the cell, whereas in transformed cells expressing the Ad5 large E 1B protein, p53 was associated with the viral protein in a cytoplasmic body which consisted of a cluster of 8-nm filaments. 3~ It was also shown that, in both types of cell line, p53 is strongly stabilized. This observation is rather paradoxical since it shows that E1B from the weakly oncogenic adenovirus interacts with p53, whereas p53 does not interact with E1B from the highly oncogenic form. Furthermore, it suggests that interaction with the viral protein is not necessary for stabilization of p53, as had been suggested for SV40 large-T antigen. A clue to this observation is found in the work of Van den Hevel et al., who showed that the oncogenicity of Ad5 is related to the level of free nuclear p53. 31'32


63

Fisher rat embryo cell line 3Y 1 could be transformed with Ad5, and a panel of Ad5-transformed 3Y 1 cells with varying E1B expression was established. In cells with high levels of E1B, all endogenous p53 was sequestered in the inactive cytoplasmic body. These cells form tumors only in nude mice after a very long latency period, and in the tumors that appear, selection has occurred in favor of cells lacking the complex and containing free nuclear p5 3. 33Moreover, Ad5-transformed 3Y1 cells which express low levels of E1B have a nuclear p53 and are highly oncogenic in nude mice. In another set of experiments, the authors showed that high expression of Ad5 large-E1B protein in Ad 12-transformed cells led to the accumulation of p53 in the cytoplasmic body and to a loss of oncogenicity. This indicates that it is not the nature of the EIB protein per se, but rather the level of free nuclear p5 3 which determines oncogenicity in nude mice, suggesting that stable p53 induces a dominant phenotype in adenovirus-transformed cells.

E. Papillomavirus Since Shope's pioneering work concerning rabbit papilloma virus in 1933, similar viruses have been isolated from different species, including humans. Papillomavirus DNA is a double-stranded, covalently closed, circular, supercoiled molecule which does not bear any sequence homology with either polyoma or SV40 genome (see Figure 1 and Table 1). More than 50 distinct human papilloma viruses (HPV) have been described, with each sharing a similar virion structure and genomic organization. A strong association exists between two groups of HPV and some anogenital cancers, including cervical cancer. 34'35 The first group, including HPV-6 and HPV- 11, is generally associated with benign anogenital warts that only rarely progress to cancer, and has been referred to as a "low-risk" virus group. The second group of "high-risk" viruses, which includes HPV-16 and HPV-18, is associated with lesions that have a strong tendency toward malignant progression. This is demonstrated by the ability of cloned viral genomes derived from the high-risk but not the low-risk HPV to transform cells in culture, suggesting that these HPV types have an etiologic role in such tumors. Among the seven early genes encoded by HPV, two of them, E6 and E7, have been shown to be involved in this transforming process. 35 E7 alone is sufficient for transformation of established rodent cell lines, and can transform primary rat kidney cells in cooperation with an activated ras gene. Both E6 and E7 are necessary and sufficient for efficient immortalization of the natural host cells of HPV, human squamous epithelial cells. 36'37 E7 proteins of HPV are acidic nuclear phosphoproteins 100 amino acids in length. They possess transcriptional modulatory and transformation properties of adenovirus E 1A. 38 E6 proteins are 150 amino acids in length and contain four CXXC motifs which may be involved in the zinc-binding property of the proteins. 39 Thus far, little is

64

THIERRY SOUSSI

known about their biochemical properties. BPV- 1 E6, HPV- 16 E6, and HPV- 18 E6 are reported to have transcriptional transactivating properties.

F. Papilloma Virus E6 Protein and p53 Studies performed on adenovirus E1B protein and the SV40 large-T antigen prompted some investigators to attempt to elucidate the behavior of the E6 proteins with the p53 protein. Werness et al.4~ have demonstrated that the E6 proteins of high-risk HPV types, but not low-risk HPV types, could associate in vitro with human p53 (see also later for E6-p53 interaction). Furthermore, it was demonstrated that this binding stimulates the degradation of p53 in vitro. This E6-promoted degradation of p53 is ATP-dependent and involves the ubiquitin, dependent protease system. 41 Analysis of p53 status in vivo shows that p53 levels are generally reduced in cell lines or in tumor cells expressing E6 and E7 protein, but numerous HPV cell lines retain significant amounts of p53. 42 Pulse-chase analysis shows that the p53 protein has decreased stability in the HPV cell line. 42 Hela cells (containing an HPV genome integrated into the host chromosome), which have been known for some time to have translated wild-type p53 mRNA and no detectable p53, 43 have recently been shown to contain a very low level of p5 3.

G. p53, RB, and DNA Tumor Virus Oncogene Products Another class of genes involved in tumorigenesis but unrelated to any viral protein is that of the tumor-suppressor genes. Inactivation of these genes has been implicated as a causal event in the generation of numerous types of human tumors. It appears that when both copies of a tumor-suppressing gene are inactivated, cells initiate uncontrolled growth. The best studied tumor suppressor gene is rbl, the inactivation of which favors the appearance of retinoblastomas, osteosarcomas, and certain soft tissue carcinomas 44 for review. The p 110-RB is a nuclear phosphoprotein of 110 kDa. Extensive work on the p 110-RB indicates that it plays a key role in cellular proliferation by regulating transcription of genes required for a cell to enter into or remain in a quiescent state, or for progression through the G 1 phase of the cell cycle45 for review. The main surprise was the finding that the p 110-RB binds to viral-transforming proteins such as SV40 large-T antigen, 46 adenovirus E1A protein, 47 and HPV 16 E7 protein 48 (Figure 2). A careful examination of the regions involved in these interactions indicates that: The three viral proteins bind to the same regions of the p 110-RB, which consists of two noncontiguous domains (aa 393 to 572 and 646 to 772, respectively). These binding sites on p 110-RB overlap with the position of naturally occurring inactivating mutations of the rbl gene.

The p53 Tumor SuppressorGene

65

Figure 2. Complexes between the oncogenes of DNA tumor viruses and cellular tumor suppressor genes. The SV40 large-T antigen binds both pl05-RB and p53. In adenoviruses and papiliomaviruses, the binding activities are on separate polypeptides. The binding domain of the DNA tumor virus oncoprotein for pl05-RB and p53 lies within a domain involved in their oncogenic process, except for E6 and p53. Black boxes correspond to protein domains involved in transforming or immortalizing properties and shaded boxes represent the region involved in binding to pl05-RB or p53. 2.

3.

The p110-RB protein binds to regions of the viral proteins which are homologous to the three proteins. These regions have been shown to be essential for their transforming properties including cooperation with an activated r a s gene and stimulation of DNA synthesis. The p 110-RB binding region of SV40 large-T antigen is quite different from those involved in p53 binding, and reflects a different structural region of SV40 large-T antigen, p53 binds to a domain involved in immortalization of the viral protein (Figure 2).

Taken together, these results indicate that viral oncogene products act through p 110-RB and p53 tumor suppressor gene products to promote cell growth. Naturally, this process leads to stimulation of cell division after virus infection since it

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THIERRY SOUSSI

generally requires an actively replicating host cell. Certain functions in viruses have evolved to repress negative regulators of cellular proliferation that prevent cell replication in order to promote cell growth and maximize virus production. Under laboratory conditions, using high multiplicity of infection and semi- or nonpermissive cells, a viral genome can integrate the host cell DNA, leading to constitutive synthesis of the viral antigen and continuous cell growth. For adenoviruses or papilloma viruses, two viral proteins are necessary to ensure this process, whereas in SV40 the large-T antigen combines both activities. Recently, it has been shown that the hepatitis Bx antigen and the EBV EBNA-5 protein binds to p53. 49'50 Furthermore, the EBNA-5 was also shown to associate with the p 110-RB. 5~ The significance of these interactions is not known actually, but it is tempting to speculate that transformation process which involve these two viruses may also include impairment of p 110-RB and p53 function. All these experiments using independent oncogene and tumor suppressor genes have strengthened the notion that cancer arises via a multistep process which requires activation and inactivation of a multiple set of cellular genes.

III.

THE

p53 GENE

A. p53 cDNA Cloning In 1983, cloning a complementary DNA (cDNA) corresponding to an unknown protein was a very difficult task, even with the help of numerous mAbs. The strategy used for cloning of mouse p53 involved immunoselection of p53 mRNA by immunoprecipitation of polysomes with p53 mAbs. The enriched mRNA fraction was used as a probe either for library screening or for library construction. In both approaches, short cDNA clones were obtained. 51-54 They were shown to contain p53 specific sequences by a hybrid selection assay. Longer cDNA and genomic clones were further isolated using more conventional approaches with the first cDNA as a probe. 55-57 A mouse probe was also used for cloning human p53 cDNA 58-6~and gene. 61'62 Both mouse and human genomes contain a single copy of a functional p53 gene per haploid genome, where it is located on chromosome 11, and the short arm of chromosome 17,63'64 respectively. This result is in agreement with the synthenic relationship already established for several other genes on human chromosome 17, and their corresponding homologs on mouse chromosome 11.65The mouse genome also contains an inactive processed pseudogene on chromosome 14. Using various DNA probes, evolutionary studies of p53 have been performed. p53 cDNAhave been cloned and sequenced in all vertebrates tested so far, including monkeys, 66 rats, 67 hamsters, 68 chickens, 69 X. laevis, 7~ and rainbow trout. 71 However, p53 has never been detected in invertebrates such as Drosophila, the sea urchin, or in yeast72 (Table 2).

Table 2. Species

Probe

Characteristics of p53 from Various Speciesa

Library Used for Screening

Mouse

Monoclonal antibody

Transformed cells

Human X. laevis Rat

Mouse p53 complete cDNA Mouse p53 coding sequence Mouse p53 complete cDNA

Transformed cells Total oocytes Transformed cells

Chicken Monkey Hamster R. trout

Spleen cells Mouse p53 complete cDNA Transformed cells Human p53 complete cDNA Transformed cells Mouse p53 complete cDNA Spleen cells X. laevis p53 domains IV and V

Gene Structure

Chromosome Localization

mRNA Size

Protein Size

References

12kb, 11 exons 1 pseudogene 20 kb, 11 exons 18 kb, 11 exons 12 kb, 10 exons, 1 pseudogene NA b

11

2.0kb

53 kDa (43 kDa)

51

17p13 NA NA

2.8 kb 2.2 and 3 kb 2.0 kb

55 kDa (44 kDa) 46 kDa (41 kDa) 54 kDa (43 kDa)

58 70 67,340

NA

1.8 kb

50 kDa (40 kDa)

69

NA NA NA

NA NA NA

NA NA 2.4 kb

55 kDa (44 kDa) 43 kDa (56 kDa) 57 kDa (44 kDa)

66 68 71

Notes: a p53 protein displays an abnormal migration in SDS-polyacrylamidegel electrophoresis leading to an apparent molecular weight higher than the theoretical value deduced from the sequence of the protein (modified from72). bNA: information not available.

68

THIERRY SOUSSI

B. p53 Gene Organization p53 genes from three species (human, mouse, and X. laevis) have been cloned and sequenced. 57'61 The genes contain 11 exons interrupted by 10 introns (Figure 3). Although the introns are variable in length, they interrupt the exons at precise homologous positions, except for a small segment of the least conserved exon 2 in X. laevis. 72 In X. laevis, there are two active p53 genes (genes A and B). These findings are not unexpected, since its ancestor, X. tropicalis, duplicated its genome 30 million years ago resulting in several Xenopus species, such as X. laevis that are tetraploid. In every p53 gene analyzed thus far, there are three striking features which may be summarized as follows: 1. Unusual transcriptional promoter. The promoter region does not contain any of the consensus sequences found in most eukaryotic promoters such as CAAT

Figure 3. Human p53 transcriptional unit. The human p53 gene contains 11 exons with an ATG start in the second exon. Human gene contain a second promoter (P2) localized in intron 1. The 3' untranslated region of the human p53 mRNA contains an Alu sequence. From the primary sequence, it is predicted that p53 contains an acidic helical amino-terminus with a high number of proline residues followed by a hydophobic central part and ending with a basic helix-coil-helix carboxy-terminus. All the p53 proteins studied thus far have a similar structure. Human, mouse, and X. laevis p53 genes are also highly similar.


69

box, TATA box, and G/C-rich sequences. This feature applies to mouse, human, rat, 57'61 and X. laevisp53 (Soussi et al., unpublished results). In the mouse promoter, a negative element has been detected, but the same region of human p53 is active. Reismann et al. have shown that there is a second promoter located at the beginning of intron I of the human p53 gene 1000 bp downstream from exon I. 73'74 This promoter was shown to be differentially regulated during terminal differentiation of the human promyelocytic leukemia cell line HL60. The product of this promotor-initiated transcript has not been characterized. Interestingly, it has been reported that human intron I is rearranged in several osteogenic sarcomas. Two protein-binding elements have been described in the 5' region of the murine p53 gene and one downstream of the transcription site. 75 The first element, the PF1 site, bears a strong homology with the consensus APl-binding site (7/8). It was demonstrated that the PF1 site was able to stimulate transcription when coexpressed with c-jun. Nevertheless, authentic AP1 was unable to bind the PF1 site, suggesting the presence of a closely related factor. The second element is an NFl-binding site. DNase I footprinting competition analysis shows that this region can bind NF1 or NFl-like factor.75 The third element was found in the first noncoding exon of the gene, 80 bp from the transcription start. DNase I protection and mobility shift assays show that a nuclear factor binds to a DNA sequence which contain a helix-loophelix recognition motif. 76 Reisman et al. showed that two transcription factors, c-myc and USF, bind to this helix-loop-helix motif. 77'78 Cotransfection of plasmids constitutively expressing c-myc or USF with the intact p53 promoter expressing the CAT gene leads to a 2- to 5-fold enhancement of expression from the p53 promoter. The physiological role of this regulation remains to be determined. Recently, Defile et al. identified a sequence in the murine p53 promoter that is responsive to expression of wild-type but not to mutant p53, suggesting that p53 could regulate its own transcription. 79 2. Non-coding sequence of exon 1. The first exon exclusively comprises 5' untranslated sequences. Alarge conserved dyad symmetry element is present in the (noncoding) first exon of mouse, rat, and human p53,57 but not in that of X. laevis p53 gene. Attempts to determine the transcriptional start site of p53 have been inconclusive, probably due to the potential stem and loop structure. S 1 nuclease analysis of the corresponding mouse mRNA indicates that two major start sites reside at the 3' and 5' ends of the dyad element, respectively, whereas primer extension analysis shows a major transcript start site at the 3' end of this element. 51 Similar conclusions were drawn for human p53, with recent studies indicating that the major start site of human p53 mRNA lies 3' of the putative stem and loop structure. 61,80 3. Unusual large intron. There exists a very large intron in the 5' part of the gene, but the biological significance (if any) of this intron is not known. It may be involved in a process related to the transcription or stability of p53 mRNA.

70

The p53 TumorSuppressorGene

71

In the mouse p53 gene, a regulatory element has been identified in intron 4. 81 The presence of intron 4 in cDNA/genomic constructs results in a high level ofp53 mRNA. Similarly, in transgenic mice, the p53 gene constructs with introns expressed 100-fold more p53 mRNA than cDNA constructs without introns. 82 DNA binding activity specific for the 5' region of intron 4 has been identified by band-shift assay and methylation interference, but its nature remains unknown. 8~ Alternative splicing has been described for both human and murine p53 transcripts. In murine cells, sequence analysis of cDNA clones from transformed cells has revealed the existence of cDNA with a 96-bp insertcorresponding to p53 intron 10 and which mapped 96 bp upstream of the 5' acceptor splicing site of p53 exon 11.83 More recent data describe a similar finding in normal mouse cell. 84 This 96-bp insert leads to the synthesis of a p53 protein 83 which is nine amino acids shorter than wild-type p53. This putative truncated p53 does not contain the epitope for mAb PAb122, the casein kinase II phosphorylation site, and is unable to form oligomers. Such proteins have not yet been directly identified, but p53 proteins (murine or human) lacking the PAb122 epitope have already been described (see below for more detail). Alternative splicing leading to an altered 5'-end coding region has also been described in human cells, 85 but its significance is not known.

IV. p53 LOCALIZATION IN THE CELL A. p53 Distribution in Normal and Transformed Cell For some time, p53 has been strictly described as a nuclear protein, a notion in agreement with the view that p53 is a dominant oncogene. 86 Using immunofluorescence studies with different mAbs, p53 has been shown to be localized in the nucleus of SV40-transformed cells and other transformed cells. Extensive work on

Figure4. Comparison ofthe predicted p53 amino acid sequences of different species. These sequences were aligned and positioned with respect to the human sequence. The five black boxes (I to V) represent the corresponding domains of high homology discussed in the text. The serine boxed in the carboxy terminus of p53 is the residue covalently linked to a small RNA in mouse p53 protein. The specific p53 sequences used for this comparison are: wild-type mouse p53337, human, 58 rat,67 hamster,68 chicken, 69 Xenopusgene A 70; Xenopusgene B (Caron de Fromentel, Soussi, and May, unpublished results); rainbow trout 71 and monkey p5366. Those amino acid identical to human p53 are indicated by dashes. For mouse p53, it is assumed that the second ATG is used as the initiation codon. The reading frame from this ATG to the termination codon predicts a 387-amino acid protein (modified from Ref. 72).

72

THIERRY SOUSSI

nuclear biochemical subfractionation revealed that p53 in normal and transformed cells is found in the chromatin, nuclear matrix fraction, and nucleoplasmic fraction, 87'88 whereas ultrastructural immunocytochemistry in combination with electron microscopy showed that p53 in situ is associated with a nuclear RNP structure containing hnRNA. 89 Most of these works were performed with various transformed cells in which p53 accumulation could be easily detected using immunofluorescence. Nevertheless, several observations had suggested that p53 localization was not strictly nuclear, since p5 3 protein was detected both in the cytoplasm and the plasma membrane of the cell. 90'91 Careful reinvestigation of subcellular distribution of p53 protein was performed. 92 Using a synchronous cell population (Balb/c 3T3) stimulated to grow by the addition of serum, it was shown that the subcellular localization of p53 varies throughout the cell cycle. In growth-stimulated cells, p53 is produced at an elevated level and the newly synthesized protein accumulates in the cytoplasm during the G 1 phase. Around the S phase, p53 migrates in the cell nucleus where it can be found for 3 hours. 92 Following DNA synthesis, p53 is no longer found in the nucleus and accumulates in the cytoplasm. Using different murine cell lines, Zerrhan et al. have shown that the cytoplasmic location of mutant p5 3 cannot be seen by immunofluorescence, but requires cell fractionation for its detection. 93 A temperature-sensitive mutant of murine p53 was shown to have wild-type properties at 32 ~ and mutant properties at 39.5 ~ Immunostaining demonstrates that this mutant p53 protein is in the nucleus of the arrested cells at 32 ~ but in the cytoplasm of the growing cells at 37 ~ Furthermore, on the basis of the use of protein synthesis inhibitors, it was suggested that a short-lived protein was responsible for retaining the mutant p53 in the cytoplasm at 37 ~ Taken together, these observations suggest that p53 localization is closely correlated with its biological function.

B. Nuclear Localization Signal in p53 Protein Examination of the carboxy-terminus of p53 protein revealed the existence of a putative nuclear localization signal (NLS) which is conserved in all p53 species. 96'97 When a peptide comprising this sequence is linked to a cytoplasmic reporter protein, it is targeted to the nucleus of the cell. 96 Furthermore, deletion of this signal led to synthesis of a cytoplasmic p53. Two other NLS with weaker activity have been reported in the carboxy-terminus of murine p5 397 (Figure 5).

Figure 5. Functional domains of the p53 protein. The hot spot of mutations in human cancer, 238'242 the cdc2 phosphorylation site106 and the domains involved in oligomerization 144 and transactivation164 have been identified in human p53. Other phosphorylation sites, nuclear localization signals,338 E1B,125 SV40 large-T anti~ gen, 118 the hsp binding site, 135 DNA binding sites154 and mdm-2 binding site149 were identified on mouse p53. The coordinate of each domain refers to the species where it was identified. The coordinate of the p53 protein and the conserved domain at the top of the figure correspond to human p53. 73

74

THIERRY SOUSSI

V. THE p53 PROTEIN A. p53 Protein Organization Comparison of all p53 protein sequences available led to the identification of five domains which have been highly conserved through evolution (domains I to V, Figure 5). 70,72 They include two stretches of 23 amino acids (domain IV) and 17 amino acids (domain V), respectively, which are almost 100% homologous. The sequences linking or flanking domains I, II, III, IV, and V are much more divergent, perhaps reflecting the fact that these regions are probably not involved in essential functions. Interestingly, domains I, III, IV, and V are specified by exons 2, 5, 7, and 8 of the p53 genes in the one exon/one block relation. The situation is slightly different for domain II specified by both exons 4 and 5 (see Figure 3); indeed, it is noteworthy that only the region of domain II encoded by exon 5 is subject to mutations in human cancer (see below). An analysis of the hydropathic profile, secondary structure potential and charge distribution of all p53 proteins under study reveals that, in contrast to the marked divergence in amino acid sequences observed for the two extremities of the molecule, a number of p53 features are highly conserved during evolution. 72 This is not unexpected, since it has been generally observed that structure is better conserved than sequence through evolution. These additional conserved features are summarized as follows (see Figure 3): 1. The amino terminus region (100 residues) contains a high number of acidic residues and very few (if any) basic residues. Furthermore, the protein displays a high proline content; this feature is thought to be involved in the abnormal migration of p53 in SDS-polyacrylamide gel electrophoresis. 2. The carboxy-terminus has a high-charge density and a very hydrophilic profile. 3. The internal amino acid sequence of all p5 3 proteins from about amino acids 100 to 300 contains very few charged residues and possesses two highly hydrophobic regions which are, in fact, domains IV and V, respectively. All these features are in agreement with a simple organization of the p53 protein where the residues of the amino- and carboxy-terminus may be located at the protein surface, and those of the hydrophobic central part may be stacked in the interior of the molecule. The four conserved domains of p53, II to V are found in the hydrophobic central part of the molecule and their role is probably crucial for the potential functions of the protein. All of the available data, including the evolutionary changes in DNA and protein sequences, and the structure prediction based on computer analysis, can be integrated into the tentative model of the anatomy of the p53 protein as represented. 72 The protein itself may be subdivided into three distinct regions of different


75

hydrophobicity, charge density, and predicted folded structure. We can also discriminate 5 core regions conserved through evolution (perhaps 6, if we consider the conservation of the particular structure of the carboxy-terminus). These regions are linked together or flanked by more divergent "auxiliary" sequences. It is reasonable to suppose that these latter sequences contribute to preserving the overall net charge and some favorable physical properties of the protein. The importance of the hydrophobic central part and, in particular, of domains II to V in p53 function, is strongly supported by the observation that they correspond to the hot spot of p53 mutation in human cancers and also in rodent tumors. Furthermore, this region of the p53 protein is involved in the interaction with viral antigens such as SV40, which may also play a role in transformation.

B. p53 Phosphorylation p53 is a multiple phosphorylated protein which is a substrate for several protein kinases. Numerous studies have been performed to map the phosphorylation site and to identify the kinase involved in this posttranslational process, but at present little is known concerning the signification of this modification, p53 expressed in a prokaryotic system can be phoshorylated in vitro using different types of kinase. Human, mouse, or X. laevis p5 3 expressed in insect cells can be phosphorylated in vivo in the same way that the X. laevis p53 can be phosphorylated in mammalian cells. All these observations suggest that p53 phosphorylation involves a common mechanism conserved throughout evolution. The majority of the phosphorylation sites were found on a serine residue. 98 One phosphorylation site on a threonine residue was observed on the amino-terminus of p53, but its exact localization is currently unknown. 98 No phosphorylation sites on tyrosine residues have been described. Regarding the localization of the phosphorylated site and the kinase involved in phosphorylation, three patterns of phosphorylation can be distinguished.

Phosphorylation at the Carboxy-Terminus of Mouse p53 Mouse p5 3 isolated from either normal or SV40-transformed NIH3T3 cells were shown to be phosphorylated at position serine 387. 98,99 This phosphorylation was identical in both cell types. It was shown by Carroll et al. l~176 that a phosphopeptide containing serine 387 was alkaline-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that serine 387 may be covalently linked to RNA. A similar result has been described for SV40 large-T antigen, but the significance of these observations is unknown. It is remarkable that this amino acid surrounded by two acidic amino acids is conserved in all p53 proteins, suggesting an essential function in p53 biological activity (see Figure 4). The phosphorylation of these penultimate residues in other species has not yet been demonstrated, with the exception of monkey p53.

76

THIERRY SOUSSI

Protein kinase activity was found to be associated with immunopurified mouse p53 protein, ~~ but it was demonstrated that this activity can be attributed to casein kinase II, 1~ which copurifies with p53. Subsequently, it was shown that this casein kinase II was involved in the phosphorylation of serine 386 on mouse p53.1~ Immunopurified p53 expressed in E. coli was phosphorylated in vitro by highly purified casein kinase II, and the stoichiometry of the reaction was 1 mole of phosphate per mole of mouse p53. Mutant p53, which had amino acid serine 387 replaced by alanine, was not phosphorylated by the same kinase. 102

Phosphorylation at Serine 315 on Human p53 p53 was shown to be associated with a 35-kDa protein in the mouse SV3T3 and 3T3 cell lines. 1~ This protein was subsequently identified as p34 cdc2 kinase, 1~ and additional work has shown that human p53 can be phosphorylated by the p 34.cdc2(106,107) This kinase was first identified in yeast Saccharomyces cerevisae as the product of the CDC28 gene and the cdc2+ gene of Schizosaccharomyces pombe. The cdc2/CDC28 protein is required at two transition points in the cell cycle: commitment to DNA replication at the start, and preparation for mitosis at the G2/M boundary. 1~ Evolutionary studies have demonstrated that higher eukaryotes possess multiple CDC2-1ike proteins, p34 cdc2 is the mammalian homolog of S. pombe cdc2. Biochemical analysis suggests that CDC2 is activated at the transition points by a posttranslational mechanism which includes phosphorylation. In addition, p34 cdc2 per se does not possess any intrinsic kinase activity, p34 cdc2 activation requires the association with regulatory subunits known as cyclins, a group of unstable proteins whose level changes during the cell cycle. 1~ Two distinct subpopulations of p34 cdc2 kinase are detectable in mammalian cells. One consists of p34 cdc2 in a complex with cyclin B (p62/p34Cdc2), and is maximally active during mitosis. The other is comprised of p34 cdc2 in a complex with a polypeptide of approximately 60 kDa (p60/p34 cdc2) and is active in the interphase. An increasingly large number of proteins have been found to serve as substrates for the p34CdC2/cyclin complex either in vitro or in vivo. These include histone H 1 and the product of a number of viral and cellular oncogenes and tumor suppressor genes, such as SV40 large-T antigen, c-abl, or p110-RB. In vitro experiments show that both p62/p34 cac2 and p60/p34 cac2 are able to phosphorylate human p53.11~176 Nevertheless, this phosphorylation is cell-cycledependent since it occurs predominantly in cells which are in S phase. Those experiments were done in vitro either on a synthetic peptide containing the phosphorylation region or on purified human p53 protein. Studies of in vivo phosphorylation of p53 during the cell cycle do not show any gross variations at the different stages. This might be due to the fact that possible cell cycle phosphorylation in vivo


77

by p34 cdc2kinase can be masked by other types ofphosphorylation involving kinase and which are not cell-cycle-regulated.

Phosphorylation Sites in the Amino-Terminal Region of p53 A multiple phosphorylation site was found in tryptic peptide, corresponding to the amino-terminus of mouse p53. 98'99 This region contains a cluster of serine residues which may be potential phosphorylation sites. They have been proposed to be sites for phosphorylation by double-stranded-DNA-dependent kinase (DNAPK) from HeLa cells, ill and can be dephosphorylated by protein phosphatase 2A. 112 Using mutagenesis experiments to investigate these potential serine residues, Wang and Eckhardt 113 identified serines 4, 6, 15, and 34 as in vivo phosphorylation sites. Furthermore, these authors showed that mouse p53 expressed in bacteria is phosphorylated by DNA-PK in the amino-terminal region, though the nature of the phosphorylated residues was not checked. In another approach, Milne et al. 114 showed that mouse p53 expressed in E. coli can be phosphorylated in vitro with highly purified casein kinase I. The sites of phosphorylation were identified as serines 4, 6, and 9, with a marked preference for serine 6. Purification ofkinase activity which copurified with mouse p53 in vivo enabled identification of a novel casein-kinase-I-like enzyme (PK270) which phosphorylated the same sites at the amino-terminal region of mouse p53. ll4 The observation that two different kinases (DNA PK- and CKI-like enzyme) are involved in the phosphorylation of the amino-terminal region of p53 is not a contradiction. Further analyses are needed to assess whether both kinases phosphorylate the same residues in vivo. Indeed, it is already known that a single site can be phosphorylated by different kinases in response to various signals. The role of phosphorylation in the regulation of p53 function is not known at present. Unlike the rbl gene product, p53 phosphorylation does not show any major variation during the cell cycle. It was demonstrated that phosphorylation of serine 312 was two-fold higher in SV40-transformed cells than in NIH 3T3 cells. 99 Analysis of the phosphorylation of free and bound forms of monkey p53 and SV40 large-T antigen during lytic infection of CV1 cells indicates that increases in specific phosphorylation in the two proteins correlate with the association of SV40 large-T antigen and p53.115 This enhanced phosphorylation may be a consequence of the complex formation (a better target for some kinases), or could reflect an increased affinity for highly phosphorylated forms of SV40 large-T antigen. It has been reported that the mutant p53 found in human cancer cell lines was underphosphorylated compared to wild-type p53, ~16but the significance of this finding is not known. These observations are in agreement with recent work published by Ulrich et al., 117 who showed that the wild-type p53 form involved in inhibition of cellular proliferation has increased phosphorylation compared to mutant p53 (see below for model).

78

THIERRY SOUSSI

C. p53 Interaction with SV40 Large-T Antigen Regions of murine p53 involved in the interaction with SV40 large-T antigen have been characterized (see Figure 5). Jenkins et al. 118defined two discontinuous regions, mapping between amino acids 168 and 202 (including highly conserved domain III) and amino acids 236 and 289 (including highly conserved domains IV and V). Tan et al. 119 defined the SV40 large-T antigen-binding region between amino acids 123 and 215 of murine p53. The evolutionary conservation of the p53 binding domain prompted some investigators to examine the behavior of several p53 with SV40 large-T antigen. It was demonstrated that X. laevis p53 is also able to interact strongly either in vivo or in vitro with SV40 large-T antigen. 12~ Like mammalian p53, X. laevis p53 when complexed with SV40 large-T antigen, exhibits a 20-fold increase in its half-life. 12~Furthermore, an in vitro association between rainbow trout p53 and SV40 large-T antigen was also observed. 71 These results confirm the correlation between the presence of highly conserved domains and the localization of the p53 region involved in binding with SV40 large-T antigen. This lends support to the hypothesis that p53 association with large-T antigen could alter the function of p53, either by blocking its interaction with some cellular "T-antigen equivalent" protein, by inducing the stabilization of p53, or both. Recently, a specific interaction has been described between p53 and the lymphtropic papovavirus (LPV) large tumor antigen. LPV grows only in monkey and human B-lymphoblastoid lines in culture. 121 The large-T antigen shares 45% sequence identity with both SV40 and polyoma large-T antigen. SV40 large-T antigen complexes with murine or X. laevis p53 are very stable, since they are not dissociated by harsh treatment (1 M NaC1, 0.5 % NP40 or 2 M urea, 0.1% SDS). However, primate p53 complexes with the viral protein are more fragile and are dissociated in the presence of 0.1% SDS. The reason for this is not known, nor is its biological significance, if any. Using in situ cell fractionation, Schmieg and Simmons 122 showed that the association between p53 and SV40 large-T antigen occurs in the nucleus of the cell after the migration of the two free proteins from the cytoplasm. The association was shown to be very rapid (5 to 15 min). Studies of the stoichiometry of SV40 large-T antigen and p53 in complexes isolated from SV40-transformed cells indicate that, in general, they are composed of four molecules of SV40 large-T antigen and four or five molecules of p53. Stabilization of p53 in the absence of SV40 large-T antigen is the result of point mutations which alter the conformation and the stability of the protein (see later). In SV40-transformed cells, it is generally considered to be the result of the association with SV40 large-T antigen. However, several studies are somewhat at variance with this view. For example: 1. Deppert et al. and Reihsaus et al. 88'123 showed that, in SV40-transformed cells, part of the metabolically stable p53 is present in a free non-T-antigen-


.

79

associated form, while in 3T3 cells abortively infected with SV40, p53 is associated with T antigen without being stabilized. Adl 2-transformed cells contain wild-type-stabilized p53 without any interaction with E 1B protein. In human tumor cells, there exist examples of p53 stabilization without any mutation. All these observations suggest that the increased p53 half-life in SV40-transformed cells is more closely related to the transformed state of the cell than to its association with T antigen, but the possibility that both phenomena contribute to the stabilization of p53 cannot be excluded. In human tumor cells in the absence of SV40 large-T antigen, the presence of a T-cell equivalent which could act as its viral counterpart cannot be precluded. The MDM2 protein may be a very good candidate for this function (see below).

D. p53 Interaction with Adenovirus EIB Protein This interaction has been subject to less intensive work. Only mild extraction procedures are able to detect a complex between adenovirus 5 Elb-58 kDa and p53 during infection of rodent cells, whereas the complex is more stable in transformed cells. 124A similar behavior with SV40 large-T antigen was reported by Duthu et al. during abortive infection of mouse cell with SV40. Using mAbs, it was shown that only mAbs reacting with amino-terminal epitopes on p53 displace the E1B protein. 124 Using a series of p53 and adenovirus 2 Elb-55-kDa-protein-mutant, Kao et al. 125 mapped the interaction domains in both proteins. The domain in murine p53 includes amino acids 11 to 123 (see Figure 5), whereas the main domain in the E1B protein lies between amino acids 224 and 354. It should be stressed that this region in the p53 protein is quite different from those reported for SV40 large-T antigen. This observation again argues for the notion that the p53 interaction with the two viral proteins leads to different biological processes in order to inactivate p53 function.

E. p53 Interaction with E6 Papilloma Virus Protein This field is currently the subject of intensive work, with some contradictory observations. As stated above, the primary work of Sheffner et al. 41 showed that only the E6 protein from HPV- 16 and 18 can form complexes with p53. In another report, Crook et al., 126 showed that both high- and low-risk E6 protein can bind in vitro to human p53, whereas only the high-risk E6 protein is able to induce p53 degradation. They assigned the p53-binding region to the carboxy-terminus of the E6 protein (conserved in all HPV types), whereas the region necessary for p53 degradation could be assigned to the amino-terminus of E6 (conserved in high-risk types HPV). The reason for this discrepancy in the behavior of the high- and

80

THIERRY SOUSSI

low-risk HPV E6 is not known, and further work will be necessary to analyze in more detail the relationship between p53 and HPV E6. The association between p5 3 and the HPV-16 or 18 E6 protein was found to be mediated via a 100-kDa cellular protein which is able to bind to the viral proteins. 127 The identity of this protein is not known at present. Using p53 mutants, a correlation between p53 binding and degradation was established. Only p53 mutants that bound to HPV 16-E6 were targeted for degradation, whereas those that did not complex HPV- 16 E6 were not degraded. 128Using a fusion protein consisting of the amino-terminal half of E7 protein (p110-RB binding domain) and the full length HPV- 16 E6 protein, Scheffner et al. 129 demonstrated that this chimera could promote the in vitro degradation of the retinoblastoma protein. Interestingly, the capacity to stimulate degradation of other proteins is found with both high- and low-risk HPV E6 proteins, suggesting that the specificity of the HPV type is mediated by p53 binding. At present nothing is known concerning the p53 domain involved in the interaction with the E6 protein.

F. p53 Interaction with hsp70 Several different cell lines expressing a stabilized p53 protein in the absence of SV40 large-T antigen have been shown to contain p53 complexed to a 68-kDa protein (termed p68). Monkey cells transiently expressing a mutant p53 protein contain a p68-p53 complex which co-immunoprecipitates when p53-specific mAb are used. In one transformed cell line, it has been shown that p68 is a heat-shock protein of the 70-kDa (hsp70) family. 13~This observation was confirmed by several other groups using antisera prepared against hsp70 and showing coprecipitation of p53.131'132 The identity of the member of the hsp70 involved in this complex is a subject of controversy, as Hinds et al. TM showed that only the constitutive form of this family, hsc70, is associated with p53, whereas Sttirzbecher et al. 132 showed that both the constitutive and the inducible (hsp70) forms bind to p53. Nonetheless, it is quite clear that wild-type p53 do not bind to hsp70, and that only certain mutant p53 with altered conformation are able to form tight complexes with hsp70. Several studies have shown that p53 mutants which bind hsp70 are more efficient in transforming cells in vitro, 133 and they are found in human tumors associated with an immune response to p53 (see later) and with poor prognosis. The interaction with hsp70 appears to involve remarkably conserved structures, since p53 also interacts with the bacterial hsp, dnaK, when expressed in E. coli. TM Furthermore, X. laevis p53, when expressed at a temperature well above its optimal temperature, binds very well to mammalian hsp70, whereas this interaction is abolished at more physiological temperatures, again suggesting that altered conformation of p53 is involved in its recognition by hsp70.12~ Using an in vitro system, Hainaut and Milner have demonstrated that hsp70 complexes with dimers and


81

possibly monomers of p53 in a manner which requires the carboxy-terminus of p53135 (see Figure 5). Heat-shock proteins belong to a class of proteins broadly defined as "molecular chaperones" involved in facilitating the transport, folding, and assembly of many proteins 136'137for review. Upon physiological stress, the synthesis of these proteins dramatically increases. The biological significance of p53-hsp70 complexes is poorly understood. It has been proposed that these complexes merely consist of aberrant p53 polypeptides whose appropriate folding and transport programs have been altered by the encoded p53 mutation. It is possible that regulation of the conformation of wild-type p53 involves a very transient interaction with hsp70 which cannot be caught by our current methods.

G. p53 Interaction with p53 Besides heterologous protein-protein interactions between p5 3 and other cellular or viral proteins, p53 forms homologous oligomers. It was first shown in vivo that murine p53 from F9 cells sediments mainly at 8S, which represent tetramers of p53.138 High molecular mass oligomers of p53 were subsequently found in most cells studied so far.139 Mouse and human p53 expressed in insect cells 140 or expressed in an in vitro translation-transcription system TM also form high molecular mass oligomers. Using gradient gel electrophoresis and chemical cross-linking, Stenger et al. 142 showed that under nondenaturing conditions, murine p53 forms mainly tetramers or multiples of tetramers. These oligomers are very stable in the presence of high salt (1M NaC1) or reducing agents. Pulse-chase analysis shows that oligomerization is a very rapid process (2 min). Truncation of the carboxyterminus of p53 prevents the oligomerization process. 143An amphipathic a-helix followed by a stretch of basic residues identified in the carboxy-terminus of all mammalian p53 was shown to be required for tetramerization TM (see Figure 5). Thus far, tentative correlations between oligomerization, phosphorylation, and conformation of p53 have not been conclusive.

H. p53 Interaction with Mdm-2 A cellular protein of approximately 90 kDa, termed p90, has been described which coprecipitates with p53 from cell extracts containing wild type or mutant p53 proteins. 133'145This protein was purified from a cell line which expressed high levels of p53 and p90.146 Sequencing of p90 showed that it is the mdm-2 (murine double minute 2) oncogene product. 146The mdm-2 gene enhances the tumorigenic potential of cells when it is overexpressed, and encodes a putative transcription factor. Expression of murine mdm-2 in cells transfected with plasmid expressing murine wild-type p53 inhibits the transactivation properties of p53.146 In the cell line containing the temperature-sensitive p53 mutant (see above), it was observed that the p53-mdm-2 complex was only detected at 32 ~ when p53 was in wild-type

82

THIERRY SOUSSI

Figure 6. The p53-mdm-2 autoregulatory loop model. In this model, p53 protein activates mdm-2 expression through a regulatory element localized in intron 1 of mdm-2 gene. On the other hand, Mdm-2 protein inhibits the transactivating properties of p53 via complex formation. This model is adapted from the work of Wu et al. 148

form. At 37 ~ with a mutant p53, the complex is not detected even though it was clear that the Mdm-2 protein binds well to mutant p53.147'148 It has been subsequently demonstrated that wild-type p53 protein stimulates the transcription of the mdm-2 gene. 147'148Furthermore, Wu et al. 148 identified that the first intron of murine mdm-2 gene contains a p5 3 DNA-binding site which, when placed adjacent to a minimal promoter, can stimulate a test gene in a p53-dependent fashion. These results have led to the proposal by Wu et al. 148 of the p53-mdm-2 autoregulatory feedback loop model (Figure 6). In this model, the p5 3 protein regulates the mdm-2 gene at the level of transcription, and the Mdm-2 protein regulates the p53 protein at the level of its activity. This model is reinforced by the identification of the p5 3 domain which interacts with p5 3.149'150 It has been mapped in the N-terminal 52 amino acid residues (Figure 5) of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by concealing the activation domain of p5 3. The mdm-2 homolog in humans was isolated and its encoded product bound to both wild-type and mutant human p53.151 The human gene was shown to be amplified in one-third of diverse sarcomas. 151 See Section X for the role of mdm-2 and p53 in human cancer.


83

VI. p53 ACTIVITIES A. p53 as a DNA Binding Protein The first biochemical activity demonstrated for p53 was nonspecific doublestranded DNA binding activity. 152 Further work has shown that both wild-type and mutant p53 bind to single-stranded or double-stranded DNA in cellulose chromatography. 153 Using either wild-type or truncated murine p53 expressed in E. coli, Foord et al. 154 demonstrated that the DNA binding domain of p53 is contained in the carboxy-terminus of the protein (Figure 5). Kern et al. 116 showed that different mutant p53 derived from human tumors or mouse-transformed cells bound calf thymus DNA more weakly than did wild-type p53. Mutant p53 expressed in insect cells showed a similar behavior, suggesting that p5 3 binding is a property intrinsic to the protein.

B. p53 as a Sequence-Specific DNA Binding Protein Using a random immunoprecipitation assay, two human sequences have been identified that specifically bind to wild-type human p53 in vitro. 155 Both sequences contain two repeats of the TGCCT motif. Using a methylation interference assay, the authors showed that guanine residues are necessary for binding. Two human p53s containing missense mutations commonly found in human cancer (p53-His 175 and p53-His 273) are unable to bind to these sequences. Examination of the primary sequence indicates that one of them contains sequences found near a putative replication origin of the ribosomal gene cluster. 155 Using "catch linker" and PCR, El-Diary et al. 156 identified 18 other human genomic clones that bind p53 in vitro. Precise mapping of the binding site revealed a consensus binding site showing internal symmetry consisting of two copies of the 10 bp motif 5'-PuPuC(A/T)(T/A)GPyPy-3' separated by 0 to 13 bp. The TGCCT motif found earlier belongs to this consensus. Mutants p53 (p53Val143, p53His175, p53Trp248, and p53His273) do not bind to the oligonucleotide corresponding to the consensus dimer. 156 Using a similar approach, Funk et al. 157 have cloned 17 human DNA sequences containing p53 binding activity. Sequence analysis indicates that they contain the consensus described above. Thus far, it is not known whether the specific DNA binding activity of p53 is also located in the carboxy-terminus of the protein. In another report, Bargonetti et al., ~58 showed that wild-type but not mutant p53 proteins bind to a sequence adjacent to the SV40 origin of replication, but that this sequence bears little homology with the consensus sequence. It has been demonstrated that the murine creatine phosphokinase (MCK) gene and its enhancer-promoter element could be regulated positively by wild-type p53159 (see below). Using a filter-binding and gel-mobility shift assay, Bargonetti et al. demonstrated that wild-type p53 binds with similar affinity to MCK and RGC

84

THIERRY SOUSSI

sites, but less tightly to SV40 sites. 16~All these studies emphasize the importance of the DNA binding properties of wild-type p53 and its loss in mutant p53 found in human cancer.

C. p53 as a Transactivating Protein via a DNA Binding Activity Careful examination of the amino acid sequence of p53 indicates that it could resemble a transcription factor. The amino-terminus is very acidic and is followed by a proline-rich stretch of amino acids. This feature has been found in the transactivating domain of several proteins including c-Fos, Gal4 and b-Jun. Furthermore, p53 DNA binding activities also suggest that it could function as a transcription factor. Using Ga14-p53 fusion proteins, it has been demonstrated that p53 protein contains a transcription-activating domain. 161-163This activity is localized in the amino-terminal 42 residues of the protein 164 (see Figure 5). More interestingly, p53 mutants are devoid of any transactivating activity. 161-164 These results were confirmed using a reporter gene located downstream of a DNA sequence that binds p53 in vitro. 165 Cotransfection of an expression vector that encoded human wild-type p53 and the reporter vector led to expression of the reporter gene, indicating that p53 binding also occurred in vivo, and that it induced transactivation. Nevertheless, p53 mutants are totally devoid of any transactivating activity, suggesting that transcriptional regulation is fundamental to wild-type p53 function. More recently, using an in vitro transcription assay, Farmer et al. 166 have been able to show that stimulation of transcription is restricted to wild-type p53. Although, all these data are rather convincing, more recent data have shown that there is a heterogeneity in the transcriptional activity of mutant p53 protein when various p53 DNA targets are used. 167

D. p53 as a Transactivating Protein via Protein Binding In addition to the ability of wild-type p53 to act as a positive regulator of certain promoters, it can often behave as a transcriptional repressor 168-172 (see also Table 3). This activity is not mediated by a DNA binding activity of p53. Seto et al. 173 have shown that this inhibition can be reproduced in cell free extracts. They were able to demonstrate that human or murine wild-type p53 could bind to the TATAbinding protein, suggesting that p53 repression is mediated by interfering with the binding of basal transcription factors to the TATA motif. This model has been confirmed by other groups. 174-178 Furthermore, it was shown that this association is mediated by the p53 transactivation domain. 174'178 The biological role of this activity remains to be elucidated.


85

Table 3. Effect of Wild-Type and Mutant p53 on Various Viral and Cellular Promoters a Promoter CMV early RSV HTLV-I LTR SV 40 early UL 9 HSV HTLV-I LTR b PCNA MCK b Rb MDR1 II-6 c-Fos B-Actin MHC Hsc70 class I M H C c-Jun c-Fos 13-Actin c-Fos c-Jun c-Jun B c-Fos c-Myc p53 OTC b-Myb DNA pol.-o~ PCNA Hstone H3

WiM-Type p53

Mutant p53

inhibition inhibition inhibition inhibition inhibition stimulation inhibition stimulation inhibition inhibition inhibition inhibition inhibition ~nhibition inhibition no effect inhibition inhibition inhibition no effect no effect no effect no effect no effect no effect no effect inhibition inhibition inhibition inhibition

no effect no effect no effect no effect no effect no effect stimulation no effect stimulation stimulation slight inhibition slight inhibition slight inhibition slight inhibition no effect no effect no effect no effect no effect no effect ND c

Cell U s e d HeLa HeLa HeLa HeLa HeLa CV 1 HeLa CV 1;HepG2; 10T 1/2 HeLa NIH3T3 HeLa HeLa HeLa HeLa REF REF REF REF REF REF REF

ND ND ND ND ND ND ND ND ND

T98G T98G T98G T98G T98G T98G T98G T98G T98G

Reference

341

180 341 159 342 170

343

169

197

Notes: "For more clarity, viral and cellular promoters have been presented separately. Some promoters have been

studied by different authors and may appear several times in this table. In several cases, there were discrepancies between results of several authors. It should be stressed that in all cases except for the work of Lin et al., 197 transactivation was tested through a CAT assay with the plasmid-containing promoter fragment linked to the CAT reporter gene. Transient assays, were performed in various cell lines. Lin et al. 197 directly measured the expression of endogenous cellular genes in response to the production of wild-type p53. bpromoter with a natural p53 DNA binding element, eND: not done.

E. Cellular Genes Regulated by Wild-Type p53 Some cellular and viral promoters have been shown to be inhibited by wild-type p53 (Table 3). Usually, mutant forms of p53 do not inhibit the activity of the reporter gene and sometimes show slight stimulation. These observations are somewhat in contradiction with the in vitro transactivating properties of p53 described above,

86

THIERRYSOUSSI

but it should be noted that there is no evidence for a direct action of p53 on these cellular and viral promoters. In fact, two promoters were found to be able to bind p53 in vitro" the MKC promoter, 179 and the human T-cell leukemia virus type I enhancer.18~ Both promoters can be activated in vivo by wild-type p53, whereas mutant p53 has no effect. All these observations can be reconciled if we assume that the positive effect of wild-type p53 is directly mediated via binding to a p53 response element, whereas its inhibitory effect can be more indirect through the binding of cellular factors.

VII.

p53 A N D THE CELL CYCLE

A. p53 Expression during the Cell Cycle Early work on p53 suggested that it may be implicated in the promotion of cell proliferation. Earlier experiments by Reich and Levine 181 showed that mouse 3T3 cell growth, when arrested by serum deprivation, exhibited very low levels of p53 mRNA and protein. When the cell was induced to grow by serum stimulation, the level of p53 mRNA and the rate of p53 protein synthesis increased markedly, reaching a peak near the G 1/S boundary just prior to initiation of DNA replication. 181 Similar experiments performed with normal resting T lymphocytes 182 and normal diploid fibroblasts 183 showed that p53 expression is always concomitant with induction of cell growth. The level of p53 mRNA and protein is somewhat constant throughout the cell cycle when the cells are growing exponentially. 2~ This observation, added to other characteristics of the p53 protein (short half-life, nuclear localization), led to the notion that wild-type p53 could play a positive role in cell proliferation. This idea was strengthened by the work of Mercer and collaborators. 184'183Microinjection of p53 antibody (200.47 and PAb122) into the nucleus of quiescent Swiss 3T3 mouse cells inhibited the subsequent entry of the cell into the S phase after serum stimulation. This inhibition was effective only when microinjection was performed at or around the time of growth stimulation, suggesting that p53 is critical for G0/G 1 transition. 184'183Recently, similar results were obtained using methylcholanthrene-transformed mouse cells which express mutant p53185'186 Also consistent with these results is an antisense experiment which showed that inhibition of p53 expression prevented cell proliferation in both nontransformed NIH3T3 cells and transformed cells. 187 All of these observations led to the notion that wild-type p53 is a positive regulator of cell proliferation.

B. Wild-Type p53 is Antiproliferative In 1984 three groups reported that cotransfection of p53 plasmids with plasmids possessing an activated c-Ha-ras oncogene could transform REF cells in a manner similar to that observed with protooncogenes such as myc or E1A. 188-19~These


87

observations resulted in the classification of p53 as a nuclear dominant oncogene. It is now well known that the murine cDNA used for these experiments contains mutations. 191 Indeed, wild-type p53 cDNA is unable to cooperate with an activated ras gene. Moreover, expression of wild-type p5 3 inhibited transformation induced by other combinations of nuclear oncogenes with r a s . 192'193 These were the first sets of experiments indicating that p53 might function as a tumor suppressor gene (see also later). Beating in mind the work on the rbl gene, several authors performed experiments on reintroduction of wild-type p53 into transformed cells. Transfection experiments in which the wild-type human p5 3 cDNA, expressed via a heterologous promoter, was introduced together with a selectable marker into human cancer cells indicated that p53 was antiproliferative, and only a small number of clones which did not express any p53 were selected 194 (Table 4). In contrast, mutant p53 lacked an antiproliferative effect. Using flow cytometry on transiently transfected cells, Diller et al. ~95 showed that growth arrest of the cells was due to the inability of the transfected cells to progress into the S phase. Using an elegant approach, Mercer et al., established different cell lines (of rodent or human origin) conditionally expressing wild-type p53 using the hormone-inducible mouse mammary tumor virus promoter. 196'168Wild-type p53 expression could be turned on by treating the cell with dexamethasone. These authors showed that expression of wild-type p53 inhibited G0/G 1 progression into the S phase and that cells accumulated with the start of replication. This growth arrest was accompanied by selective downregulation of a subset of late G 1 phases genes such as B-myb, proliferative cell nuclear antigen (PCNA) and DNA polymerase-o~ 197 (see also Table 4). Using the temperature-sensitive murine p53 mutant described above, it was shown that this mutant behaved like wild-type p53 at 32 ~ and suppressed growth, whereas at the higher temperature (39.5 ~ it behaved like a mutant and promoted growth. 94 Most of these experiments were performed using p53 cDNA expressed under the control of a strong heterologous promoters and it is possible that only wild-type p53 overexpression is antiproliferative. In line with this observation, Chen et al. 198'199performed experiments using a retroviral construct expressing either the wild-type or mutant p53 (Table 4). Infection of recipient cells led to integration of only one copy of the exogenous genome. Using the osteosarcoma cell line Saos-2, the authors showed that expression of wild-type p53 produced several viable clones with 50% reduction in growth rate, loss of tumorigenicity, and soft agar colony formation. 198Using A673 cells (human peripheral neuroepithelioma) which do not express detectable amounts of p53, the authors observed that expression of wildtype p53 through the retrovirus vector suppressed the tumorigenicity of cells but not their growth rate. 199 The discrepancy between these observations and those described above is unexplained, but it could reflect the experimental conditions used for reintroduction of wild-type p53 into the cells. In several cases, reintroduction of wild-type p53 into a murine myeloid leukemic cell line 2~176 and into a human colon tumor-derived cell line 2~ was shown to induce

Table 4. Suppression of Cell Growth by Human p53 a

Cells

Cell Origin

p53 Status

SW837

Human colorectal carcinoma

1 mutated p53 gene

SW480

Human coiorectal carcinoma

1 mutated p53 gene

RKO

Human colorectal carcinoma

VACO235

SAOS-2

Human colorectal adenoma Human osteosarcoma

Wild-type p53 low level expression Wild-type p53 normal level of expression No p53 gene

SAOS-2

Osteosarcoma

No p53 gene

HR8

SV40 transformed hamster

T98G

Human glioblastoma

Assume to be wild-type but stabilized Mutated p53

MCF7

Human breast carcinoma

Wild-type (exon4-8)

MDAMB468 T47D


One mutated p53 allele



DP 16-1

Human peripheral neuroepithelioma Murine friend erythroleukemia

Wild-type p53 but low level of expression No p53 gene

K562

Human CML

No p53 gene

A673

Vector Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in retroviral vector Human p53 cDNA with inductible promotor Human p53 cDNA with inductible promotor Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in retrovirus vector Murine p53 gene in plasmid with SV40 promoter Human p53 cDNA in plasmid (S V40 promoter)

Phenotype

Ref

Growth suppression

194

Growth suppression

194

Growth suppression

194

No growth suppression

194

Growth suppression

195

Suppression of neoplastic phenotype/no loss of growth Lower saturation densities increased doubling time Reversible growth suppression

198

344

No growth suppression

345

Growth suppression

345

Growth suppression

345

Suppression of neoplastic phenotype no loss of growth Growth suppression

199 346

Growth suppression

346

196

(continued)

Table 4. (continued) Cells

Cell Origin

p53 Status

SKOV-3

Human ovarian adenocarcinoma

No p53 gene

TSU

Human prostate carcinoma


PC-3

Human prostate carcinoma


SW480 NCI-H358

Human colorectal carcinoma Human lung carcinoma

1 mutated p53 gene No p53 gene

NCI-H358

Human lung carcinoma


M KNI

Human gastric carcinoma


MKN25

Human gastric carcinoma


Be-13

Human T-ALL

No expression

Vector Human p53 cDNA in plasmid (S V40 promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Chromosome 17 transfer Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in plasmid (CMV promoter) Human p53 cDNA in retroviral vector

Phenotype

Ref.

Growth suppression

346

Growth suppression

347

Growth suppression

347

Growth suppression Growth suppression

348 349

Growth suppression

349

Growth suppression

350

Growth suppression

350

Reduced growth rate loss of tumorigenicity

351

Notes: "In most of the experiments, wild type human p53 cDNA was expressed through a strong heterologous promoter in a plasmid introduced together with a selectable marker into

human cancer cells. After several weeks of selection, clones were numbered. In most cases, wild-type p53 gave rise to a 10-fold-decreased number of clones compared to mutant p53. Nevertheless, in some experiments, no antiproliferative effect of wild-type p53 was observed.

90

THIERRY SOUSSI

apoptosis. This behavior was not observed with mutant p53. The reason why wild-type p53 is antiproliferative in some cell lines but induces apoptosis in others is unknown. Taken together, the above two paragraphs are seemingly in contradiction since they suggest that wild-type p53 could act either to enhance or to inhibit cell proliferation. Several works on p53 protein, its posttranslational modifications, and its modifications in conformation might reconcile the two set of observations.

C. p53 Adopt Distinct Conformational States At present, a large panel of p53 mAbs is available and epitopes recognized by these mAbs have been extensively mapped. Most of them recognize native or denatured p53 and their epitopes correspond to short stretches of linear amino acids. Nevertheless, two classes of antibodies with conformational epitopes have been identified: (1) unstable epitopes that are destroyed by protein denaturation (and which have not been mapped), and (2) stable epitopes that can be exposed or masked on the native protein (such epitopes positions have been mapped). MAbs such as PAb2462~ and PAb16202~ belong to the first class, whereas mAbs such as PAb2402~ belong to the second. It has been shown that cell growth stimulation induces a change in the conformation of the p53 protein which can be probed using various mAbs. 2~ Using temperature-sensitive mutants and an in vitro expression system, it was demonstrated that the different conformations represent alternative structures for the same polypeptide. These observations led to the proposal that two conformational states of p53 protein are present in the cell, 2~ one with a suppressor form and one with a promoter form. The equilibrium between the two forms regulates the function of p53 in the cell cycle. Based on localization studies of p53 in various cell lines, it has been shown that there is a correlation between the conformational phenotype of p53 and its subcellular location. Furthermore, it has been shown that the ability of wild-type p53 to exert its antiproliferative effect correlates with the presence of a unique conformational state which is characterized by increased phosphorylation and the loss of the PAb421 epitope, llv Mutant p53 found in human cancer cannot assume this conformational state (Figure 7).

D. Wild Type and Mutant p53 As stated earlier in this chapter, the p53 gene is frequently mutated in human cancer. Most of the mutations are point mutations, which has led to the synthesis of a mutated protein with properties different from those of wild-type p53 (Table 5). Briefly, three aspects of p53 are altered by these mutations: (1) the conformation of the protein (binding to hsp70, oligomerization or interaction with specific mAbs); (2) some biochemical properties (specific DNA binding, transactivation of reporter gene); and (3) some biological properties (inhibition of cellular growth arrest). There are several reasons which make it difficult to correlate all these


91

NH 2

Q

NH 2

PROTEIN KINASE

,,.=

BLOCKED BY MUTATION OR BY COMPLEX FORMATION WITH VIRAL OR CELLULAR PROTEINS

PHOSPHATASE ?

COOH

COOH

PROLIFERATIVE FORM PAb421 + SUPPRESSOR PAb1620 + PAb240 -

ANTI PROLIFERATIVE FORM PAB421PROMOTER PAb1620 PAb 240 +

Figure 7. A conformational model for p53 function. The two forms of p53 are distinguishable by their reactivities with specific mAbs. In transformed cells, the proliferative form is blocked either by mutations or by a specific interaction with viral oncoproteins (SV40 large-T antigen, adenovirus E1 B protein) or cellular proteins such as mdm-2. This model was first proposed by Milner et al. 2~ and redefined more recently by Ulrich et al. 339

properties of mutant p53. First, not all mutants exhibit the same properties, suggesting that there are several classes of mutant 2~ (also Ref. 208 for a review). Second, not enough mutants have been fully tested thus far, and we await more data in order to draw a clear picture of the p53 function(s) targeted by the mutations. Third, we do not know at present whether the different properties tested so far really correspond to the main function of p53, or whether what we are seeing are side effects of the mutation. A more clear-cut test will enable discovery of p5 3 biological activity. It is interesting to note that several regions of the p53 protein with known biological functions are never mutated in human cancer; the transactivating domain in the amino-terminus and the region containing the nuclear localization signal and oligomerization domain in the carboxy-terminus of the protein. These observations suggest that some p53 protein properties are necessary for the transformed phenotype of the cell and that mutations do not simply abolish the activity of the protein.

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THIERRY SOUSSI

Table 5. Properties of Wild-Type and Mutant p53 a Binding to hsp 70

Wild Type

143 Ala

175 His

248 Trp

273 His

No ND c

No 7h

No

Yes

Yes

20 min

2h

4-6 h

Cooperation with Ha-ras

No

Yes, weak

Yes, strong

ND

Yes

Recognition by PAb 1620

Yes

ND

No

ts in vitro

ND

Recognition by PAb240

No

No

Yes

ts in vitro No in vivo

No

Oiigomerization

Yes

NAb

NA

No

ND

Sequence specific DNA binding

Yes

No

No

No

No

Transactivation

Yes

No

No

No

No

Dominant negative activity

NA

Yes

Yes

Yes

Yes

Inhibition of cell proliferation

Yes

No

No

No

No

Half life

Notes." aTypicalbiochemical or biological activities either lost or gained by mutant p53 are listed. Mutantp53 Ala 143 corresponds to a widely used mutated allele first found in a colorectal carcinoma by Baker et al. 194Mutants His 175,Trp248, and His273correspond to the hot spot amino acids commonly found in human cancers. 242 bNA: information not available. ~ND: not done.

This hypothesis is reinforced by the discovery of the negative dominant effect of the mutant protein over the wild-type protein. Coexpression of the wild type and mutant together (either in vitro or in vivo) led to the production of p53 heterooligomers which have the mutant conformation. 2~ Such situation (expression o f wild-type and mutant p53 together) may correspond to a tumor cell which have recently acquired a p53 gene mutation, but which still retain the wild-type allele. The negative dominant effect of mutant p53 over its wild-type counterpart could be a major contributing factor to tumor progression.

E. p53 and the Cellular Response to DNA Damage: A Final Model for p53 Function? In 1992, Donehower et al. 21~created a surprise with the publication of their article describing mice which, though lacking both p53 alleles, are viable and able to produce offspring. Nevertheless, null mice have a very high incidence of cancers over a three- to six-month period. This suggests that the p53 gene is clearly not essential for cellular viability, but may be involved in a more subtle set of regulations of cell proliferation. An important step in the cellular response to DNA damage is the shutoff of DNA replication, which is assumed to enable optimal repair of damage before the cell reinitiates DNA synthesis and begins mitosis. This transient inhibition of cell division occurs via both G 1 and G2 arrest.

Figure 8. Model for p53 function in the pathway induced by DNA damage. In this

model, DNA damage leads to stabilization of the p53 protein, possibly by a post-translational modification of the protein. The p53 protein alters the transcription activity of target genes leading to G1 arrest of the cell, causing either successful repair of the damage or a suicide response. Alteration in p53 function via different mechanisms does not enable such arrest of cellular division and leads to multiplication of cells with increasing numbers of alterations. This model is derived from the work of Kastan et al.212,213 and is adapted from Lane.216 93

94

THIERRY SOUSSI

In 1984, Maltzman and Czyzyk TM showed that irradiation of nontransformed mouse cells induced an increase in the p53 protein level. More recently, Kastan and co-workers 212 demonstrated that G 1 arrest of human hematopoietic cells induced by gamma irradiation is concomitant with an increased level of p53 protein (Figure 8). Drugs which inhibit DNA synthesis without any DNA damage do not cause p53 accumulation. On the other hand, hematopoietic cells that either lack p53 gene expression or express a mutant form of the p53 gene do not exhibit G 1 arrest after irradiation. These observations were extended to nonhematopoietic cells, such as the colorectal carcinoma cell line SW480 which expresses a mutant p53, and the osteosarcoma cell line Saos-2 which does not express any p53. Furthermore, reintroduction of wild-type p53 in the Saos-2 cell line restores G1 arrest after gamma irradiation. 213 Using another system, Yin et al. and Linvingston et al. 214'215 showed that exposure of normal human fibroblasts to the drug N-(phosphonacetyl)-L-aspartate (PALA) led to growth arrest of the cell at G1 and G2, and failed to generate PALA-resistant colonies by gene amplification. Cells with mutant p5 3 failed to stop growing when placed in the drug, and displayed the ability to amplify at high frequency and thus generate PALA-resistant colonies. Taken together, these data show that p53 is an important component of a subset of signaling pathways by which cells regulate G 1-S transition in response to various physical and metabolic perturbations. As proposed by Lane, 216 p53, "the guardian of the genome", could act as a molecular sensor which monitors the integrity of the genome and triggers a switch so as to inhibit replication when DNA is damaged (Figure 8).

VIII. p53 IN DIFFERENTIATION AND EMBRYOGENESIS Several lines of evidence indicate that p53 is involved in early embryonic development and during differentiation. The p53 protein was detected in primary cells of 10- to 14-day-old mouse embryo, but not in 16-day-old mouse embryo. 217Asimilar observation was made in primary cultures of rat and hamster embryos at mid-gestation showing that p53 decreases with the development stage. 217'218 A marked reduction in the amount of mouse p53-specific mRNA was observed from day 11 onward, which is correlated with progress in differentiation. 219 Using in situ hybridization, Schmid et al. showed expression of p53 mRNA in all cells of an 8.5to 10.5-day-old mouse embryo. 22~Upon differentiation, the amount of p5 3 mRNA declined sharply. Louis et al. reported a similar finding during embryonic development of chicken. 221 In X. laevis, large amounts of p53 mRNA are stored in oocytes and behave like a maternal RNA. 222 More recently, the group of V. Rotter has shown that p53 expression is very high during spermatogenesis. 223'224 Using in-situ hybridization, they determined that this expression is specific for the tetraploid primary spermatocytes. Using specific


95

p53 monoclonal antibodies, they also found an accumulation of the p53 protein at this specific phase. 224 This expression at a phase that involved pairing of chromosomes, recombination, and repair of DNA suggest that p53 could play a direct role in these activities. This view is reinforced by the notion which involve p53 as a guard which monitored the integrity of the genome. 216 Undifferentiated embryonic carcinomas cells, thought to be analogous to normal embryonic stem cells, contain relatively high levels of p53 mRNA and protein. Upon differentiation in vitro, there is a marked decrease in mRNA, and it was shown that a posttranscriptional mechanism was involved is this regulation since the rate of p53 expression did not change upon differentiation, ee A similar observation was made in another model of cellular differentiation, the virus-transformed murine erythroleukemia cell line. 23'24 Using another approach, V. Rotter and collaborators found another differentiation pathway regulated by wild-type p53. 225 The L12 cell line is an Abelson murine leukemia virus-transformed lymphoid pre-B-cell line in which the p53 gene is rearranged by integration of a Moloney murine leukemia virus in the first intron of the p53 gene. This cell line does not express any p5 3 mRNA or protein and is highly tumorigenic. Reconstitution of wild-type p53 in this cell line gives rise to stably growing clones which have undertaken differentiation in vitro, as tested by the specific expression of the cytoplasmic m heavy chain or by increased levels of a B-cell-specific surface marker, B220, both of which are absent in the parental L 12 cell line. 226'227 Furthermore, these p53 producer clones failed to produce lethal tumors in syngenic mice. Taken together, these data suggest that p53 expression may be involved during either embryonic development or cell differentiation. On the other hand, null mice without p53 expression apparently have normal embryonic development and are able to produce normal progeny. 21~ There is no clear explanation for correlating these two types of observations. It is easy to invoke "alternative pathways" or "redundancy of function" so as to hide our ignorance, but it is clear that more knowledge of p5 3 function is needed in order to build a model capable of integrating all available data.

IX. p53 AS A TUMOR SUPPRESSOR GENE As stated above, cotransfection experiments using the activated Ha-ras gene and p53 cDNA which were assumed to be the wild type led to the classification of p53 as a nuclear oncogene. 188-~9~Nevertheless, several observations have cast doubt on these interpretations. The first has come from the work of Benchimol and co-workers using murine erythroleukemia induced by the Friend virus as a model. 228-231 They showed that, in cells transformed by this virus, the p53 gene is a frequent target for mutation.

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THIERRY SOUSSI

Most of the leukemic cell clones either expressed a truncated (or mutated) p53 or failed to express any p53 protein. The second observation has come from the work of Jenkins et al. 232 who report that cellular immortalization was stimulated when using an artificial mutant p53 construct. The finding that one murine p53 cDNA clone isolated from the F9 cell failed to cooperate with an activated Ha-ras gene was another clue that the p53 cDNA clones differ from one another in their behavior.191 Examination of all murine p53 cDNA clones available revealed several codon changes which were primarily assumed to be due to polymorphism. However, comparison of these sequence differences with p53 from lower species indicated that some of them occur in highly conserved regions and do not lead to a conserved amino acid. Careful reinvestigation of all sequences led to the conclusion that the F9 cDNA clone was a wild type, while most of the others used in transfection experiments contain point mutations which activate their transforming properties. A new set of experiments has shown that cotransfection of a plasmid encoding wild-type p53 reduced the transformation potential of plasmids encoding p53 and an activated Ha-ras gene. 192'193 Furthermore, wild-type p53 was shown to suppress transformation by a mixture of E1A or myc and an activated Ha-ras gene. These transformation experiments indicate that wild-type p53 is a suppressor of cell transformation in vitro. The final set of observations leading to the notion of p53 as a tumor suppressor gene has come from the high rate of p53 mutations found in human cancers (see Section X), and the fact that it is implicated in human hereditary cancers via germline mutations.

X. p53 ALTERATION IN H U M A N CANCER For some time, molecular biology has been describing alterations in numerous oncogenes in human cancer. These studies were facilitated by identification of these genes as DNA sequences which could be transduced by oncogenic retroviruses. The isolation and characterization of their cellular counterparts led to intense investigation, with the description of dominant alterations found in a wide variety of human tumors (see other chapters in this volume). Nevertheless, several lines of studies suggest that inactivation of tumor suppressor genes is also an important event in the development of human malignancies. Such inactivation (through deletion or mutation) is generally linked to the loss of the other allele via various mechanisms. 233,234

A. Frequencyof the p53 Mutations in Human Cancer Using colorectal carcinoma as a model of tumor progression, Vogelstein et al. showed that LOH of the short arm of chromosome 17 (17p) was a frequent event


97

(75%) in this cancer. 235 Using the new development of PCR technology, they were able to show that point mutations in the p53 gene were present in DNA from the two tumors examined, one at position 143 and the second at position 175. 236 In another report, Takahashi et al. 237 demonstrated the presence of point mutations in the p53 gene from lung carcinoma in which 17p LOH was also a frequent event. Finally, Nigro et al. 238 published a series of 20 point mutations found in the p53 gene DNA extracted from various tumors (colon, lung, brain, and breast). In that report, the authors revealed that the point mutations did not occur randomly, but were "clustered in four hot spots which exactly coincided with the four most highly conserved regions of the gene." Two cancer types, osteosarcoma 239 and chronic myeloid leukemia (CML), 24~show a high proportion of gene rearrangement which are not seen in other cancer types. Since these initial reports, more than 150 publications have shown that p53 mutations are found in the majority of human tumors (Table 6). 241'242 In several reports, examination of normal tissue from patients shows an absence of mutation, reinforcing the idea that modifications seen in tumoral tissues are tree mutations and not polymorphism. Concerning the p53 mutation rate in specific cancers, several independent reports from various laboratories describe mutation frequencies that may vary greatly within a given cancer type. It is important to keep in mind that many parameters can affect the frequency of mutation. Some of them derive from the strategy or the technical approach used for the study. Most of the sequencing studies were focused on exons 4 to 8 which contain the hot spot of mutations. It is now established that at least 10% of mutations lie outside this region. This value is higher for certain specific cancers such as the squamous cell carcinoma of the skin. Amplification of exon region may also miss some splicing mutations, which account for 5% of total mutations. Furthermore, use of indirect molecular analysis such as SSCP, DGGE, CDGE, or HOT technology can speed up the process of mutation detection, but none of these attained a 100% detection value. The second reason for the discrepancy in the mutation rate among various authors may be due from a bias in the selection of patients at various stages of development in tumor progression. Finally, we cannot exclude the possibility that some geographical variations may occur (see paragraph on breast carcinoma243). In 1989, Bos 244 published a review on the incidence of ras gene mutation in human tumors. Ha-ras, K-ras, and N-ras mutations were shown to be present in a wide variety of tumor types, although the incidence varied greatly. The highest incidence was found in adenocarcinomas of the pancreas (90%), colon (50%), lung (30%), thyroid tumors (50%), and in myeloid leukemia (Table 6). Among the 10 most frequent cancers throughout the world, ras gene alterations are present in 10 to 15% of all cancer patients, p53 alterations are remarkably more frequent since they occur in 40 to 45% of total cancers with an incidence which varies from one tumor type to the other (Table 6). No specific correlation between p5 3 and the ras alteration could be drawn from these data.

Table 6.

Rank

Frequency of p53 and ras Mutations in the 12 Most Frequent Human Cancers Worldwide a

CancerType SCLC

Frequency of ras Mutation 30%

1

NSCLC

low (1-5%)

2

GASTRIC

low (1-5%)

3

4

5

6

BREAST CA.

low (1-5%)

COLORECTAL 50%

CERVIX

low (1-5%)

NASOlow PHARYNGEAL

Frequencyof p53 Alteration

Method

Ref

d' Amico et al.

16/20

Dir. seq.

352

Hensel et al.

6/10

SSCP (4-9)

353

Miller et al.

20/27

Dir. seq. (4-8)

354

Sameshima et al.

23/27

SSCP (2-11)

355

Takhashi et al.

11/15

Dir. seq.

356

Lohmann et al.

18/28

SSCP(5,7-8)

357

Chiba et al. Kishimoto et al. Mitsudomi et al.

23/51 60/115 57/77

RNase Prot/Dir. seq. 358 SSCP 359 SSCP (5-9) 360

Suzuki et al.

14/30

Dir. seq.

361

Kim et al. Matozuki et al.

6/10 7/12

Dir. seq. Dir. seq.(5-9)

362 363

Seruca et al.

3/9

CDGE (5-9)

364

Tamura et al.

9/24

SSCP (no seq.)

365

Yamada et al.

6/12

SSCP (5-11)

366

Renault et al. 15/29 Borensen et al. 11/32 Chen et al. 2/13 Davidoff et al. 7/49 Coles et al. 41/137 Kovach et al. 4/11 Mazars et al. 18/96 Osborn et al. 11/26 Runnenbaum et al. 10/59 Sommer et al. 14/44

DGGE (5-8) CDGE (5-9) Dir. seq. (5, 7-8) Dir. seq. (5-8) HOT (5-9) Dir. seq. (5-9) SSCP (2, 5-9) SSCP (4-9) SSCP (5-9) Dir. seq. (5-9)

367 368 369 274 261 370 277 371 276 243

Moll et al. Thorlacius et al. Baker et al. Cunninghan et al. Ishioka et al.

7/27 18/109 23/33 10/15 8/14

Dir. Seq. (1-11) CDGE (5,7-8) Dir. seq. (5-9) Dir. seq. (5-9) Dir. seq. (4-9)

372 373 245 374 375

Kikuchi et al. Lothe et al. Rodrigues et al. Shaw et al.

45/96 14/33 5/7 16/24

SSCP (5-8) CDGE (5-9) Dir. seq. (5-9) Dir. seq. (5-8)

273 376 377 378

Crook et al. Crook et al.

2/8 (a) 3/24 (a)

Dir. seq. Dir. seq.

294 296

Sheffner et al

2/2 (a)

Dir. seq.

295

Fujita et al.

2/36(a)

SSCP (5-8)

300

No data available (continued) 98

Table 6. (continued) Rank 7

Cancer Type LYMPHOMA

Frequency of ras Mutation variable b

Frequency of p53 Alteration

Method

Ref.

Farell et al.

10/12 (c,d) SSCP (5-8)

379 380

Gaidano et al.

9/27 (c)

381

Bhatia et al.

10/27 (c)

SSCP (5-8) Dir. seq.

17/37 (c,d) 8

LIVER

15

ESOPHAGEAL low (1-5%)

10

11

12

PROSTATE

BLADDER

LEUKEMIA

Bressac et al.

5/10

Dir. seq. (5-8)

382

Hsu et al.

8/16

Dir. seq. (5-8)

285

Murakami et al.

7/43

SSCP(2-11)

383

Oda et al.

17/26

SSCP (5-8)

384

Ozturk et al.

12/79

Special (249)

286

Scorsone et al.

21/36

Special (249)

385

Sheu et al.

20/61

Dir. Seq. (5-8)

386

Oda et al.

49/169

SSCP (5-8)

287

Hollstein et al.

2/15

Dir. Seq. (5-8)

387

Buetow et al.

10/51

SSCP (5-8)

388

Bennett et al.

5/10

Dir. seq. (5-8)

389

Casson et al.

6/24

SSCP (5-8)

290

Hollstein et al.

15/34

Dir. seq. (5-8)

288

Hollstein et al.

7/18

Dir. seq. (5-9)

289

Wagata et al.

15/32

SSCP (2-11 )

390

Huang et al.

14/25

SSCP (5-8)

391

low (1-5%)

No data available

10-15%

low rate of mutation Fujimoto et al.

8/23

SSCP (4-11)

392

Sidranski et al. Spruck et al.

11/18 29/80

Dir. seq. (5-9) SSCP (5-8)

393 394

variable b

variable but less than 10%

395,38 1,396398

Notes: aAIIdata concerning p53 were compiled from 200 communications published before August 1993 (T. Soussi,

unpublished data) and include all type of mutational events (point mutations, deletions, insertions and splice mutations). It is essential to keep in mind that, in more than 50% of the studies performed, only exons 4 to 8 were examined. Furthermore, several molecular studies were performed using SSCP, DGGE, CDGE, or the HOT approach for prescreening of the region to be analyzed and this is not fool proof. Thus, all the information described in this table is underestimated by 5 to 15% (seeTM for discussion). bRas data were taken from the work of BosTM and Rodenhuis.335 CRank of cancer throughout the world is from Parkin et al.336 aOnly tumor or cell lines without HPV (or with a low copy number) have p53 mutations (see text for more details). eFor leukemia and iymphoma, the rate of ras mutation is usually very low except for specific cancers such as acute leukemias (mainly of the myeloblastic type) and for myeleodysplastic syndrome. fOnly Burkitt lymphomas are described as they have been the subject to extensive study. gOnly cell lines have been studied.

99

O ..,.a

Figure 9. Distribution of p53 mutations in human tumors. Conserved regions (I to V) through evolution are indicated in the schematic representation of the p53 protein in each diagram. Data were compiled from 230 communications published before August 1 993 (T. Soussi, unpublished data). Only point mutations are taken into account in this figure.

102

THIERRY SOUSSI

Table 7. Distribution of Mutational Events in Different Types of Cancer a

Note:

aAll data were compiled from 200 communications published before July 1993 (T. Soussi, unpublished data) and correspond to the point mutation shown in the figure 9. Hatched box in GC to AT transitions corresponds to CpG mutation.

B. Distribution of p53 Mutations in the Molecule A compilation of published data on more than 300 human tumors with p5 3 point mutations has been published, 241'242 and at present more than 1000 mutations of thep53 gene have been described. Distribution of the mutations is shown in Figure 9. As reported by Nigro et al., 238 95% of the mutations are clustered in the central part of the molecule and 57% can be found in a hot-spot region (HSR). A total of 3% of the mutations occur at amino acids specific for human p53 (27/987), 13% at amino acids specific for mammalian species (129/987), and 84% at amino acids conserved in all vertebrate p53 species.


103

Four HSRs (A to D) were initially described by Nigro et al. 238 and Baker et al. 245 on the basis of mutations found primarily in colorectal carcinomas. Later, Caron de Fromentel and Soussi 242 described a new HSR found predominantly in lung carcinomas (HSR A') (Figure 9). Among the five HSRs defined above, the three amino acids that are the main targets for mutation are Arg 175,Arg 248, and Arg 273. They are hit 55, 98 and 65 times, respectively (22% of total missense mutations). It should be noted that the codon for Arg 213 is hit 26 times, codon Gly 245 44 times and codon Arg 282 34 times; thus they can also be considered as hot-spot codons. Codon Arg 249 (hit 61 times) will be discussed later in this chapter. All codons described above contain a CpG dinucleotide which has a high rate of mutation (see below).

C. Mutational Events, p53 Mutations, and Cancer Types It is generally assumed that a decrease in genetic stability of the genome is involved in the accumulation of a multitude of genetic changes leading to the selection of a neoplastic cell. There are two types of mutagenic events affecting DNA: external (exogenous) events, involving environmental factors; and internal (endogenous) events, resulting from errors in the mechanisms involved in nucleic acid metabolism. In the first case, the mutagenic agent determines the nature of the lesion: thymine dimer formation follows UV irradiation; GC/AT conversion occurs in the presence of nitric acid, etc. In the second case, mutations (depurination, replication errors, etc.) appear to be spontaneous. CpG dinucleotides are frequently subject to this type of mutation, explaining their under-representation in vertebrates. The high mutability of CpG dinucleotides is well documented and is attributed to the presence of 5-methylcytosine residues in these dinucleotides in the mammalian genome. Deamination of 5-methylcytosine can generate a C to T or G to A transition. The CpG transition found in neoplastic cells can be provoked either by a higher deamination rate of 5-methylcytosine, or by a lack of reparation of the GT mismatch obtained after deamination (Table 7).

D. Immunohistochemical Analysis of p53 Accumulation in Tumor Cells Molecular analysis of the p53 alteration generally involves PCR amplification of tumoral DNA and sequencing of either the HSR (exon 4 to 8, 800 bp) or the entire coding region (2000 bp). Such PCR-based methods are not suitable for routine practice since they are expensive and time-consuming, require special equipment, and necessitate the handling of radiolabeled molecules. As stated above, most alterations found in human cancers are missense mutations leading to the expression of a mutant p53 which accumulates in the nucleus of the tumor cell. This observation has encouraged intensive investigation of the expression of the p53 protein via immunohistochemistry on a large panel of tumors, as there appears to be a good correlation between p53 gene mutation and protein

104

THIERRY SOUSSI

accumulation. Several thousand samples have already been analyzed, due to the rapidity and simplicity of the assay 246--259 (see also Ref. 260 for discussion). Typically, immunostaining of the p53 protein is confined to the nucleus. This implies that nuclear localization is rarely, if ever, the site of mutation in tumors and that mutant proteins, particularly those that have acquired a dominant transforming activity, remain in the nucleus to exercise this function. There is good agreement, for a given type of cancer, between the frequency of positive samples found by immunohistochemical analysis performed without screening for DNA mutations, and the frequency of tumors with mutations detected directly by DNA sequencing. This concordance is particularly clear in the case of colorectal or lung cancers. Studies in which both analyses were conducted in parallel on the same sample are especially informative. As expected, p53 overexpression is usually accompanied by the presence of mutated p53. Exceptions do exist, but these are generally consistent with a molecular basis for the mutation. Tumors with nonsense or frameshift mutations that result in production of an unstable truncated protein are immunostain-negative, as expected, as are cells with mutations in the RNA splice that cannot be correctly transcribed. These categories of mutations are estimated to account for less than 15% of human tumor p53 mutations, depending on the cancer type. Overexpression without the p53 mutation has been observed, especially in breast carcinomas where there is some discrepancy between the rate of p53 mutation (between 30 and 40%) and p53 overexpression (60%). 261 Wild-type p53 stabilization through its interaction with the mdm-2 gene product which is amplified in one-third of sarcoma, has been reported. 151 Immunostaining studies of tumor cells require antibodies with very high specificity, an absence of cross-reactivity with another cellular protein, a high affinity for the antigen regardless of the method of tissue fixation, and an unlimited supply. This has led to the production of a new panel of mAb specific for human p5 3. 262,263

E. Serological Analysis In 1979, DeLeo et al., 264 showed that the humoral response of mice to some methylcholanthrene-induced tumor cell line such as MethA was directed toward the p53 protein. Later, it was found that animals bearing several types of tumors elicited an immune response specific for p53. 265-267In 1982, Crawford et al. 268 first described antibodies against human p53 protein in 9% of breast cancer patient sera. No significant clinical correlation was reported, and at that time no information was available concerning mutations of the p53 gene. Caron de Fromentel et al. later found that such antibodies were present in sera of children with a wide variety of cancers. 269 The average frequency was 12%, but the figure was 20% in Burkitt lymphoma. Davidoff et al. 270 showed that the presence of p53 autoantibodies in patients with breast carcinomas is associated with a specific subset of p53 mutants located in exons 5 and 6 of the gene, and which bind tightly to hsp70. These mutants are known

The p53 TumorSuppressorGene

105

to have enhanced transforming properties in vitro. In lung carcinomas, Winter et al. 271 showed that development of antibodies in lung cancer patients was dependent on the type of p53 mutation. They showed that the immune response was found only when p53 accumulation was detected in the tumor cell, but mutation or overexpression did not automatically lead to a p5 3 immune response and there was no correlation with the location of the mutation. For example, a mutation at codon 248 (exon 7) was tested in one breast carcinoma and four lung carcinomas, p53 antibodies were found in only one patient with lung carcinoma. 271 Other parameters therefore appear to be involved in this immune response. They may depend on several factors, such as the stage of the tumor, the immune response of the patient, or its MHC group. Schlichtholz et al. 272 performed a detailed analysis of patients with breast carcinomas. They showed that 15% (15/100) of primary breast cancer patients had circulating antibodies to p53 protein when tested either by immunoprecipitation or immunoblot. They found a close correlation between the presence of such antibodies and a poor prognosis, such as a high histological grade and the absence of hormone receptors. Similar correlations were noted with p53 mutations found by sequencing or with p53 expression detected by immunocytochemical analysis. Studies of the antibodies present in sera indicated that they recognized wild-type or mutant p5 3. It was found that the B-cell response to p5 3 protein was induced by two immunodominant regions located at the carboxy- and amino-terminus of the protein, outside the central mutational HSR. 272 These observations were extended to other carcinomas, such as lung or prostate, and also to lymphoma and leukemia. A similar immune response was found in animals immunized with human p5 3. All these results suggested that the p53 immune response in patients was due to the accumulation of mutant p53 in the nucleus of the cell. The protein may either have been released during tumor cell necrosis, or else translocated to the surface of the cell, inducing a B-cell response as a result of a breakdown in immune system tolerance. 272 Serological analysis of p53 alterations in human cancers is in fact at an initial stage, and is currently being undertaken in several laboratories. As stated in Table 8, such an analysis presents certain advantages.

Colorectal Carcinoma p53 mutations have been described in about 50 to 60% of colorectal carcinomas (see Table 6). LOH of the short ann of chromosome 17 is also found in most of these tumors and are associated with aggressive tumors. 236'245Analysis ofcolorectal adenomas shows that the p53 mutation is rare in such tumors. Studies at various tumor stages in colorectal carcinomas demonstrated that p53 mutations are generally a late event which is followed by loss of the remaining wild-type allele. In a study of 274 colorectal tumors of four histopathological grades, Kikuchi-Yanoshita, et al. 273 showed that the p53 mutation and LOH of the 17p chromosome were found

Table 8. Multifactorial Analysis of p53 Alterations in H u m a n C a n c e r Moleculctr Analy~is Diracr

2

a cn

Resulrs

DNA sequencing of exon 4-8 DNA sequencing of p53 gene DNA sequencing of thc coding region of the cDNA Identification of mutation

hldirecr

Detection of p53 protein in lrnmunoblot SSCP tumor cells using either ImmUnoprecipitation polyclonal or monoclonal CDGE antibodies HOT Knoulcdgc of thc prcscncc of Accumulation of p53 protein Presence of p53 antibodies in a mutation in the tumor cell patient sera

YCS

Yes

Nonsense mutation in exon Deleliontinsertion out of frame in exon Splice muration

Yes

Yes Yes

Gene deletion (2 allelcs) Promoter mutation

p53 alteration (via stabilization ?) without mutation

Ycs Yes Depend of the primer used for sequencing NO

Semlogical Analysis

DGGE

Missense mutiltivn in exon (80 to 90% ofthe mutation)

Yes

Imnnuuor,v~ochemiculAnalysis

Depends on the primer used for amplification Yes Depends on the primer used for amplification No

Found in most

Found in some

No

No

No No

No No

Yes

Yes

Table 8. (continued) Molecular Atlalysis Direct

In~munocyfochetnicalAnalysis

Serological Analysis

Indirect

- Exact knowledge of the mutation - Very rapid event - Can identify all mutation events

- Can be used to screen a

Cannot be performed in routine diagnosis at present

Accuracy of the methods is between 80and90%

Tumor tissue required

Cannot be performed in routine diagnosis Tumor tissue required

large number of patients

Can be performed in routine Can be performed in routine diagnosis diagnosis Can identify p53 stabilization Do not require tumor tissue without mutation Can be easily used for patient followup Can identify p53 alteration (stabilization?) without mutation Some mutations do not Some mutations do not induce p53 overexpression induce the production of p53 antibodies Tumor tissue required

108

THIERRY SOUSSI

Figure 10. Pattern of p53 mutations in colorectal tumors from FAP and non-FAP

patients. Four histopathological grades were tested: Ad md: adenomas with moderate dysplasia. Ad sd: adenomas with severe dysplasia. Int. ca: intramucosal carcinoma. Inv. ca: invasive carcinoma. Dark and light shades correspond to FAP and non-FAP patients, respectively. All data are taken from the work of Kikushi-Yanoshita et al. 273

more frequently in carcinomas than in early adenomas (Figure 10) in both familial and nonfamilial adenomatous polyposis patients. All these data suggest that p53 alterations are associated with the conversion from colorectal adenoma to early carcinoma. Analysis of the mutational event leading to p53 gene mutations in this carcinoma indicates that 80% of the mutations are GC->TA transversions which are predominantly located at the CpG dinucleotide. On the other hand, GC->TA transversions, deletions, insertions, or splice mutations are a very rare event.

Breast Carcinoma p53 mutations are found in about 30% of breast carcinomas (see Table 6). There is a high rate of missense mutations (50%) and LOH of the short arm of chromosome 17. Mutations at the CpG dinucleotide are a much less frequent event (25%, 31/123) than in colorectal carcinoma, while G->T transversion is a more frequent event (20%). The reason for this high rate of transversion is not known. In a study of breast cancer in women in the midwestern United States, Sommer et al. 243 found alterations in the p53 gene in 32.6% (14/44) of patients, but some of them were


109

microdeletions (4) or splice mutations (1) which have never been described elsewhere for this kind of cancer. The authors suggest that this pattern of mutation could reflect the presence of a mutagen or genetic predisposition which would influence p53 to a greater extent in this geographical region, but it could also be due to a selection bias or to a small sample size. Some studies have shown that the p53 mutation occurs relatively early in the development of breast cancer, 274,275 before the malignant cells have developed the ability to invade tissue. In a recent report, Coles et al. 261 reviewed all data concerning the p53 alteration in breast carcinoma (molecular and immunohistochemical analyses). The authors described a significant discrepancy between results of molecular analysis (30 to 40%) and those of immunocytochemistry (60%), suggesting that p53 stabilization can occur via some mechanisms other than mutation in the gene and which might be an important mechanism in breast carcinoma. p53 alterations in breast carcinoma analyzed via molecular, 276'261'277'243 immunohistochemical, 246'278-28~and serological methods 272 are always associated with pathobiological factors such as estrogen receptor negativity and high nuclear grade, which are known to be very bad prognostic factors. Multivariate survival analysis indicates that p53 alteration is associated with a shorter survival period.

LungCarcinoma p53 mutations in lung carcinomas have been extensively studied and reveal surprising features. The p53 mutation rate is slightly smaller in non-small cell lung carcinomas (NSCLC) (45 to 50%) than in small cell lung carcinomas (SCLC) (60 to 70%) (see Table 6 and references therein). In NSCLC, p53 mutations were not significantly associated with tumor stage, nodal status, or sex, and were found in all histological types, suggesting that the p53 mutation is an earlier event in this type of cancer. In SCLC, p53 alterations were also found at early and late clinical stages. LOH of the short arm of chromosome 17 was found in only 25 to 35% of the tumors for both SCLC and NSCLC. Analysis of the mutational event leading to p53 mutation revealed a predominance of GC->TA transversions which were not found in other tumor types (except for hepatocarcinoma, see below). Lung cancer is known to occur in smokers, and some carcinogens such as benzo(a)pyrene induce this substitution. There was also a remarkable bias in the distribution of the GC->TA transversions. In 95% (83/88) of cases, the guanine residue was located on the nontranscribed strand (a similar observation has been made for most GC->TA transversions in all tumor types). This observation is generally attributed to the preferential repair of the transcribed strand or increased accessibility of the opposite strand to electrophilic attack. A fifth HSR, (designated HSR A') was identified when mutations from all cancer types were evaluated. 242 A total of 49% (29/59) of the mutations observed in this HSR were found in lung carcinomas and 93% (27/29) of them were G-> T

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transversions. No mutations were observed in this region in colorectal tumors. Of the 30 nucleotides in this region, 25 were G or C, supporting the notion that this region is highly susceptible to chemical carcinogens A study of radon-associated lung carcinoma from uranium miners showed a high proportion of p53 gene alterations (37%, 7/19). None of the mutations were GC->TA transversions; they were not found in the HSR, and they were predominantly other transversion types. They were clustered in regions 146-161 and 195-208, suggesting that this may be a hot spot for mutations by radon-induced ionizing alpha radiation. Using immunohistochemistry analysis, several studies have shown that accumulation of p53 protein correlates with a poor prognosis in human lung cancer.281-283,259

Hepatocellular Carcinoma In 1991, two reports described mutations of the p53 gene in hepatocellular carcinomas (HCC) from southern Africa and eastern Asia. 284'285 In both cases, a high predominance of G->T transversions was found. Furthermore, Bressac et al. 284 showed that four of five mutations were clustered at codons 249, whereas Hsu et al. 285 found that all mutations (eight) were clustered at that same position. It was then suggested that one of the risk factors endemic to these geographical regions, the common food contaminant aflatoxin B 1, was responsible for the mutations. This hypothesis was reinforced by the work of Ozturk et al., 286 which showed that HCC from low-aflatoxin-exposure areas only rarely contained the Arg249 mutation, whereas HCC from areas with high-aflatoxin exposure had a high rate of Arg249 mutations. More recent work on HCC in an aflatoxin B 1 low-exposure area (mainly Japanese patients) revealed that 29% (49/169) tumors show p53 mutations which were distributed in the central part of the molecule. 287 HCD IV and V contained 65% of all mutations and codon 249 was the most frequent mutation site (7/49) but only two of them have a mutational event similar to those found in an aflatoxin B 1 high exposure area. The spectrum ofp53 mutation did not differ among HCCs in relation to the type of hepatitis virus infection, sex, age, and background liver disease, but incidence and site were significantly associated with the degree of differentiation of cancer cells.

Esophageal Carcinoma The pattern of substitution found in esophageal carcinoma is intermediate. Transversions GC->TA are frequent (25%, 8/33), 288'289 suggesting the occurrence of carcinogenic risk factors such as tobacco or alcohol consumption which have been found to be associated with such carcinomas, p53 mutations were also found in Barret's epithelium, 29~which is considered to be a precursor of adenocarcinoma of the esophagus or in preinvasive lesions in esophageal squamous cell carcinoma.


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All these observations suggest that p53 mutations in esophageal carcinoma are an intermediate event which could confer a growth advantage upon the pre-invasive cells, thereby contributing to malignant conversion.

Gastric Carcinoma Gastric carcinoma is one of the most frequent tumors worldwide; paradoxally, it has rarely been studied in the context of detection of p53 mutations. Only 21 point mutations have been reported, and they show a predominance of GC->AT transitions (see Table 6); however, more data are necessary in order to assess the significance of mutational events in this cancer. Mutations have been detected in aneuploid tumors, but not in diploid tumors. No correlation has been found between p53 mutations and the degree of histological differentiation of the tumors.

Skin Carcinoma Epidemiological studies have identified causal agents for many human cancers; in squamous cell carcinoma of the skin, one of these is UV light. This mutagen produces specific mutations which are predominantly C->T transitions at dipyrimidine sites, including CC->Tr double-base mutations. More than 50% of skin SCC studied showed p53 mutations, with all of them at the dipyrimidine sites. 291-293Furthermore, some tumors contained the double-change base CC->TT. Such events have not been found in other cancer types tested so far. This observation strongly indicates that the p53 gene is the direct target of mutations produced by UV in such cancers. Cervix Cancer

There is recent evidence associating specific human papillomaviruses (HPV) with certain human anogenital cancers, most notably cervical cancer. 3'34 Recent studies have demonstrated that 84% of cervical carcinomas contained DNA from a high risk HPV (mostly HPV 16 and 18 and to a lesser extent, HPV 31,33, 35, 39, 45, 51, 52, and 56). The DNA is usually found to be integrated, but there are some cases where it is apparently extrachromosomal. The finding that E6 protein from high-risk HPV can induce the degradation of p53 either in vitro or in vivo has led to the proposal that such an inactivation pathway could be involved in the neoplastic process leading to a cervix cancer. This observation prompted some investigators to study the distribution of p53 mutations in human primary cervical carcinoma (or cell lines) with and without HPV infection. In a first report, Crook et al. 294 showed that six HPV-positive cervical cell lines expressed wild-type p53, whereas two apparently HPV-negative lines expressed mutant p53. Scheffner et al., 295 reported that two other HPV-negative cervical cell lines expressed mutant p5 3 (see Table 6). More recently, analysis of tumor samples from 28 women with primary cancer of

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the cervix showed that 25 were HPV (16 or 18) positive but sequencing of the entire coding region of the p53 gene failed to reveal any mutation. 296 By contrast, sequencing has revealed point mutations in p53 from the three HPV-negative tumors. The fact that HPV-negative carcinomes have a worse prognosis than HPV-positive ones reinforces these results. Inactivation of p53 by E6 protein only leads to the loss of functinal p53, whereas somatic mutation results in the expression of an altered p53 protein, which interfering with wild-type p53 can elicit positive transforming activity. This very attractive model is still subject to some controversy as several authors do not find such inverse correlation between HPV-positive cancer and p53 mutation. 297-299 A recent report from Fujita et al. 3~176 suggests that the number of HPV copies present in the transformed cell could be a more critical paramater.

Cerebral Tumors Brain tumors progress through three histopathologically defined stages: the premalignant stage of low grade astrocytoma; and two malignant stages, that of anaplastic astrocytomas and that of multiform glioblastomas. Using a conventional approach involving direct sequencing of the p53 gene from tumor cell, p53 mutations were found predominantly in anaplastic astrocytomas and glioblastomas. 3~176 No mutations were found in low-grade astrocytoma. Most of the mutations were correlated with LOH on chromosome 17p. Using a highly sensitive assay, Sidransky et al. 3~ were able to show that a subpopulation of cells was present in low grade astrocytomas (8 - 20%) which contained the same p53 gene mutation predominant in the cells of recurrent tumors that had progressed to glioblastoma. This result unequivocally indicates that cells with a mutation in the p53 gene are selected during tumor progression.

Hematological Disorders

p53 in Chronic Myeloid Leukemia. Chronic myeloid leukemia (CML) is a clonal disorder of pluripotent hematopoetic stem cells with a biphasic clinical course. The initial chronic phase, with the Philadelphia chromosome anomaly (Ph), is usually followed by an acute blast crisis phase characterized by an increase in cell proliferation, arrest of maturation, and new karyotypic abnormalities (+8, +Ph, i(17q) (14). An isochromosome 17q[i(17q)] (i.e., deletion of 17p and duplication of 17q) occurs in about 20% of patients with Ph 1-positive CML in the acute phase, mainly of a nonlymphoblast type. 3~ Initial studies have shown that 20 to 30% of patients in blastic crisis exhibit rearrangements of the p53 gene, whereas it remains a rare event in patients in a chronic phase. 24~176 More recent studies using PCR amplification and DNA sequencing have also shown that p53 inactivation via point mutations can also be detected in patients in blastic crisis. An attempt to correlate


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p53 mutations with chromosome 17p abnormalities (consistent with the recessive model of tumor suppressor activity of the p53 gene) has been carried out on four cases, but more cases must be examined to confirm this correlation. Nevertheless, a clear correlation has been observed between the p53 mutation and myeloid blast crisis in CML, suggesting that alterations in p53 are involved in the progression of CML. On the other hand, studies of lymphoid blast crisis have failed to detect any p53 alteration.

p53 in Lymphoid Malignancies. AnalysisofT-celltumorsshowsthatthep53 mutation is a very rare event. On the other hand, numerous mutations were found in established malignant T-cell lines, This discrepancy can be explained either by selection of the cells that carry a p53 mutation during establishment of the cell line, or by alterations in the gene during this process. Analysis of B-CLL (chronic lymphocytic leukemia) shows that p53 mutations occur at a frequency of 10 to 15%. In Richter's syndrome, which corresponds to the aggressive evolution of CLL, 3 out of 7 cases (40%) presented a p53 mutation. This situation can be compared to that of blastic evolution in CML, where p53 mutations seem to be correlated with evolution of the neoplasia. The most frequent lymphoid malignancies which is subject for p53 mutations are the Burkitt type of ALL (L3) and Burkitt's lymphoma (BL). The two neoplasias share similar cell morphology and chromosomal translocation leading to Myc activation. Thus far, no correlation between p53 inactivation and Myc activation has been revealed, but the role of the two proteins in regulation of cell proliferation suggests that these two events may act together in the establishment of the transformed phenotype. A discrepancy has been observed in the number of mutations in BL tumors (35%) and cell lines (70%) which can be discussed as above for T cells. Statistical analysis of available data from BL tumors indicates that regions from codon 209 to 216 and from codons 234 to 243 contain a statistically significant large proportion of mutations compared to the proportion in the same region of other tumors. In contrast, the region from codons 270 to 286 contains a statistically significant lower proportion of mutations in BL relative to other tumor types. In other hemopathies, such as myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), the number of p53 mutations found was very low despite the large panel of patients studied. However, the p53 gene mutations detected were often associated with several chromosome changes, prominent myelodysplastic features and poor response to treatment, suggesting advanced disease, probably resulting from a multistep process. Mutational events leading to p53 mutation in hematological disorders were found to be very similar to those found for colorectal carcinomas. More than 50% of the mutations were GC->TA transitions and 70% of them occurred at CpG dinucleotides.

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Hereditary Cancer Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder that predisposes individuals to multiple forms of cancer, including breast carcinoma, soft tissue sarcoma, osteosarcoma, leukemia and adrenoc0rtical carcinoma. 314'315 These diverse tumor types develop at unusually early ages. Transgenic mice harboring a mutant p53 gene have been shown to have an increased incidence of osteosarcomas, soft tissue sarcomas, adenocarcinomas of the lung, and adrenal and lymphoid tumors. 316 This observation led to the notion that p53 might be one of the hereditary components of LFS. The first study published by Malkin et al. 317 showed that germline p53 mutations could be observed in all five LFS families analyzed. Mutations were found to be clustered in the highly conserved domain (HCD) IV (codon 248 for three independent families; codon 252 and 258 for the two others). Segregation studies showed that the p53 mutations were correlated with family members who developed various sarcomas. Unaffected carriers of the mutation could be predicted to be at high risk for developing cancer. Analysis of the p53 gene in tumoral DNA showed that the normal p53 allele had been lost in two cases examined. 317In another independent report, Srivastava et al. 318 also found a germline mutation in one LFS family. The mutation was found at position 245 in the same area described by Malkin et al. Subsequent studies have found germline mutations of the p53 gene in some LFS families, but not in others, 319 suggesting that this syndrome may be heterogeneous with p53 mutations accounting for only a fraction of LFS families. On the other hand, germline mutations on the p53 gene have been found in young adults with a second primary cancer whose family history was not indicative of LFS 32~ or in other cancer prone family. 3~ Taken together, these reports have led to the notion that: (1) p53 mutations represent the main component which predisposes family members with LFS to increased susceptibility to cancer; (2) germline p53 mutations cannot be identified solely by reviewing the family's history of cancer; and (3) most mutations in LFS are clustered in HCD IV (see Figure 8). This third observation is of interest, since it is well known that not all p53 mutations are fully equivalent. Some mutations induce synthesis of p53 proteins which show drastic changes in their properties (binding to heat-shock protein, cooperation with activated Ha-ras oncogene, a sharp increase in their half-life, induction of p53 antibodies in patient sera, and formation of heterooligomers with wild-type p53), whereas other mutations present only some or none of these properties. 133'2~ Mutations found in LFS seem to have slight changes in their properties. Analysis of the p53Arg 248 mutation showed that it was unable to form heterooligomers with wild-type p53. 2~ It was therefore assumed that the p53 mutations selected in LFS cells were clustered in a region which enabled coexpression of normal and mutant p53 in the cell with no major alterations in regulation of cellular proliferation. Nevertheless, the presence of this mutated p53 led to in-


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creased susceptibility to cell transformation. Tumor cells were found only for the mutated allele of p53, but nothing is currently known concerning events leading to inactivation of the wild-type p53 allele and the transformation process; indeed, other steps are clearly necessary. Mutational spectra found for germline mutations were very similar to those found for colorectal carcinoma, with more than a 50% GC->AT transition at the CpG dinucleotide.

p53, mdm-2, and Sarcomas Sarcomas consist of tumors originating from bone, cartilage, and various types of connective tissues. In 1987, Masuda et al. 239 found that p53 gene was rearranged in human osteosarcomas. This figure was later confirmed by Miller et al. 328 which described a preferential rearrangement of p53 intron one in 18% (11/60) osteosarcomas. More recently, with the notion of p53 as a tumor suppressor gene, p53 mutations were found in different subtypes of human sarcomas. 329-332 The mdm-2 gene is amplified in human sarcomas. 151 As described above, the mdm-2 product inactivates the p53 transactivation function. Then, one would expect that those tumors with mdm-2 gene amplification would be devoid of p53 mutation. This hypothesis was confirmed by Leach et al. 333 Analysis of 24 human soft tissue sarcomas (11 malignant fibrous histiocytomas and 13 liposarcomas) showed that p53 alteration could be found in 24 of the sarcomas and mdm-2 amplification was detected in another 8 tumors; however, no tumor contained an alteration in both genes. Also in brain tumors, mdm-2 amplification was observed in 10% of glioblastomas and astrocytomas. TM These results suggest strongly that mdm-2 amplification can be an alternative molecular mechanism by which p53 is inactivated.

Other Neoplasias p53 mutations have been found in a number of other human neoplasias (see Table 6). Indeed, tumors showing no p53 alterations are rare. A low frequency of p53 mutations has been observed in prostate carcinoma, thyroid carcinoma, meduloblastoma, and T-cell leukemia. However, it is not known whether this reflects a bias in selection of the patients, or is in fact a true underrepresentation of the p53 alteration.

XI. CONCLUSIONS AND PERSPECTIVES In the 13 years since p53 was first discovered, a long list of potential p53 functions has been proposed but often eliminated. Originally, p53 was found to be stably associated with viral antigens in virally transformed cells. Later, this ubiquitous

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protein was potentially considered to be the elusive "tumor antigen" expressed in all transformed cells. Cloning of a mutant p53 gene, assumed to be the wild type, led to the misinterpretation that p53 could function as a dominant oncogene. The finding that diverse sets of tumors have associated point mutations in the p53 gene eventually led to recognition of p53 as a tumor suppressor gene whose functional inactivation is vital to deregulated growth in many tissues. The wild-type protein is expressed in most normal tissues, but is not essential for the viability of the cell. Its function involves a very subtle set of regulations with posttranslational modifications and conformational changes in the protein. Like Dr. Jekyll and Mr. Hyde, there are two p53 isoforms with opposite properties, and an equilibrium between the two forms is essential in order to maintain cellular growth control.'The model describing p53 as a regulator in the cell cycle pathway induced by DNA damage is an attractive idea, since it reconciles several observations made during the past year. One of the most attractive features of p53 is its clinical aspect. Identification of the p53 alteration as the most frequent molecular event in human cancer has led to intensive work on the analysis of p53 status in a wide variety of cancers and the description of more than 2000 mutations. In addition to this knowledge of the p53 status of the patient and its clinical consequences, these studies provide considerable information and material concerning p53 function. It is clear, at present, that all of these mutations are not equivalent in terms of biological activity. It is now necessary to perform more basic research work for the dissection of such p53 mutant activity and its relationship with the transformed phenotype. All these works highlight one of the most exciting aspects of the p53 studies, i.e. the constant exchange between basic research and clinical studies. The finding of germline p53 mutations in families with Li-Fraumeni syndrome or in other patients has raised the possibility of testing at-risk relatives who have not had cancer. This possibility raises important questions concerning biological and medical aspects, but also ethical considerations. Predictive testing for germline p53 mutations among cancer-prone individuals has been recommended. As stated before, the p53 alteration occurs in more than 45% of human cancers. Thus, treatment for malignancies caused by the alteration in p53 (and other tumor suppressor genes) could and should be developed, and it is hoped that, in the near future, the very close relationship between basic and clinical studies will lead both to an improvement in the diagnosis of the p53 alteration and to the development of specific protocols adapted to p53 alterations in human cancers. Another aspect which should be developed concerns the germline mutation of the p53 gene.

ACKNOWLEDGMENTS I am very grateful to J. Brams for her important contribution to the design of this manuscript. I wish to thank R. Berger, C. Larsen, Y. Legros, and K. Ory for critical discussion and to A.


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Braithwaite, E. Brambilla, C.C. Harris, D. Lane, J. Milner, J. Minna, M. Montenarh, B. Vogelstein, and D. Windford-Thomas for sending manuscripts prior to publication.

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284. Bressac, B.; Kew, M.; Wands, J.; Ozturk, M. Selective G-mutation to T-mutation of p53 gene in hepatocellular carcinoma from southern Africa. Nature 1991, 350, 429-431. 285. Hsu, I. C.; Metcalf, R. A.; Sun, T.; Welsh, J. A.; Wang, N. J.; Harris, C. C. Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 1991, 350, 427-428. 286. Ozturk, M., et al. p53 mutation in hepatocellular carcinoma after aflatoxin exposure. Lancet 1991, 338, 1356-1359. 287. Oda, T.; Tsuda, H.; Scarpa, A.; Sakamoto, M.; Hirohashi, S. p53 gene mutation spectrum in hepatocellular carcinoma. Cancer Res. 1992, 52, 6358-6364~ 288. Hollstein, M. C.; Metcalf, R. A.; Welsh, J. A.; Montesano, R.; Hams, C. C. Frequent mutation of the p53 gene in human esophageal cancer. Proc. Natl. Acad. Sci. USA 1990, 87, 9958-9961. 289. Hollstein, M. C.; Peri, L.; Mandard, A. M., et al. Genetic analysis of human esophageal tumors from two high incidence geographic areas - frequent p53-base substitutions and absence of ras mutations. Cancer Res. 1991, 51, 4102-4106. 290. Casson, A. G.; Mukhopadhyay, T.; Cleary, K. R.; Ro, J. Y.; Levin, B.; Roth, J. A. p53 gene mutations in barrett's epithelium and esophageal cancer. Cancer Res. 1991, 51, 4495-4499. 291. Brash, D. E.; Rudolph, J. A.; Simon, J. A., et al. A role for sunlight in skin cancermUV-induced p53 mutations in squamous cell carcinoma. Proc. Natl. Acad. Sci. USA 1991, 88, 10124-10128. 292. Pierceall, W. E.; Mukhopadhyay, T.; Goldberg, L. H.; Ananthaswamy, N. Mutations in the p53 gene tumor suppressor gene in the human cutaneous cell carcinomas. Mol. Carcinog. 1991, 4, 445-449. 293. Kress, S.; Sutter, C.; Strickland, P. T.; Mukhtar, H.; Schweizer, J.; Schwarz, M. Carcinogen-Specific mutational pattern in the p53 gene in Ultraviolet-B Radiation-Induced squamous cell carcinomas of mouse skin. Cancer Res. 1992, 52, 6400-6403. 294. Crook, T.; Wrede, D.; Vousden, K. H. p53 point mutation in HPV negative human cervical carcinoma cell lines. Oncogene 1991, 6, 873-875. 295. Scheffner, M.; Munger, K.; Byme, J. C.; Howley, P. M. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc. Natl. Acad. Sci. USA 1991, 88, 5523-5527. 296. Crook, T.; Wrede, D.; Tidy, J. A.; Mason, W. P.; Evans, D. J.; Vousden, K. H. Clonal p53 mutation in primary cervical cancer - association with human-papillomavirus-negative tumours. Lancet 1992, 339, 1070-1073. 297. Borresen, A. L.; Helland, A.; Nesland, J.; Holm, R.; Trope, C.; Kaern, J. Papillomaviruses, p53, and cervical cancer. Lancet 1992, 339, 1350-1351. 298. Busby-Earle, R. M. C.; Steel, C. M.; Williams, A. R. W.; Cohen, B.; Bird, C. C. Papillomaviruses, p53, and cervical cancer. Lancet 1992, 339, 1350. 299. Mcgregor, J. M.; Levison, D. A.; Macdonald, D. M.; Yu, C. C. Papillomaviruses, p53, and cervical cancer. Lancet 1992, 339, 1351. 300. Fujita, M.; Inoue, M.; Tanizawa, O.; Iwamoto, S.; Enomoto, T. Alterations of the p53 gene in human primary cervical carcinoma with and without human papillomavirus infection. Cancer Res. 1992, 52, 5323-5328. 301. Chung, R.; Whaley, J.; Kley, N., et al. TP53 gene mutation and 17p deletions in human astrocytomas. Genes Chrom. Cancer 1991, 3, 232-331. 302. Hayashi, Y.; Yamashita, J.; Yamaguchi, K. Timing and role of p53 gene mutation in the recurrence of glioma. Biochem. Biophys. Res. Commun. 1991, 180, 1145-1150. 303. Mashiyama, S.; Murakami, Y.; Yoshimoto, T.; Sekiya, T.; Hayashi, K. Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 1991, 6, 1313-1318. 304. Ohgaki, H.; Eibl, R. H.; Wiestler, O. D.; Yasargil, M. G.; Newcomb, E. W.; Kleihues, P. p53 mutations in nonastrocytic human brain tumors. Cancer Res. 1991, 51, 6202--6205. 305. Frankel, R. H.; Bayona, W.; Koslow, M.; Newcomb, E. W. p53 mutations in human malignant gliomas - comparison of loss of heterozygosity with mutation frequency. Cancer Res. 1992, 52, 1427-1433.

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306. Fults, D.; Brockmeyer, D.; Tullous, M. W.; Pedone, C. A.; Cawthon, R. M. p53 mutation and loss of heterozygosity on chromosome-17 and chromosome-10 during human astrocytoma progression. Cancer Res. 1992, 52, 674--679. 307. Von Deimling, A.; Eibl, R. H.; Ohgaki, H., et al. p53 mutations are associated with 17p allelic loss in grade-II and grade-Ill astrocytoma. Cancer Res. 1992, 52, 2987-2990. 308. Sidransky, D.; Mikkelsen, T.; Schwechheimer, K.; Rosenblum, M. L.; Cavanee, W.; Vogelstein, B. Clonal expansion of p53 mutant cells is associated with brain tumour progression. Nature 1992, 355, 846-848. 309. Alimena, G.; De Cuia, M. R.; Diverio, D.; Gastal, D. R.; Nanni, M. The karyotype of blastic crisis. Cancer Gener Cytogener 1987, 26, 39--45. 310. Ahuj a, H.; Bar-Eli, M.; Clark, D., et al.p53 gene alterations in the evolution of chronic myelocytic leukemia. In: Cancer Cells. CSH Laboratory. Cold Spring Harbor, N.Y. 1989, 7, 117-120. 311. Kelman, Z.; Prokocimer, M.; Peller, S., et al. Rearrangements in the p53 gene in philadelphia chromosome positive chronic myelogenous leukemia. Blood 1989, 74, 2318-2324. 312. Mashal, R.; Shtalrid, M.; Talpaz, M., et al. Rearrangement and expression of p53 in the chronic phase and blast crisis of chronic myelogenous leukemia. Blood 19911, 75, 180--189. 313. Feinstein, E.; Cimino, G.; Gale, R. P., et al. p53 in chronic myelogenous leukemia in acute phase. Proc. Natl. Acad. Sci. USA 1991, 88, 6293-6297. 314. Li, E P.; Fraumeni, Jr. J. F. Soft tissuse sarcomas, breast cancer and other neoplasms: a familial syndrome? Ann. Intern. Med. 1969, 71,747-752. 315. Li, E P.; Fraumeni, Jr. J. E; Mulvihill, J. J., et al. A cancer family syndrome in twenty-four kindreds. Cancer Res. 1988, 48, 5358-5362. 316. Lavigueur, A.; Maltby, V.; Mock, D.; Rossant, J.; Pawson, T.; Bernstein, A. High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene. Mol. Ceil. Biol. 1989, 9, 3982-3991. 317. Malkin, D.; Li, E P.; Strong, L. C., et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990, 250, 1233-1238. 318. Srivastava, S.; Zou, Z. Q.; Pirollo, K.; Blattner, W.; Chang, E. H. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature 1990, 348, 747-749. 319. Santibanez-Koref, M. E; Birch, J. M.; Hartley, A. L., et al. p53 germline mutations in Li-Fraumeni syndrome. Lancet 1991, 338, 1490-1491. 320. Malkin, D.; Jolly, K. W.; Barbier, N., et al. Germline mutations of the p53 tumor-suppressor gene in children and young adults with second malignant neoplasms. N. Engl. J. Med. 1992, 326, 1309-1315. 321. Toguchida, J.; Yamaguchi, T.; Dayton, S. H., et al. Prevalence and spectrum of germline mutations of the p53 gene among patients with sarcoma. N. Engl. J. Med. 1992, 326, 1301-1308. 322. Law, J. C.; Strong, L. C.; Chidambaram, A.; Ferrell, R. E. A germ line mutation in exon-5 of the p53 gene in an extended cancer family. Cancer Res. 1991, 51, 6385-6387. 323. Metzger, A. K.; Sheffield, V. C.; Duyk, G.; Daneshvar, L.; Edwards, M. S. B.; Cogen, P. H. Identification of a germ-line mutation in the p53 gene in a patient with an intracranial ependymoma. Proc. Natl. Acad. Sci. USA 1991, 88, 7825-7829. 324. Felix, C. A.; Nau, M. M.; Takahashi, T., et al. Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia. J. Ciin. Invest. 1992, 89, 640--647. 325. Iavarone, A.; Matthay, K. K.; Steinkirchner, T. M.; Israel, M. A. Germ-line and somatic p53 gene mutations in multifocal osteogenic sarcoma. Proc. Natl. Acad. Sci. USA 1992, 89, 4207-4209. 326. Sameshima, Y.; Tsunematsu, Y.; Watanabe, S., et al. Detection of novel germ-line p53 mutations in diselecting patients with childhood adrenocortical carcinoma. J. Natl. Cancer Inst. 1992, 84, 703-707. 327. Sidransky, D.; Tokino, T.; Helzlsouer, K., et al. Inherited p53 gene mutations in breast cancer. Cancer Res. 1992, 52, 2984-2986.

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328. Miller, C. W.; Aslo, A.; Tsay, C., et al. Frequency and structure of p53 rearrangements in human osteosarcoma. Cancer Res. 1990, 50, 7950-7954. 329. Mulligan, L. M.; Matlashewski, G. J.; Scrable, H. J.; Cavenee, W. K. Mechanisms of p53 loss in human sarcomas. Proc. Natl. Acad. Sci. USA 1990, 87, 5863-5867. 330. Stratton, M. R.; Moss, S.; Warren, W., et al. Mutation of the p53 gene in human soft tissue sarcomas--association with abnormalities of the rbl gene. Oncogene 1990, 5, 1297-1301. 331. Toguchida, J.; Yamaguchi, T.; Ritchie, B., et al. Mutation spectrum of the p53 gene in bone and soft tissue sarcomas. Cancer Res. 1992, 52, 6194-6199. 332. Andreassen, A.; Oyjord, T.; Hovig, E., et al. p53 abnormalities in different subtypes of human sarcomas. Cancer Res. 1993, 53, 468-471. 333. Leach, E S.; Tokino, T.; Meltzer, P., et ai. p53 mutation and mdm-2 amplification in human soft tissue sarcomas. Cancer Res. 1993, 53, 2231-2234. 334. Reifenberger, G.; Liu, L.; Ichimura, K.; Schmidt, E. E.; Collins, V. P. Amplification and overexpression of the mdm-2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res. 1993, 53, 2736-2739. 335. Rodenhuis, S. ras and human tumor. Sere. Cancer Biol. 1992, 3, 241-247. 336. Parkin, D. M.; Pisani, P.; Ferlay, J. Estimates of the worldwide incidence of eighteen major cancers in 1985. Int. J. Cancer 1993, 54, 594-606. 337. Hinds, P.; Finlay, C.; Levine, A. J. Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation. J. Virol. 1989, 63, 739-746. 338. Shaulsky, G.; Goldfinger, N.; Benzeev, A.; Rotter, V. Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol. Ceil. Biol. 1990, 10, 6565--6577. 339. Ullrich, S. J.; Anderson, C. W.; Mercer, W. E.; Appella, E. The p53 Tumor Suppressor Protein, a Modulator of Cell Proliferation. J. Biol. Chem. 1992, 267, 15259-15262. 340. Hulla, J. E.; Schneider, R. P. Structure of the rat p53 tumor suppressor gene. Nucleic Acids Res. 1993, 21,713-717. 341. Deb, S.; Jackson, C. T.; Subler, M. A.; Martin, D. W. Modulation of cellular and viral promoters by mutant human p53-proteins found in tumor cells. J. Virol. 1992, 66, 6164-6170. 342. Shiio, Y.; Yamamoto, T.; Yamaguchi, N. Negative regulation of Rb expression by the p53 gene product. Proc. Natl. Acad. Sci. USA 1992, 89, 5206-5210. 343. Santhanam, U.; Ray, A.; Sehgal, P. B. Repression of the interleukin-6 gene promoter by p53 and the retinoblastoma susceptibility gene product. Proc. Natl. Acad. Sci. USA 1991, 88, 7605-7609. 344. Mercer, W. E.; Shields, M. T.; Lin, D.; Appella, E.; Ulrich, S. J. Growth suppression induced by wild-type p53 protein is accompanied by selective downregulation of proliferating-cell nuclear antigen expression. Proc. Natl. Acad. Sci. USA 1991, 88, 1958-1962. 345. Casey, G.; Lohsueh, M.; Lopez, M. E.; Vogelstein, B.; Stanbridge, E. J. Growth suppression of human breast cancer cells by the introduction of a wild-type p53 gene. Oncogene 1991, 6, 1791-1797. 346. Johnson, P.; Gray, D.; Mowat, M.; Benchimol, S. Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells. Mol. Ceil. Biol. 1991, 11, 1-11. 347. Isaacs, W. B.; Carter, B. S. Genetic changes associated with prostate cancer in humans. Cancer Stow. 1991, 11, 15-24. 348. Goyette, M. C.; Cho, K.; Fasching, C. L., et al. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol. Cell. Biol. 1992, 12, 1387-1395. 349. Takahashi, T.; Carbone, D.; Takahashi, T., et ai. Wild-type but not mutant p53 suppresses the growth of human lung cancer cells bearing multiple genetic lesions. Cancer Res. 1992, 52, 2340-2343.


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350. Matozaki, T.; Sakamoto, C.; Suzuki, T., et al. p53 gene mutations in human gastric cancer wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. Cancer Res. 1992, 52, 4335-4341. 351. Cheng, J.; Yee, J. K.; Yeargin, J.; Friedmann, T.; Haas, M. Suppression of acute lymphoblastic leukemia by the human wild-type p53 gene. Cancer Res. 1992, 52, 222-226. 352. D'Amico, D.; Carbone, D.; Mitsudomi, T., et al. High frequency of somatically acquired p53 mutations in small-cell lung cancer cell lines and tumors. Oncogene 1992, 7, 339-346. 353. Hensel, C. H.; Xiang, R. H.; Sakaguchi, A. Y.; Naylor, S. L. Use of the single strand conformation polymorphism technique and PCR to detect p53 gene mutations in small cell lung cancer. Oncogene 1991, 6, 1067-1071. 354. Miller, C. W.; Simon, K.; Aslo, A., et al. p53 mutations in human lung tumors. Cancer Res. 1992, 52, 1695-1698. 355. Sameshima, Y.; Matsuno, Y.; Hirohashi, S., et al. Alterations of the p53 gene are common and critical events for the maintenance of malignant phenotypes in small-cell lung carcinoma. Oncogene 1992, 7, 451--457. 356. Takahashi, T.; Takahashi, T.; Suzuki, H., et al. The p53 gene is very frequently mutated in small-cell lung cancer with a distinct nucleotide substitution pattern. Oncogene 1991, 6, 17751778. 357. Lohmann, D.; Putz, B.; Reich, U.; Bohm, J.; Prauer, H.; Hofler, H. Mutational spectrum of the p53 gene in human small-cell lung cancer and relationship to clinicopathological data. Am. J. Pathol. 1993, 142, 907-915. 358. Chiba, I.; Takahashi, T.; Nau, M. M., et al. Mutations in the p53 gene are frequent in primary, resected non-small-cell lung cancer. Oncogene 1990, 5, 1603-1610. 359. Kishimoto, Y.; Murakami, Y.; Shiraishi, M.; Hayashi, K.; Sekiya, T. Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung. Cancer Res. 1992, 52, 4799-4804. 360. Mitsudomi, T.; Steinberg, S. M.; Nau, M. M., et al. p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene 1992, 7, 171-180. 361. Suzuki, H.; Takahashi, T.; Kuroishi, T., et al. p53 mutations in non-small-cell lung cancer in Japan association between mutations and smoking. Cancer Res. 1992, 52, 734-736. 362. Kim, J. H.; Takahashi, T.; Chiba, I., et al. Occurrence of p53-gene abnormalities in gastric carcinoma tumors and cell lines. J. Natl. Cancer Inst. 1991, 83, 938-943. 363. Matozaki, T.; Sakamoto, C., Matsuda, K., et al. Missense mutations and a deletion of the p53 gene in human gastric cancer. Biochem. Biophys. Res. Commun. 1992, 182, 215-223. 364. Seruca, R.; David, L.; Holm, R., et al. p53 mutations in gastric carcinomas. Bt: J. Cancer 1992, 65, 708-710. 365. Tamura, G.; Kihana, T.; Nomura, K.; Terada, M.; Sugimura, T.; Hirohashi, S. Detection of frequent p53 gene mutations in primary gastric cancer by cell sorting and polymerase chain reaction single-strand conformation polymorphism analysis. Cancer Res. 1991, 51, 3056-3058. 366. Yamada, Y.; Yoshida, T.; Hayashi, K., et al. p53 gene mutations in gastric cancer metastases and in gastric cancer cell lines derived from metastases. Cancer Res. 1991, 51, 5800-5805. 367. Renault, B.; Vandenbroek, M.; Fodde, R., et al. Base transitions are the most frequent genetic changes at p53 in gastric cancer. Cancer Res. 1993, 53, 2614-2617. 368. Borresen, A. L.; Hovig, E.; Smithsorensen, B., et al. Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc. Natl. Acad. Sci. USA 1991, 88, 8405-8409. 369. Chen, L. C.; Neubauer, A.; Kurisu, W., et al. Loss of heterozygosity on the short arm of chromosome-17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer. Proc. Natl. Acad. Sci. USA 1991, 88, 3847-385 I. -

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370. Kovach, J. S.; Mcgovern, R. M.; Cassady, J. D., et al. Direct sequencing from touch preparations of human carcinomas- analysis of p53 mutations in breast carcinomas. J. Natl. Cancer Inst. 1991, 83, 1004-1009. 371. Osborne, R. J.; Merlo, G. R.; Mitsudomi, T., et al. Mutations in the p53 gene in primary human breast cancers. Cancer Res. 1991, 51, 6194--6198. 372. Moll, U. M.; Riou, G.; Levine, A. J. Two distinct mechanisms alter p53 in breast cancer - mutation and nuclear exclusion. Proc. Natl. Acad. Sci. USA 1992, 89, 7262-7266. 373. Thorlacius, S.; Borresen, A. L.; Eyfjord, J. E. Somatic p53 mutations in human breast carcinomas in an Icelandic population- a prognostic factor. Cancer Res. 1993, 53, 1637-1641. 374. Cunningham, J.; Lust, J. A.; Schaid, D. J., et al. Expression of p53 and 17p allelic loss in colorectal carcinoma. Cancer Res. 1992, 52, 1974-1980. 375. Ishioka, C.; Sato, T.; Gamoh, M., et al. Mutations of the p53 gene, including an intronic point mutation, in colorectal tumors. Biochem. Biophys. Res. Commun. 1991, 177, 901-906. 376. Lothe, R. A.; Fossli, T.; Danielsen, H. E., et al. Molecular genetic studies of tumor suppressor gene regions on chromosome-13 and chromosome-17 in colorectal tumors. J. Natl. Cancer Inst. 1992, 84, 1100-1108. 377. Rodriges, N. R.; Rowan, A.; Smith, M. E. F., et al. p53 mutations in colorectal cancer. Proc. Natl. Acad. Sci. USA 1990, 87, 7555-7559. 378. Shaw, P.; Tardy, S.; Benito, E.; Obrador, A.; Costa, J. Occurrence of Ki-ras and p53 mutations in primary colorectal tumors. Oncogene 1991, 6, 2121-2128. 379. Bhatia, K. G.; Gutierrez, M. I.; Huppi, K.; Siwarski, D.; Magrath, I. T. The pattern of p53 mutations in Burkitt's lymphoma differs from that of solid tumors. Cancer Res. 1992, 52, 4273-4276. 380. Farrell, P. J.; Allan, G.; Shanahan, E; Vousden, K. H.; Crook, T. p53 is frequently mutated in Burkitt's lymphoma cell lines. EMBO J. 1991, 10, 2879-2887. 381. Gaidano, G.; Ballerini, P.; Gong, J. Z., et al. p53 mutations in human lymphoid malignancies association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 1991, 88, 5413-5417. 382. Bressac, B.; Galvin, K. M.; Liang, T. J.; Isselbacher, K. J.; Wands, J. R.; Ozturk, M. Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc. Natl. Acad. Sci. USA 1990, 87, 1973-1977. 383. Murakami, Y.; Hayashi, K.; Hirohashi, S.; Sekiya, T. Aberrations of the tumor suppressor-p53 and retinoblastoma genes in human hepatocellular carcinomas. Cancer Res. 1991, 51, 55205525. 384. Oda, T.; Tsuda, H.; Scarpa, A.; Sakamoto, M." Hirohashi, S. Mutation pattern of the p53 gene as a diagnostic marker for multiple hepatocellular carcinoma. Cancer Res. 1992, 52, 3674-3678. 385. Scorsone, K. A.; Zhou, Y. Z.; Butel, J. S.; Slagle, B. L. p53 mutations cluster at codon-249 in hepatitis-B virus-positive hepatocellular carcinomas from China. Cancer Res. 1992, 52, 16351638. 386. Sheu, J. C.; Huang, G. T.; Lee, P. H., et al. Mutation of p53 gene in hepatocellular carcinoma in Taiwan. Cancer Res. 1992, 52, 6098-6100. 387. Hollstein, M. C.; Wild, C. P.; Bleicher, E, et al. p53 mutations and Aflatoxin-B 1 exposure in hepatocellular carcinoma patients from Thailand. Int. J. Cancer 1993, 53, 51-55. 388. Buetow, K. H.; Sheffield, V. C.; Zhu, M. H., et al. Low frequency of p53 mutations observed in a diverse collection of primary hepatocellular carcinomas. Proc. Natl. Acad. Sci. USA 1992, 89, 9622-9626. 389. Bennett, W. P.; Hollstein, M. C.; He, A., et al. Archival analysis of p53 genetic and protein alterations in Chinese esophageal cancer. Oncogene 1991, 6, 1779-1784. 390. Wagata, T.; Shibagaki, I.; Imamura, M., et al. Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas. Cancer Res. 1993, 53, 846-850.


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391. Huang, Y.; Meltzer, S. J.; Yin, J., et al. Altered messenger RNA and unique mutational profiles of p53 and rb in human esophageal carcinomas. Cancer Res. 1993, 53, 1889-1894. 392. Fujimoto, K.; Yamada, Y.; Okajima, E., et al. Frequent association of p53 gene mutation in invasive bladder cancer. Cancer Res. 1992, 52, 1393-1398. 393. Sidransky, D.; Voneschenbach, A.; Tsai, Y. C., et al. Identification of p53 gene mutations in bladder cancers and urine samples. Science 1991, 252, 706-709. 394. Spruck, C. H.; Rideout, W. M.; Olumi, A. F., et al. Distinct pattern of p53 mutations in bladder cancer--relationship to tobacco usage. Cancer Res. 1993, 53, 1162-1166. 395. Fenaux, P.; Jonveaux, P.; Quiquandon, I., et al. p53 gene mutations in acute myeloid leukemia with 17p monosomy. Blood 1991, 78, 1652-1657. 396. Jonveaux, P.; Fenaux, P.; Quiquandon, I., et al. Mutations of the p53 gene in myelodysplastic syndromes. Oncogene 1991, 6, 2243-2247. 397. Soussi, T.; Jonveaux, P. p53 gene alterations in human hematological malignancies: a review. Nouv. Rev. Fr. Hematol. 1991, 33, 477-480. 398. Fenaux, P.; Preudhomme, C.; Lai, J. L., et al. Mutations of the p53 gene in B-cell chronic lymphocytic leukemia: a report on 39 cases with cytogenetic analysis. Leukemia 1992, 6, 246-250.

UPDATED MATERIAL ADDED IN PROOFS Since completion of this review in August, 1993, more than 2000 articles have been published dealing with either fundamental or clinical aspects of p53. Due to space and time limitations, this addendum will focus only on breakthrough discoveries which have led to new insights into this overcrowded field.

p53 and Cell Cycle Regulation Several target genes of wild-type p53 have recently been identified. (i) the WAF1-CIP1 gene, which codes for a 21 kDa protein that specifically inhibits the kinase activity of the cdk2-cyclin complex required for the G 1 to S transition of the cell cycle, l In addition, p21 binds and inhibits the proliferating cell nuclear antigen (PCNA), a regulatory subunit ofDNA polymerase 5. (ii) The GADD45 gene, which codes for a protein which stimulates DNA repair. 2 (iii) The Bax gene, whose product is involved in induction of apoptosis. 3 These findings strengthen the role of p53 in the response to genotoxic stress induced by ultraviolet light, ), rays or genotoxic chemicals. Upon DNA damage, the p53 pathways allow normal cells to undergo a transient cell arrest in G1, thus enabling DNA repair (via p21Wafl and cdk2 interaction),4 blocking ongoing DNA replication (via p21Wafl and PCNA interaction), 5 and stimulating DNA repair (via the GADD45/PCNA interaction). For unknown reasons, some cells do not take such pathways, but are eliminated by apoptosis. This appears to occur by the up-regulation of Bax and the down-regulation of Bcl2, both resulting from p53 activity. Analyses of the various p53 cancer mutants show that most of them are unable to transactivate any of these target genes leading to impaired response to DNA damage.

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The p53 Partner In 1993, B. Vogelstein w r o t e in an editorial in Nature that there w a s " n o m o r e r o o m at the p53 inn," m e a n i n g that the n u m b e r o f proteins that can c o m p l e x with p53 was e v e r increasing. 6 In v i e w of the literature since that time, the inn is g o i n g to h a v e to b e c o m e a hotel. At least 19 viral and cellular proteins has b e e n s h o w n to bind to p53 (see Table 1). Since m a n y o f these interactions h a v e b e e n identified either in vitro or in s y s t e m s involving the o v e r e x p r e s s i o n o f p53, it r e m a i n s to be p r o v e n that they truly play a role in vivo in the various signalling p a t h w a y s i n v o l v i n g p53.

Table 1. p53-Associated Proteins Protein

WiM type p53 domain p53 Mutantp53 im,olved

AgT (SV40) E I b (Ad5)

+ +

vary vary

HBx (HBV)

+

vary

E6 (HPV) EBNA 5 (EBV) BZL 1 (EBV)

+ + +

vary + +

IE84 (CMV) E6-AP hsp70

+ + -

9 9 vary

mdm2

+

+

TPB

+

WT1 CBF RPA

+ + +

9 +

S 100 ERCC3 BPI

+ + +

9 + -

BP2

+

TAFII40 and TAFII60

central amino

Effect

p53 stabilization induce a cytoplasmic localization of p53 9 inhibits p53 dependent transactivation inhibits p53/ERCC3 interaction ? induce p53 degradation 9 9 carboxy inhibits p53 dependent transactivation 9 p53 stabilization? central mediate p53 E6 interaction? amino and stabilize mutant p53? or carboxy amino ter inhibits p53 dependent transactivation amino and inhibit TFIID transcriptional carboxy activity repress WT1 activity 9 repress transcription amino and inhibit DNA-binding or carboxy activity of RPA 9 9 9 ? central inhibit DNA-binding activity of p53 central inhibit DNA-binding activity of p53 amino activate p53 transcriptional activity

References 21, 22 23 24, 25

26 27 28 29 30 31 32 33, 34 35 36 37 38 25 39 39 40


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p53 Structure Careful analysis of the DNA binding activity of p5 3 has led to the identification of two domains. The carboxy-terminus of p5 3, which is able to bind nonspecifically to DNA, but cannot bind to the RGC or CONS sequences and the central region which contains the specific DNA-binding activity (Figure 1). The first indication of the role of the central region in DNA binding came from Halazonetis and Kandil, who demonstrated that the highly conserved evolutionary blocks IV and V were directly involved with DNA contact. 7 Proteolytic digestion of wild-type p53 by enzymes such as thermolysin or subtilysin generates a 27-kDa fragment containing the entire central portion of the protein (amino acids 92/102 to 306/292, according to the protease used). This fragment of p5 3 is able to specifically bind to the RGC or CONS sequence if it comes from wild-type p53, whereas digestion fragments from mutant p53 can no longer do so. 8'9 Using truncated p53 produced in insect cells, Wang et al. defined a similar region (aa 80-290) necessary and sufficient for specific DNA binding. 1~More recently, the crystal structure of the DNA binding domain of p53 has been elucidated. 11 This core region has been shown to include the following motifs: (i) two antiparalle113 sheets composed of 4 and 5 13-strands, respectively; these two sheets form a rather compact sandwich that holds the other elements" (ii) a loop-sheet-helix motif (LSH) containing 3 [3-strands, an co-helix and the L1 loop; (iii) an L2 loop containing a small helix; and (iv) an L3 loop mainly composed of turns. It is quite remarkable to note the very good agreement between these various structural elements and the four evolutionarily conserved blocks (II to V). The LSH motif and the L3 helix are involved in direct DNA interaction (LSH with the major groove and L3 with the minor groove). The L2 loop is presumed to provide stabilization by associating with the L3 loop. These two loops are held together by a zinc atom tetracoordinated to the following amino acids" Cys 176 and His 179 on the L2 loop and Cys 278 and Cys 242 on the L3 loop. Analysis of the distribution of mutations in p53 shows that they are essentially clustered in the central region of the protein, and especially in the four blocks II-V which have been identified as the DNA binding region (Figure 1). In view of the 3-dimensional structure of the protein, it has been proposed that two classes of mutations can be predicted: class I mutations which affect the amino acids directly involved in the protein-DNA interaction (residues in the LSH and L3), and class II, which affect the amino acids involved in stabilization of the 3-dimensional structure of the protein (residues in L2). Indeed, the study of the biological and biochemical activity of more than 30 p53 mutants has revealed that not all p53 are equivalent and could be classified into the two classes described previously. 12The oligomerization domain has also been the subject of extensive studies. Wang et al., show that segments of p53 consisting of amino acid 323 to 355 are sufficient for assembly of stable tetramers. 13 Furthermore, high-resolution structures of a small p53 segment (residues 319 to 360) have been studied by multidimensional NMR. 14-16 or by crystal structure analysis (residues 320 to 356). 17 These studies confirm that

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THIERRY SOUSSI

9 NLSSignal I ~ 1 0 l i g o m e r i z a t i o n domain

Figure 1. Relationship of structural elements to p53 function. residues 325 to 356 are essential for tetramerization. This domain contains a 13-strand (aa 326-333) and an (~-helix (aa 335-354). They form a V shaped structure, with the helix axis being antiparallel to the direction of the 13-strand. Gly 334is critical for the stability of this structure. This region forms a stable tetramer with a very unusual topology which has not been observed in other multimeric proteins. Each subunit forms a dimer through antiparallel interaction of their 13-strand and two dimers interact through hydrophobic and electrostatic contact between their (~helice to form a tetramer.

p53, Apoptosisand Therapy It is generally agreed that common anti-tumor agents such as ionizing radiation, fluorouracil and etoposide can act by inducing tumor cells toward an apoptosis program. Lowe et al. have shown that wild type p53 is involved in this process. 18 Tumor cells bearing mutant p53 are resistant to these agents, raising the exciting prospect that p53 mutations may provide a genetic basis for drug resistance. Using


139

an animal model, these authors showed that transplantable p53 deficient tumors treated with g a m m a radiation or adriamycin continued to enlarge and contained few apoptotic cells. 19 In contrast, tumors expressing wild type p53 contained a high proportion of apoptotic cells and regressed after similar treatment. Acquired mutations in p53 were associated with both treatment resistance and relapse in p53 expressing tumors. These observations have fundamental impact in clinical practice and suggest that p53 status may be an important determinant of tumor response to therapy. Using recombinant adenovirus vectors which express wild type p53, Fujiwara et al. have been able to restore chemosensitivity in human lung cancer cells which were deficient for wild type p53. 2~ Because p53 mutations are among the most c o m m o n alterations observed in human cancers, and since they are usually associated with more aggressive tumors and resistance to treatment, it will be of fundamental importance in the near future to be able to develop early diagnostic procedures and new therapy for targetting mutant p5 3.

REFERENCES 1. E1-Deiry, W. S.; Tokino, T.; Velculescu, V. E.; et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993, 75, 817-825. 2. Smith,M. L.; Chen, I. T.; Zhan, Q. M.; et al. Interaction of the p53-regulated protein gadd45 with proliferating cell nuclear antigen. Science 1994, 266, 1376-1380. 3. Miyashita, T.; Reed, J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995, 80, 293-299. 4. Dulic, V.; Kaufmann, W. K.; Wilson, S. J.; et al. p53-Dependent inhibition of Cyclin-Dependent kinase activities in human fibroblasts during Radiation-Induced gl arrest. Cell 1994, 76, 10131023. 5. Li, R.; Waga, S.; Hannon, G. J.; Beach, D.; Stillman, B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 1994, 371,534-537. 6. Pietenpol, J. A.; Vogelstein, B. No room at the p53 inn. Nature 1993, 365, 17-18. 7. Halazonetis, T. D.; Davis, L. J.; Kandil, A. N. Wild-Typep53 adopts a Mutant-Like conformation when bound to DNA. EMBO J. 1993, 12, 1021-1028. 8. Bargonetti, J.; Manfredi, J. J.; Chen, X. B.; Marshak, D. R.; Prives, C. A proteolytic fragment from the central region of p53 has marked Sequence-Specific DNA-Binding activity when generated from Wild-Type but not from oncogenic mutant p53-Protein. Gene Develop. 1993, 7, 2565-2574. 9. Pavletich, N. P.; Chambers, K. A.; Pabo, C. O. The DNA-Binding domain of p53 contains the 4 conserved regions and the major mutation hot spots. Gene Develop. 1993, 7, 2556-2564. 10. Wang, Y.; Reed, M.; Wang, P.; et al. p53 domains--identification and characterization of 2 autonomous DNA-Binding regions. Gene Develop. 1993, 7, 2575-2586. 11. Cho, Y. J.; Gorina, S.; Jeffrey, P. D.; Pavletich, N. P. Crystal structure of a p53 tumor suppressor DNA complex: understanding tumorigenic mutations. Science 1994, 265, 346-355. 12. Ory, K.; Legros, Y.; Auguin, C.; Soussi, T. Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation. EMBO J. 1994, 13, 3496-3504. 13. Wang, P.; Reed, M.; Wang, Y.; et al. p53 domains: structure, oligomerization, and transformation. MoL Cell. Biol. 1994, 14, 5182-5191.

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14. Clore, G. M.; Omichinski, J. G.; Sakaguchi, K.; et al. High-resolution structure of the oligomerization domain of p53 by multidimensional NMR. Science 1994, 265, 386-391. 15. Lee, W.; Harveey, T. S.; Yin, Y.; Yau, P.; Litchfield, D.; Arrowsmith, C. H. Solution structure of the tetrameric minimum transforming domain of p53. Nature Structural Biology 1994, 1, 877-890. 16. Clore, G. M.; Omichinski, J. G.; Sakaguchi, K.; et al. lnterhelical angles in the solution structure of the oligomerization domain of p53 (vo1265, pg 386, 1994). Science 1995, 267, 1515-1516. 17. Jeffrey, P. D.; Gorina, S.; Pavletich, N. P. Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science 1995, 267, 1498-1502. 18. Lowe, S. W.; Ruley, H. E.; Jacks, T.; Housman, D. E. p53-Dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993, 74, 957-967. 19. Lowe, S. W.; Bodis, S.; Mcclatchey, A.; et al. p53 status and the efficacy of cancer therapy in vivo. Science 1994, 266, 807-810. 20. Fujiwara, T.; Grimm, E. A.; Mukhopadhyay, T.; Zhang, W. W.; Owenschaub, L. B.; Roth, J. A. Induction of chemosensitivity in human lung cancer cells in vivo by Adenovirus-Mediated transfer of the Wild-Type p53 gene. Cancer Res. 1994, 54, 2287-2291. 21. Lane, D. P.; Crawford, L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature 1979, 278, 261-263. 22. Linzer, D. I. H.; Levine, A. J. Characterization of a 54 K dalton cellular SV40 tumor antigen present in SV40-transformed cells and in infected embryonal carcinoma cells. Cell 1979, 1, 43-52. 23. Sarnow, P.; Ho, Y. S.; Williams, J.; Levine, A. J. Adenovirus EIB-58Kd tumor antigen and SV40 large tumor antigen physically associated with the same 54 Kd cellular protein in transformed cells. Cell 1982, 28, 387-394. 24. Feitelson, M. A.; Zhu, M.; Duan, L. X.; London, W. T. Hepatitis-B x-Antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma. Oncogene 1993, 8, 1109-1117. 25. Wang, X. W.; Forrester, K.; Yeh, H.; Feitelson, M. A.; Gu, J. R.; Harris, C. C. Hepatitis B virus X protein inhibits p53 Sequence-Specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc. Natl. Acad. Sci. USA 1994, 91, 2230-2234. 26. Wemess, B. A.; Levine, A. J.; Howley, P. M. Association of human papillomavirus type-16 and Type-18 E6 proteins with p53. Science 1990, 248, 76-79. 27. Szekely, L.; Selivanova, G.; Magnusson, K. P.; Klein, G.; Wiman, K. G. EBNA-5, an Epstein-Barr Virus-Encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proc. Natl. Acad. Sci. USA 1993, 90, 5455-5459. 28. Zhang, Q.; Gutsch, D.; Kenney, S. Functional and physical interaction between p53 and BZLFl--Implications for Epstein-Barr virus latency. Mol. Cell. Biol. 1994, 14, 1929-1938. 29. Speir, E.; Modali, R.; Huang, E. S.; et al. Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 1994, 265, 391-394. 30. Huibregtse, J. M.; Scheffner, M.; Howley, P. M. A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus type-16 or type-18. EMBO J. 1991, 10, 4129-4135. 31. Pinhasi-Kimhi, O.; Michalovitz, D.; Ben-Zeev, A.; Oren, M. Specific interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature 1986, 320, 182-184. 32. Momand, J.; Zambetti, G. P.; Olson, D. C.; George, D.; Levine, A.J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 1992, 69, 1237-1245. 33. Seto, E.; Usheva, A.; Zambetti, G. P.; et al. Wild-type p53 binds to the TATA-binding protein and represses transcription. P roc. Natl. Acad. Sci. USA 1992, 89, 12028-12032. 34. Horikoshi, N.; Usheva, A.; Chen, J. D.; Levine, A. J.; Weinmann, R.; Shenk, T. Two domains of p53 interact with the TATA-Binding protein, and the adenovirus 13S E1A protein disrupts the


35. 36. 37. 38.

39. 40.

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association, relieving p53-mediated transcriptional repression. Moi. Cell. Biol. 1995, 15, 227234. Maheswaran, S.; Park, S.; Bernard, A.; et al. Physical and functional interaction between WT1 and p53 proteins. Proc. Natl. Acad. Sci. USA 1993, 90, 5100-5104. Agoff, S. N.; Hou, J.; Linzer, D. I. H.; Wu, B. Regulation of the Human hsp70 Promoter by p53. Science 1993, 259, 84-87. Dutta, A.; Ruppert, J. M.; Aster, J. C.; Winchester, E. Inhibition of DNA replication factor RPA by p53. Nature 1993, 365, 79-82. Baudier, J.; Delphin, C.; Grunwald, D.; Khochbin, S.; Lawrence, J. J. Characterization of the tumor suppressor protein-p53 as a protein Kinase-C substrate and a S 100b-Binding protein. Proc. Natl. Acad. Sci. USA 1992, 89, 11627-11631. Iwabuchi, K.; Bartel, P. L.; Li, B.; Marraccino, R.; Fields, S. Two cellular proteins that bind to wild-type but not mutant p53. Proc. Natl. Acad. Sci. USA 1994, 91, 6098-6102. Thut, C. J.; Chen, J. L.; Klemm, R.; Tjian, R. p53 transcriptional activation mediated by coactivators TAF(II)40 and TAF(II)60. Science 1995, 267, 100-104.


GENETIC ASPECTS OF TUMOR SUPPRESSOR GENES

Bernard E. Weissman and Kathleen Conway

Abstract

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. I n t r o d u c t i o n

II.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

H i s t o r y o f Cell F u s i o n

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

144 144 145 145 147

III.

C o n t r o l of T u m o r i g e n i c i t y in H y b r i d Cells

IV. V.

Complementation Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . M o n o c h r o m o s o m e T r a n s f e r Studies . . . . . . . . . . . . . . . . . . . . . . .

VI.

O t h e r T r a n s f o r m e d P h e n o t y p e s w h i c h B e h a v e as R e c e s s i v e G e n e t i c Traits . . 150 A. Cellular I m m o r t a l i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 B. C.

VII.

GeneAmplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metastatic P r o g r e s s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . .

148

153 153

Corr elation o f K n o w n T u m o r S u p p r e s s o r G e n e s with S o m a t i c Cell G e n e t i c s Studies . . . . . . . . . . . . . . . . . . . . . . . . . . A.

Retinoblastoma Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. p53 C. VIII.

. . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other Tumor Suppressor Genes

Conclusions References

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154 154 155 155

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156

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157

Advances in Genome Biology Volume 3A, pages 143-162. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

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144

BERNARD E. WEISSMAN and KATHLEEN CONWAY

ABSTRACT In this chapter, we will review the history of genetic studies of the control of tumorigenicity, metastasis, genetic instability, and cellular senescence focusing mainly on somatic cell genetic studies of human tumor cells. After a brief consideration of the development of somatic cell fusion techniques, we will examine the experimental evidence supporting the concept of the recessive genetic nature of tumorigenic potential. We will also synopsize the literature showing the existence of multiple tumor suppressor genes. After the examination of the suppression of in vivo tumor growth, we will consider the genetics of other transformed cell phenotypes. We will then attempt to correlate features of known tumor suppressor genes such as rb and p53 with the data from the functional cell fusion experiments. Finally, we will explore some of the mechanisms by which tumor suppressor genes might function to control the in vitro and in vivo growth of human tumor cells.

I. I N T R O D U C T I O N During the last 25 years, research on human cancer has increasingly focused on the molecular basis of this disease. Although these studies have identified the many facets of cellular transformation, many of the key genetic events responsible for their appearance remain elusive. In order to investigate the processes of malignant transformation, investigators have developed several models including transformation of cells both in vitro and in the animal by chemical carcinogens or oncogenic viruses. Based on the earliest studies, two general models of cancer development arose. Huebner and Todaro as well as Temin developed theories based on the activation of genetic information called oncogenes leading to cell transformation. 1'2 Most of the support for their hypotheses came from studies on RNA tumor viruses which could transform normal cells in culture and form tumors in animals. By definition, these types of apparently single hit events implied a dominant genetic nature for human cancer. In contrast, Alfred Knudson in 1970 developed a "two-hit" hypothesis for the development of human cancer based on his observations on the development of retinoblastoma in pediatric patients. 3 The disease, which occurs in both sporadic and hereditary forms, could involve either one or both eyes. Based on a mathematical analysis of the characteristics of the cancer in a patient population, Dr. Knudson opined that loss of two pieces of genetic information must take place in order for this tumor to develop. In 1973, Comings combined many of the elements of both these hypotheses into a somatic mutation theory of cancer, suggesting that both activation of oncogenes and loss of suppressor genes contribute to the development of human malignancies. 4 The appearance of these two different schools of thought on the fundamental basis of human cancer energized the scientific community into studying human cancer on a variety of levels. A clear need arose for an understanding of the genetics

Genetic Aspects of Tumor Suppressor Genes

145

of human cancer. In this review, we will cover the history of genetic studies on human cancer with an emphasis on the use of somatic cell genetics. After reviewing our present understanding and knowledge on this topic, we will integrate the functional studies from somatic cell genetics with the recent advances in molecular studies. Finally, we will explore some of the potential mechanisms by which these genes exert their effects on human tumor cells.

il. HISTORY OF CELL FUSION Some of the most interesting studies have involved the use of somatic cell hybrids formed between tumorigenic cells and their normal counterparts. In 1960, Barski et al. first observed somatic cell hybrids between two cells as a spontaneous event in cell culture. 5 These cell hybrids arose by coculturing the two parental cell types together for an extended period. Identification of the hybrid cells rested on their overgrowth with the concomitant disappearance of one of the parental cell populations. However, viable hybrid cell formation occurred as a relatively rare event in tissue culture requiting refinements of somatic cell hybridization techniques. These included the development of selective media for hybrid cells as well as the use of fusogens to increase the formation of the hybrid cells. 6-9 These improved procedures for the isolation of somatic cell hybrids has led to a variety of studies aimed at determining the genetic behavior of tumorigenicity. For the purposes of this chapter, tumorigenicity is defined as the ability of a cell to form a progressively growing tumor in an appropriate host animal, usually a newborn syngeneic or an immunosuppressed mouse. We will only briefly consider a tumor cell's ability to metastasize in the animal as genetic studies on the behavior of this parameter still continue. At this time, the majority of studies based on somatic cell hybrids between tumorigenic and normal cells support the concept that tumorigenicity behaves as a recessive genetic trait. We will only mention these studies in an historical context as recent comprehensive reviews exist in the literature. ~'12 This review will focus on what new information has arisen from a study of these somatic cell hybrids both in vivo and in vitro, and what these results imply about the process of malignant transformation in human cells.

Iil. CONTROL OF TUMORIGENICITY IN HYBRID CELLS In 1960, Barski et al. first examined the genetic behavior of tumorigenicity in somatic cell hybrids between a highly tumorigenic cell line, NCTC 2472, and a poorly tumorigenic cell line, NCTC 2555.12'13 Their studies, showing that the hybrid cells were as tumorigenic as the parental NCTC 2472 cells, suggested that tumorigenicity acted as a dominant genetic trait. Following these initial studies, several other investigators confirmed these findings using a variety of other cell lines as the tumorigenic parent, including polyoma-transformed mouse fibroblasts

146


and mouse melanoma cells. By the end of the decade, the combined evidence from a number of laboratories supported the notion of a dominant nature for tumorigenicity in rodent cells. In 1969, Harris et al. decided to reevaluate these data with an emphasis on the chromosomal complements of the hybrid populations before and after inoculation. Their findings on hybrid cells formed between different highly tumorigenic mouse cell lines and an L cell line of low tumorigenic potential showed suppression of tumorigenicity when the hybrid cells retained the full chromosomal complement of both parental cells. 14 Tumors formed by the hybrid cells had invariably lost a large number of chromosomes in comparison to the pre-injection cell lines. 14'15 These results were later extended to include hybrid cell lines derived from tumorigenic mouse cells and normal diploid mouse fibroblasts. 16 Thus, Harris and Klein concluded that: (1) tumorigenicity behaved as a recessive genetic trait in somatic cell hybrids, and (2) reexpression of tumorigenic potential correlated with a loss of chromosomes from the cells. Since these early studies, other investigators have used cells from different species to investigate the genetics of tumorigenic expression in mammalian cells including intraspecific Chinese hamster or human cell hybrids as well as interspecific cell hybrids between different rodent species or rodent and human cell lines. A review of these studies supports the concept that tumorigenicity behaves as a recessive genetic trait. 17 The human intraspecific cell hybrid system has proved especially useful for the study of the control of expression of tumorigenicity. Initial studies by Stanbridge in 1976 demonstrated that cell hybrids formed between HeLa, a cervical carcinoma cell line, and normal human fibroblasts showed absolute suppression for tumorigenic potential. 18Other reports have expanded these initial studies to establish suppression of tumorigenicity in human cell hybrids as a generalized phenomenon. One group of investigations has shown that normal cells from different human tissues can suppress the tumorigenic potential of the HeLa cell line. 19'2~Conversely, fusion of different types of human tumor cell lines to normal human fibroblasts also results in suppression of tumorigenicity. 21-23 These data clearly establish the recessive genetic behavior of tumorigenicity in human cells. Although tumorigenicity behaves as a recessive genetic trait in most systems, two clear exceptions exist in systems which used either lymphoid cells or virally transformed cells as the tumorigenic parent. In the case of lymphoid cells, hybrid cells between myeloma and normal bone marrow cells resulted in the formation of the well-characterized hybridoma cell lines. 19 Injection of hybridoma cells into an appropriate animal results in tumor formation without the apparent loss of chromosomes observed in other systems. 19 Jonasson and Harris have also shown that normal human lymphoid cells do not suppress the tumorigenic potential of mouse tumor cell lines as well as normal human fibroblasts. 25 Miller and Ruddle have reported that hybrid cells between a mouse teratocarcinoma cell line and normal mouse thymocytes are tumorigenic when assayed in nude mice. 26These results may indicate that transformation of lymphoid cells, which are normally capable of


147

dividing throughout the lifetime of the animal, follows a different course than other types of nondividing normal tissue. These findings raise obvious questions about differences in the etiologies of human leukemias and lymphomas with those of carcinomas and sarcomas. Studies using virally transformed cell lines present a less clear picture. Several investigators have reported no differences in the behavior of tumorigenicity between hybrids using either simian virus (SM)40-transformed or polyomatransformed cell lines and those cases involving spontaneously or chemically transformed tumorigenic cell lines. 27-29 However, Croce and his colleagues have demonstrated that hybrid cells formed between SV40-transformed human fibroblasts and normal mouse peritoneal macrophages remained highly tumorigenic. 3~ Weissman and Stanbridge have reported a range of tumorigenicity in hybrids between SV40-transformed human fibroblasts and normal human fibroblasts from completely suppressed to highly tumorigenic. 31 They observed one case where a nontumorigenic clone of the SV40-transformed cell line fused to a normal human fibroblast resulted in a tumorigenic hybrid cell. Investigators have relayed similar results in studies involving retrovirus-transformed cells. 32 Therefore, one finds it difficult to come to a conclusion about the genetic behavior of tumorigenicity in virally transformed cells. Several factors contribute to the problems with defining the genetics of tumorigenic potential in virally transformed cell lines. Obviously, virally transformed cells have incorporated exogenous genetic information into their genomes consistent with a dominant genetic effect. Different levels of viral gene products, as well as the instability of the integration site of the viral genome, may further render the interpretation difficult. The interaction of viral-transforming gene products with known tumor suppressor genes further complicates the picture by raising the caveat of gene dosage effects.

IV. COMPLEMENTATION ANALYSES The observed suppression of tumorigenicity in cell hybrids between tumorigenic and normal cells raises the intriguing possibility of genetic complementation between different tumorigenic cell lines. Thus, if more than one genetic defect can lead to the expression of tumorigenicity, two tumorigenic cell lines with different defects could complement each other resulting in a non-tumorigenic hybrid cell. When Harris and his co-workers investigated this possibility in the mouse intraspecific hybrid system, they found only one case of apparent complementation in crosses involving 12 different tumorigenic cell lines characterized by a reduction in take incidence from 100 to 67%. 33 The stability of human intraspecific hybrid cells has provided more dramatic evidence for the existence of multiple tumor suppressor genes. Weissman and Stanbridge have shown complete suppression of tumorigenic potential for hybrid cells derived from human tumor cell lines of different developmental lineages. 34

148


More recently, Pasquale et al. showed that hybrids between HeLa and a variety of pediatric tumor cell lines showed suppression of tumorigenicity. 35 Choi et al. also reported that hybrids between two SV40-transformed human fibroblasts and HeLa cells became totally suppressed for tumorigenicity. 36 In contrast, hybrids between a HeLa and other adult carcinoma cell lines failed to show suppression of tumorigenicity. 34 A second report also showed that hybrids between a human peripheral neuroepithelioma cell line and five other soft tissue sarcomas remained tumorigenic. 23 Thus, several complementation groups for the control of expression of tumorigenicity in mammalian cells exist that act in a recessive genetic manner. In general, the tumor suppressor gene inactivated in these tumor cell lines appears related to the developmental lineage of the tumor. However, Geiser et al. have reported at least one exception to this generality. 37

V. M O N O C H R O M O S O M E TRANSFER STUDIES While whole cell hybrids have provided initial data about the existence and location of tumor suppressor genes, they lack the sensitivity required for mapping studies. Precise chromosome mapping analyses require a method for the transfer of smaller amounts of genetic material from the normal parental cell line. The development of the microcell hybridization procedure fills this need by its ability to introduce a single chromosome into a recipient cell line. 38'39Thus, suppression of tumorigenicity in a tumor cell line after introduction of a single human chromosome provides functional evidence for the location of a tumor suppressor gene. Therefore, this technique augments the previous cytogenetic studies as well as furnishing a novel method for the mapping of tumor suppressor genes. The first report of suppression of tumorigenicity after transfer of a single human chromosome appeared in 1986. 40 Using HeLa x normal fibroblast hybrid cells which had regained tumorigenic expression, Saxon et al. demonstrated complete loss of tumorigenic expression after addition of a human t(X; 11) chromosome. 4~ Introduction of a human chromosome X had no effect on the tumorigenic potential of the cells limiting the location of this gene from 11 q23 to 1l pter. This finding agrees with the mapping of this gene to the long arm of chromosome 11 by restriction fragment-length polymorphism (RFLP) markers. 41 Both cytogenetic and RFLP studies have implicated a loss of genetic information on the short arm of chromosome 11 in the development of the pediatric cancer, Wilms' tumor. 42'43 To test for the presence of a functional tumor suppressor gene, Weissman et al. transferred a normal human chromosome 11 into a Wilms' tumor cell line. 44 Upon inoculation into animals, these microcell hybrids failed to form tumors providing functional evidence for a tumor suppressor gene on this chromosome. 44 Using smaller fragments of chromosome 11, Dowdy et al. localized this tumor suppressor gene to the region of l lp14 to 11p15.5. 45 This mapping study


149

virtually eliminated the wt-1 gene as the operative tumor suppressor gene in this s y s t e m . 46,47

Microcell hybridization studies have now identified tumor suppressor genes on nine different human chromosomes (summarized in Table 1). The majority of studies have mapped genes to chromosome 11. However, evidence from several of these studies suggests that at least two different tumor suppressor genes map to this chromosome. 41'45 Similar ambiguities appear on chromosome 17 where at least four different tumor suppressor genes have been identified. 48-52 Whether these genes function independently of each other or participate in a common pathway remains an intriguing area for further investigation.

Table 1. Mapping of Human Tumor Suppressor Genes by Monochromosome Transfer Human Chromosome 1

3 5 6 9 11

13

17 18

Cell Line/Tumor Origin

Reference

HHUA/uterine endometrial carcinoma

97

HT1080/fibrosarcoma COKFu/colorectal carcinoma YCR/renal cell carcinoma A549/lung carcinoma COKFu/colorectal carcinoma SW480/colorectal carcinoma C8161/melanoma HHUA/uterine endometrial carcinoma HHUA/uterine endometrial carcinoma SiHa/cervical carcinoma A204/rhabdomyosarcoma HHUA/uterine endometrial carcinoma HT1080/fibrosarcoma HeLa/cervical carcinoma G401/Wilms' tumor A388/squamous cell carcinoma A549/lung carcinoma A 1698/bladder carcinoma MCF-7/breast carcinoma Y79/retinoblastoma HTB 9/bladder carcinoma DU 145/prostate carcinoma

98 99 100 101 85 82 102 97 97 103 104 97 98 40 44 96 101 105 106 81

NGP/neurobl as tom a A673/peripheral neuroepithelioma COKFu/colorectal carcinoma SW480/colorectal carcinoma

107 23 85 82

150


VI. OTHER TRANSFORMED PHENOTYPES WHICH BEHAVE AS RECESSIVE GENETIC TRAITS Many investigators have used somatic cell hybridization and microcell fusion to dissect the genetic controls which govern other transformed phenotypes of tumor cell lines. These studies have shown that a number of transformed phenotypes including immortality, morphology, density-dependent inhibition of growth, serum or growth factor requirements, and anchorage-independence become suppressed upon fusion with normal cells or following transfer of a specific chromosome. 11'17 In certain cases, these hybrids retain their tumorigenic potential, indicating that some transformed properties answer to separate genetic controls from tumorigenicity.

A. Cellular Immortality Whole cell and microcell fusion studies have yielded a great deal of information concerning the basis of cellular senescence. Normal human fibroblasts in culture have a limited life span; that is, after a defined number of population doublings, the cells cease proliferating, become refractory to mitogenic stimulation, and enlarge, but remain viable and metabolically active for a prolonged period before cell death results. 53 Many reports have documented this phenomenon of cellular senescence in a variety of normal cell types. 54 In contrast, most tumor cells grow indefinitely in culture, having escaped senescence and are termed immortal. 55 For this reason, restoration of the control for cellular senescence has been proposed as one of the mechanisms by which tumor suppression occurs. 55Two main hypotheses have been proposed to explain the phenomenon of cellular senescence. One suggests that the loss of proliferative potential originates by a random accumulation of damage, such as mutations or errors in protein or RNA synthesis, while the other proposes that senescence results from active genetic processes. 54 Cell fusion studies strongly support the latter theory. A number of studies have utilized the fusion of two normal cell populations of different in vitro ages to analyze proliferation potential. Studies by Littlefield and colleagues showed that fusion of normal human cells at high population doublings with young cells could result in small clones unable to grow to any significant extent, suggesting that senescence or cellular aging behaves as the dominant phenotype. 56 Pereira-Smith and Smith fused clonal populations of either early or late life-span cells and compared the proliferative capacity of the hybrids with each parental population. 57 They found the division potential of the hybrids similar to that of the older parent, indicating again the dominant nature of senescence. Additionally, when clones at the end of their in vitro life span were fused with each other, no hybrids were obtained having life spans greater than either parent. Cell fusion studies have demonstrated that hybrids obtained from the fusion of normal cells with immortal cells exhibit limited division potential. 58-63 These


151

results indicate a dominant nature for the phenotype of cellular senescence. Therefore, immortality appears to result from recessive changes in normal growth regulatory genes, a genetically programmed process, rather than the result of random accumulation of damage. Bunn and Tarrant demonstrated that some hybrids obtained from the fusion of HeLa cells with normal human diploid fibroblasts yielded hybrids with limited division capacity. 58 They also observed that maintenance of these nondoubling hybrid populations for varying periods of time in culture yielded foci of dividing cells at a frequency of I to 2 in 105 cells. These cells had regained the immortal phenotype and could grow indefinitely. MuggletonHarris and DeSimone fused normal cellswith immortal SV40-transformed cells by micromanipulation, and reported that the majority of the fusion products (98%) had an extremely limited division potential of 40%) at two sites: 12q13 and 12q22, suggesting the presence of two candidate tumor suppressor genes at these regions (Murty et al., 1992). The presence of candidate tumor suppressor genes on 12q and the high frequency of i(12p) in these tumors highlight the central role of chromosome 12 in the development of male GCTs.

IV. MOLECULAR CYTOGENETICS OF GERM-CELL TUMORS A simpler method than Southern blot analysis to determine abnormalities involving chromosome 12, mainly 12p, in GCTs utilizes the fluorescence in situ hybridization (FISH) technique. In this procedure, a biotinylated probe is hybridized directly on to metaphase chromosomes and/or nuclear preparations and visualized by fluorescence microscopy following incubation with fluoresceinated avidin and biotinylated goat antiavidin antibody and staining with an appropriate fluorochrome (Pinkel et al., 1986). Analysis by FISH with a chromosome 12 centromere-specific satellite DNA probe showed that the centromeres of the i(12p) chromosomes could be reliably distinguished from those of the normal chromosomes 12 by virtue of their larger or smaller sizes in tumor cells at metaphase as well as at interphase (Mukherjee et al., 1991; Rodriguez et al., 1992b). Parallel cytogenetic and FISH studies of a panel of tumors showed an excellent correlation between the two methods, thereby providing a rapid method of detection of this important marker in tumors and eliminating the limitations of conventional cytogenetic analysis (Rodriguez et al., 1992b). A further enhancement of the FISH technique is chromosome painting in which the probe comprises pooled DNA fragments derived from an entire chromosome or chromosome arm. FISH analysis using such pooled probes recognizes ("paints") the corresponding chromosome or chromosome region (Figure 1). Using the painting technique, a high proportion of GCTs without i(12p) were shown to be characterized by an increased copy number of 12p incorporated into marker chromosomes proving that excess copy number of 12p is more frequent than was indicated by conventional cytogenetic or FISH analysis using the centromeric probe (Rodriguez et al., 1993; Suijkerbuijk et al., 1993) (Figure 1). Such studies also enhance the diagnostic value of this marker.

A

Figure 1. Identification of chromosome 12 material in marker chromosomes by chromosome painting using a mixed probe comprising DNA sequences isolated from chromosome 12. A is a partial metaphase from tumor 225A hybridized to a 12p painting probe showing i(12p) signal (big arrow) and normal chromosome 12 signals (small arrow). B is a partial metaphase from tumor 240A hybridized to a whole chromosome 12 painting probe showing signal in two markers (big arrows) and two normal 12 (small arrows). C is a partial metaphase from tumor 268A hybridized to 12p painting probe showing signal in two markers (arrows). 420

13

Figure 1. (continued) 421

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Male Germ-Cell Tumors

423

V. APPLICATION OF CYTOGENETIC FINDINGS IN THE DIAGNOSIS A N D PROGNOSIS OF GERM-CELL TUMORS Because of its high incidence in GCTs, i(12p) has been shown to be a valuable diagnostic marker in several types of histologically ambiguous situations. Thus, a syndrome of acute myeloid leukemia (AML) associated with a very poor prognosis resulting form malignant hematopoietic transformation of i(l 2p)-bearing teratomatous cells has been identified (Chaganti et al., 1989; Ladanyi et al., 1990; Nichols et al., 1990). Therefore, AML arising in patients with a prior or concomitant history of GCT must be evaluated for germ-cell origin of the leukemia. Aprimary diagnosis of GCT can also be established by this marker in patients with a diagnosis of poorly differentiated carcinomas or adenocarcinomas of unknown primary sites. Such patients generally do poorly with systemic chemotherapy (Didolkar et al., 1977; Woods et al., 1980). In a minority of these patients who also presented with the clinical features of the unrecognized extragonadal germ-cell cancer syndrome (UEGCCS) (age

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