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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

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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

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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

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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

Department of Clinical Genetics Karolinska Hospital Stockholm, Sweden

Bernard E. Weissman

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

Jan Zedenius

Department of Clinical Genetics Karolinska Hospital Stockholm, Sweden

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

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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.

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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

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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

BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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

Oncogenes in Tumor Progression

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

BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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.

Oncogenes in Tumor Progression

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

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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.

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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

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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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

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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

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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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

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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

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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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.

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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

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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

Oncogenes in Tumor Progression

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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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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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

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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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~

Oncogenes in Tumor Progression

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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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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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

Oncogenes in Tumor Progression

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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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BRUCE P. HIMELSTEIN and RUTH J. MUSCHEL

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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181. Kashani-Sabet, M.; Lu, Y.; Leong, L.; Haedicke, K.; Scanlon, K. Differential oncogene amplification in tumor cells from a patient treated with cisplatin and 5-fluorouracil. Eur. J. Cancer 1990, 26, 383-390. 182. Schonthal, A.; Herrlich, P.; Rahmsdorf, H.J.; Ponta, H. Requirement forfos gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters. Cell 1988, 54, 325-334. 183. Scanlon, K. J.; Kashani-Sabet, M.; Tone, T.; Funato, T. Cisplatin resistance in human tumors. Phalrnac. Ther. 1991, 52, 385--406. 184. Scanlon, K. J.; Jiao, L.; Funato, T., et al. Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc. Natl. Acad. Sci. USA 1991, 88, 10591-10595. 185. SEar, M. D.; Prochownik, E. V. Modulation of cis-platinum resistance in Friend erythroleukemia cells by c-myc. Cancer Res. 1991, 51, 2118-2123. 186. Barry, M. A.; Behnke, C. A.; Eastman, A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins, and hyperthermia. Biochem. Pharm. 1990, 40, 23532362. 187. Eastman, A. Mechanisms of resistance to cisplatinum. Cancer Res. Treat 1991, 57, 233-249. 188. Miyashita, T.; Reed, J. C. Bcl-2 gene transfer increases relative resistance of $49.1 and WEH 17.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res. 1992, 52, 5407-5411. 189. Miyashita, T.; Reed, J. C. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 1993, 81, 151-157. 190. Sentman, C. L.; Shutter, J. R.; Hockenberry, D.; Kanagawa, O.; Korsmeyer, S. J. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 1991, 67, 879-888. 191. Strasser, A.; Harris, A. W.; Cory, S. Bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell 1991, 67, 889-899. 192. Siegel, R. M.; Katsumata, M.; Miyasata, T.; Louie, D. C.; Greene, M. I.; Reed, J. C. Inhibition of thymocyte apoptosis and negative antigenic selection in bci-2 transgenic mice. Proc. Natl. Acad. Sci. USA 1992, 89, 7003-7007. 193. Benz, C. C.; Scott, G. K.; Sarup, J. C., et al. Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Rev. Treat. 1992, 24, 85-95. 194. Rimoldi, D.; Srikantan, V.; Wilson, V. L.; Bassin, R. H.; Samid, D. Increased sensitivity of nontumorigenic fibroblasts expressing ras or myc oncogenes to malignant transformation induced by 5-aza-2'-deoxycytidine. Cancer Res. 1991, 51,324-330. 195. Fertil, B.; Malaise, E. P. Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy, h~t. J. Radiat. Oncol. Biol. Phys. 1981, 7, 621-629. 196. Fertil, B.; Malaise, E. P. Intrinsic radiosensitivity of human cell lines is correlated with radioresponsiveness of human tumors: analysis of 101 published survival curves. Int. J. Radiat. Oncol. Biol. Phys. 1985, 11, 1699-1707. 197. Weichselbaum, R. R.; Dahlberg, W.; Little, J. B. Inherently radioresistant cells exist in some human tumors. Proc. Natl. Acad. Sci. USA 1985, 82, 4732-4735. 198. Weichselbaum, R. R.; Dahlberg, W.; Beckett, M., et al. Radiation-resistant and repair-proficient human tumor cells may be associated with radiotherapy failure in head- and neck- cancer patients. Proc. Natl. Acad. Sci. USA 1986, 83, 2684-2688. 199. Weichselbaum, R. R.; Beckett, M. The maximum recovery potential of human tumor cells may predict clinical outcome in radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 1987, 13, 709-713. 200. Kasid, U.; Pfeifer, A. S.; Weichselbaum, R. R.; Dritschilo, A.; Mark, G. E. The raf oncogene is associated with radiation-resistant human laryngeal cancer. Science 1987, 237, 1039-1041. 201. FitzGerald, T. J.; Rothstein, L. A.; Daugherty, C.; McKenna, M.; Kase, K.; Greenberger, J. S. The activated human N-ras oncogene enhances X-irradiation repair of mammalian cells in vitro less

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202. 203.

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216.

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effectively at low dose-rate: implications for increased therapeutic ratio of low dose-rate irradiation. Am. J. Clin. Oncol. 1985, 8, 517-522. Sklar, M. D. The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation. Science 1988, 239, 645-647. Samid, D.; Miller, A. C.; Rimoldi, D., Gafner, J.; Clark, E. P. Increased radiation resistance in transformed and nontransformed cells with elevated ras protooncogene expression. Radiat. Res. 1991, 126, 244-250. Chang, E. H.; Pirollo, K. E; Zou, Z. Q., et al. Oncogenes in radioresistant, noncancerous skin fibroblasts from a cancer-prone family. Science 1987, 237, 1036-1039. Kasid, U.; Pfeifer, A.; Brennan, T., et al. Effect of antisense c-raf-I on tumorogenicity and radiation sensitivity of a human squamous cell carcinoma. Science 1989, 243, 1354-1356. Alexandropoulos, K.; Qureshi, S. A.; Bruder, J. T.; Rapp, U.; Foster, D. A. The induction of egr- 1 expression by v-fps is via a protein kinase C-independent intracellular signal that is sequentially dependent upon Ha-ras and raf-1. Cell Growth Differ. 1992, 3, 731-737. Wood, K. W.; Sarnecki, C.; Roberts, T. M.; Blenis, J. Ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf- 1, and ESK. Cell 1992, 68, 1041-1050. Dickson, B.; Sprenger, E; Morrison, D.; Hafen, E. Raf functions downstream of rasl in the Sevenless signal transduction pathway. Nature 1992, 360, 600-603. Miller, A. C.; Kariko, K.; Myers, C. E.; Clark, E. P.; Samid, D. Increased radioresistance of EJras-transformed human osteosarcoma cells and its modulation by lovastatin, an inhibitor of p21 ras isoprenylation. Int. J. Cancer 1993, 53, 302-307. Kato, K.; Cox, A. D.; Hisaka, M. M.; Graham, S. M.; Buss, J. E.; Der, C. J. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc. Natl. Acad. Sci. USA 1992, 89, 6403-6407. Sinensky, M.; Beck, L. A.; Leonard, S.; Evans, R. Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J. Biol. Chem. 1990, 265, 19937-19941. Kohl, N. E.; Mosser, S. D.; deSolms, S. J., et al. Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 1993, 260, 1934-1937. James, G. L.; Goldstein, J. L.; Brown, M. S., et al. Benzodiazepine peptidomimetics: potent inhibitors of ras farnesylation in animal cells. Science 1993, 260, 1937-1942. Grant, M. L.; Bruton, R. K.; Byrd, P. J., et al. Sensitivity to ionising radiation of transformed human cells containing mutant ras genes. Oncogene 1990, 5, 1159-1164. Alapetite, C.; Baroche, C.; Remvikos, Y.; Goubin, G.; Moustacchi, E. Studies on the influence of an activated ras oncogene on the in vitro sensitivity of human mammary epithelial cells, h~t. J. Radiat. Biol. 1991, 59, 385-396. Harris, J. E; Chambers, A. E; Tam, A. S. K. Some ras-transformed cells have increased radiosensitivity and decreased repair of sublethal radiation damage. Somatic Cell MoL Gen. 1990, 16, 39-48. McKenna, W. G.; Weiss, M. C.; Endlich, B., et al. Synergistic effect of the v-myc oncogene with H-ras on radioresistance. Cancer Res. 1990, 50, 97-102. Carmichael, J.; Degraff, W. G.; Gamson, J., et al. Radiation sensitivity of human lung cancer cell lines. Ettr. J. Cancer 1989, 25, 527-534. Ling, C. C.; Endlich, B. Radioresistance induced by oncogenic transformation. Radiat. Res. 1989, 120, 267-279. FitzGerald, T. J.; Santucci, M. A.; Das, I.; Kase, K.; Pierce, J. H.; Greenberger, J. S. The v-abl, c-fins, or v-myc oncogene induces gamma radiation resistance of hematopoietic progenitor cell line 32D cl 3 at clinical low dose rate. Int. J. Radiat. Oncoi. Biol. Phys. 1991, 21, 1203-1210. Santucci, M. A.; Anklesaria, P.; Anderson, S. M., et al. The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src. Radiat. Res. 1992, 129, 297-303.

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222. Shimm, D. S.; Miller, P. R.; Lin, T.; Moulinier, P. P.; Hill, A. B. Effects of v-src oncogene activation on radiation sensitivity in drug-sensitive and in multidrug-resistant rat fibroblasts. Radiat. Res. 1992, 129, 149-156. 223. Iliakis, G.; Metzger, L.; Muschel, R. J.; McKenna, W. G. Induction and repair of DNA double strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc. Cancer Res. 1990, 50, 6575--6579. 224. McKenna, W. G.; Iliakis, G.; Weiss, M. C.; Muschel, R. J. Increased G2 delay in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc. Radiat. Res. 1991, 125, 283-287. 225. Su, L.; Little, J. B. Prolonged cell cycle delay in radioresistant human cell lines transfected with activated ras oncogene and/or Simian Virus 40 T-antigen. Radiat. Res. 1993, 133, 73-79. 226. Muschel, R. J.; Zhang, H.-B.; Iliakis, G.; McKenna, W. G. Cyclin B expression in Hela cells during the G2 delay induced by ionizing radiation. Cancer Res. 1991, 51, 5113-5117. 227. Cunningham, J. M.; Francis, G. E.; Holland, M. J.; Pirollo, K. E; Chang, E. H. Aberrant DNA topoisomerase II activity, radioresistance and inherited susceptibility to cancer. Br. J. Cancer 1991, 63, 29-36. 228. Schwab, M.; Varmus, H. E.; Bishop, J. M., et al. Chromosome localization in normal human cells and neuroblastomas of a gene related to c-myc. Nature 1984, 308, 288-291. 229. Amler, L. C.; Schwab, M. Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differently recombined DNA. Mol. Cell Biol. 1989, 9, 4903-4913. 230. Keim, D. R.; Hailat, N.; Kuick, R., et al. PCNA levels in neuroblastoma are increased in tumors with an amplified N-myc gene and in metastatic stage tumors. Clin. Exp. Metastasis 1993, 11, 83-90. 231. Schwab, M. Amplification of N-myc as a prognostic marker in neuroblastoma. Semin. Cancer Biol. 1993, 4, 13-18. 232. Wada, A.; Sakamoto, H.; Katoh, S., et al. Two homologous oncogenes, hstl and int2, are closely located in human genome. Biochem. Biophys. Res. Comm. 1988, 157, 828-835. 233. Lammie, G. A.; Peters, G. Chromosome 1l ql3 abnormalities in human cancer. Cancer Cells 1991, 3, 413-420. 234. Pines, J. Cyclins: wheels within wheels. Cell Growth Differ. 1991, 2, 305-310. 235. Henry, J. A.; Hennessy, C.; Levett, D. L.; Lennard, T. W. J.; Westley, B. R.; May, E E. B. Int-2 amplification in breast cancer: association with decreased survival and relationship to amplification of c-erbB-2 and c-myc. Int. J. Cancer 1993, 53, 774-780. 236. Borg, A.; Sigurdsson, H.; Clark, G. M., et al. Association of INT2/HST! coamplification in primary breast cancer with hormone-dependent phenotype and poor prognosis. Bl: J. Cancer 1991, 63, 136-142. 237. Kitagawa, Y.; Ueda, M.; Ando, N.; Shinozawa, Y.; Shimizu, N.; Abe, O. Significance ofint-2/hst-1 coamplification as a prognostic factor in patients with esophageal squamous carcinoma. Cancer Res. 1991, 51, 1504-1508. 238. Slamon, D.J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.; McGuire, W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987, 235, 177-182. 239. Slamon, D. J.; Godolphin, W.; Jones, L. A., et al. Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 1989, 244, 707-712. 240. Hetzel, D. J.; Wilson, T. O.; Keeney, G. L.; Roche, E C.; Cha, S. S.; Podratz, K. C. HER-2/neu expression: a major prognostic factor in endometrial cancer. Gynecoi. Oncol. 1992, 47, i 79-185. 241. Levine, M. N.; Andrulis, I. Editorial: The Her-2/neu oncogene in breast cancer: so what is new? J. Clin. Oncol. 1992, 10, 1034-1036. 242. Toikkanen, S.; Helin, H.; Isola, J.; Joensuu, H. Prognostic significance of HER-2 oncoprotein expression in breast cancer: a 30-year follow-up. J. Ciin. Oncol. 1992, 10, 1044-1048.

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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.

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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

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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-

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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

The p53 Tumor Suppressor Gene

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

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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

The p53 Tumor Suppressor Gene

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

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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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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.

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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.

The p53 Tumor SuppressorGene

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).

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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

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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

The p53 Tumor SuppressorGene

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

The p53 Tumor Suppressor Gene

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).

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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-

The p53 Tumor SuppressorGene

.

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

The p53 Tumor Suppressor Gene

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

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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.

The p53 Tumor Suppressor Gene

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

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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.

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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,

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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

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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

Human breast carcinoma

One mutated p53 allele

Human breast carcinoma

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

One mutated p53 allele

PC-3

Human prostate carcinoma

One mutated p53 allele

SW480 NCI-H358

Human colorectal carcinoma Human lung carcinoma

1 mutated p53 gene No p53 gene

NCI-H358

Human lung carcinoma

One mutated p53 allele

M KNI

Human gastric carcinoma

One mutated p53 allele

MKN25

Human gastric carcinoma

One mutated p53 allele

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

The p53 Tumor Suppressor Gene

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.

92

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

The p53 Tumor Suppressor Gene

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.

96

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

The p53 Tumor SuppressorGene

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.

The p53 Tumor SuppressorGene

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

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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

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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

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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

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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-

The p53 Tumor Suppressor Gene

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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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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.

1 36

THIERRY SOUSSI

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

The p53 Tumor Suppressor Gene

1 37

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

138

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

The p53 Tumor Suppressor Gene

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

The p53 Tumor Suppressor Gene

35. 36. 37. 38.

39. 40.

141

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.

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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

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

154 154 155 155

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

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

143

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

BERNARD E. WEISSMAN and KATHLEEN CONWAY

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

Genetic Aspects of Tumor Suppressor Genes

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

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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

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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

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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

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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 <6 pd. 6~ Pereira-Smith and Smith fused immortal SV40-transformed cells with normal human cells using a biochemical selection system for hybrids. 6~ Approximately 70% of the hybrids had a limited division potential of <7 pd, while about 30% of the hybrids proceeded through 16 and 62 pd before eventually senescing. The hybrids continued to express the SV40 T antigen at the time they had ceased doubling. They then determined that the functional ability of the expressed SV40 T antigen to induce DNA synthesis in senescent normal cell nuclei in heterokaryons remained intact. 62 Pereira-Smith and Smith further showed that the dominant nature of cellular senescence occurred as a general phenomenon in a variety of immortal cells fused with normal human cells. 59 They observed suppression of cellular immortality regardless of whether the cells arose after viral transformation or from naturally occurring tumors. Further evidence for the genetic basis of senescence came from the identification of four complementation groups for indefinite division in human cells. 63 In a similar fashion to the complementation studies for tumor suppression, some hybrids between different human tumor cell lines became senescent after short periods in culture. These results implicated at least four sets of genes or genetic pathways in the control of cellular senescence whose modification led to immortal human cells. Pereira-Smith and Smith analyzed the proliferative potential of hybrids from various fusions involving normal, virally transformed and tumor-derived human cells. They used 26 different immortal human cell lines to identify four complementation groups for indefinite division. 63 Certain hybrids between different immortal human cell lines senesced, indicating that different complementation groups exist for the senescence function lost in these cells. To determine which chromosomes were involved in senescence, microcell fusion was used to introduce single human chromosomes into immortal human cell lines representing the various complementation groups (Table 2). Ning et al. demonstrated that microcell-mediated transfer of a normal human chromosome 4 into three immortal cell lines--HeLa, J82, and T98G--assigned to the senescence complementation group B, resulted in loss of proliferation potential and reversal of the immortal phenotype. 64 They observed no effect of chromosome 4 transfer on the growth of representative cell lines of the other three complementation

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BERNARD E. WEISSMAN and KATHLEEN CONWAY Table 2. Mapping of Human Cellular Immortality

Suppressor Genes by Monochromosome Transfer

Human Chromosome

Cell Line

4 11 17 X

10W-2 Syrian hamster cells BHK Baby hamster kidney cells HeLa cervical carcinoma cells RD embryonal rhabdomyosarcoma cells MCF-7 breast carcinoma cells Ni-2 nickel-transformed Chinese hamster cells

Reference 65 108 64 68 70 67

groups, suggesting that chromosome 4 carries a gene involved in cellular senescence and normal growth regulation specific for complementation group B. 64 T h e control of cellular senescence by specific human chromosomes was also examined in interspecies cell hybrids between diploid human fibroblasts and an immortal Syrian hamster cell line. 65 Most such hybrids exhibited a limited life span similar to that of the human fibroblasts, indicating that cellular senescence was dominant in these hybrids. Karyotypic analyses of hybrid clones that did not senesce revealed that these had lost both copies of human chromosome 1. Introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion induced typical signs of cellular senescence, while transfer of chromosome 11 had no effect on the growth of these cells. These findings indicate that a gene involved in cellular senescence resides on human chromosome 1. The frequent deletion of chromosome 1 in many types of human cancer may underscore the importance of this observation. Klein et al. also reported evidence for an immortality suppressor gene on the X chromosome of Chinese hamster cells. 66 Transfer of a normal Chinese hamster chromosome X into nickel-transformed Chinese hamster fibroblasts resulted in suppression of growth. 66 This group later extended these studies to demonstrate a similar activity on a human chromosome X. 67 One provocative finding which arose during these investigations suggests that methylation plays a role in the expression of the gene. Transfer of the chromosome X from early passage mouse cells into the hamster cell line yielded few or no microcell hybrids. However, transfer of the same chromosome after multiple passages of the donor cell line produced large numbers ofmicrocell hybrids. 67 Pretreatment of the late-passage donor cells with 5-azacytidine restored the ability of the normal chromosome X to suppress the growth of the recipient hamster cell line. Thus, genomic imprinting may provide another mechanism to abrogate tumor suppressor gene function. Other investigators have also shown a suppression of cellular proliferation after transfer of single human chromosome. Loh et al. demonstrated that introduction of chromosome 11 into a human embryonal rhabdomyosarcoma cell line caused

Genetic Aspects of Tumor Suppressor Genes

1 53

suppression of growth. 68 Koi et al. further narrowed this region to 11p15.5 by transferring smaller fragments of chromosome 11 into the same cells. 69 Casey et al. have shown that transfer of chromosome 17 into the MCF-7 breast carcinoma cell line also results in apparent cellular senescence. 7~ Thus, locations exist for candidate genes underlying the original complementation groups reported by Pereira-Smith and Smith. 63

B. Gene Amplification A central hypothesis outlined by Peter Nowell in 1976 suggested that genomic instability may lead to the development of the multiple genetic alterations, the underlying basis of neoplasia. 71 Gene amplification, an increase in the number of copies of a particular gene, provides one marker of genomic instability. 72 It occurs at a high frequency in tumor cells, but remains undetectable in normal diploid fibroblasts. 73'74 In order to investigate the genetic control of gene amplification, Tlsty et al. measured amplification frequency as determined by resistance to PALA in hybrids formed by the fusion of normal diploid fibroblasts with either HT1080 fibrosarcoma or HeLa cervical carcinoma cells. 75 The hybrids derived from both fusions could not amplify the cad gene, indicating that amplification potential behaves as a recessive trait. If genomic instability denotes a phenotypic consequence of the neoplastic state, then tumorigenicity might cosegregate with genomic instability with tumorigenic segregants regaining the ability to undergo gene amplification. However, a comparison of the amplification potential in the tumorsuppressed hybrids and their tumorigenic segregants determined that amplification ability segregated independently of tumorigenicity and immortality. 75

C. Metastatic Progression To determine if progression of cancer from nonmetastatic to highly metastatic ability involves the loss of a metastasis suppressor gene, nonmetastatic and highly metastatic Dunning rat prostatic cancer cells were fused. 76 Hybrids retained their tumorigenic phenotype, but did not form distant metastases at either lung or axillary lymph node sites. These nonmetastatic hybrids were passaged in vivo and some animals developed metastases. Cytogenetic analysis of the metastases revealed a consistent loss of one copy of normal rat chromosome 2, suggesting that this chromosome carries a metastasis suppressor gene. In a subsequent study, human chromosome 11 was found to partially suppress metastatic ability of the highly metastatic rat prostatic cancer cells. 77 Spontaneous deletion of portions of chromosome 11 in some microcell hybrids further delineated the region of chromosome 11 capable of metastatic suppression as being between 1l p 11.2 and p 13, but not including the Wilms' tumor-1 gene. Zajchowski et al. generated somatic cell hybrids by fusion of MCF-7 human breast cancer cells and immortalized mammary epithelial cells. 78 The hybrids were suppressed for their ability to form tumors in

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nude mice, growth factor independence, sensitivity to tumor necrosis factor, and expression of pS2, a breast cancer marker. Using monochromosome transfer, Welch et al. recently investigated the role of genes on chromosome 6 in the control of tumorigenicity and metastasis in a human melanoma cell line. Transfer of a normal human chromosome 11 into the C8161 melanoma cell line had little effect on its tumorigenic potential or its metastatic ability in nude mice. 79 Introduction of human chromosome 6 also had little effect on the ability of these cells to form a tumor at the site of inoculation. However, these microcell hybrids had lost their ability to metastasize in these animals from either the primary site of inoculation or in the experimental metastasis model. 79 Thus, chromosome 6 contains a metastasis suppressor gene whose activity does not overlap with tumor suppression.

VII. CORRELATION OF KNOWN TUMOR SUPPRESSOR GENES WITH SOMATIC CELL GENETICS STUDIES Since the first demonstration of tumor suppression in mouse intraspecific hybrids, investigators have sought the identification of specific tumor suppressor genes. Due to many technical reasons, including lack of a reliable in vitro assay for tumor suppressor activity, somatic cell genetic studies have not led to the isolation of any functional tumor suppressor genes. However, during the past seven years, other approaches including molecular mapping and positional cloning have yielded candidate tumor suppressor genes from the human genome. Yet, functionality played no role in the identification of these genes leading to a retrograde search for their places in the regulation of cellular activities. In this chapter, we will examine whether any of the known tumor suppressor genes match the functions delineated by whole cell and microcell hybrid studies.

A. Retinoblastoma Gene The retinoblastoma gene (rb) was the first isolated tumor suppressor gene. 80 One report compared the effects on in vitro and in vivo properties of a variety of rb-deficient cell lines after introduction of a rb cDNA clone under the control of a heterologous promoter versus a normal chromosome 13. 81 Given the caveat of the presence of other genes on the transferred chromosome 13, this report showed similar changes in cell morphology, growth in cell culture, and tumorigenicity of the cell lines after transfer of either genetic unit. Thus, the observed changes in the cellular parameters delineated by somatic cell genetics appeared attributable to the action of the rb gene.

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155

B. p53 Several reports have examined the effects on the growth of p53-deficient cell lines of chromosome 17 transfer or whole cell fusion. In one report, transfer of chromosome 17 into a p53 mutant containing colorectal carcinoma cell line failed to produce viable microcell hybrids. 82 In the second publication, investigators found that all viable microcell hybrids after transfer of chromosome 17 into a p53-deficient peripheral neuroepithelioma cell line had lost the portion of the chromosome containing the p53 gene. 23 Several groups have reported similar results when they transfected a p53 gene into p53-deficient cell lines. 83'84 Thus, in these cases, the monochromosome 17 transfer and the p53 gene transfer studies yielded similar results. However, other studies using chromosome 17 transfers point to the danger of equating whole chromosome transfers with single gene transfections. Casey et al. also reported a suppression of growth after transfer of human chromosome 17 into the human breast carcinoma cell line MCF-7. 7~ In this case, the transferred chromosome 17 remained intact in the microcell hybrids. They also demonstrated that transfection of the wild-typep53 gene alone failed to produce a similar effect. Thus, other genetic information on this chromosome controlled the in vitro growth of the cells. A related situation occurred upon transfer of chromosome 17 into a peripheral neuroepithelioma cell line A673. The presence of the wild-type p53 gene prevented the introduction of a whole chromosome into these cells. 23 However, transfer of two different chromosome 17s containing mutant p53 proteins into these cells caused suppression of tumorigenicity. 23 Again, investigators must always remain aware of the potential presence of different tumor suppressor genes on the same chromosome.

C. Other Tumor Suppressor Genes At this time, little information exists about the effects on tumor cell properties upon transfection of other tumor suppressor genes into the appropriate cell lines. Monochromosome transfer experiments have shown that transfer of a normal chromosome 5 into two different colorectal carcinoma cell lines results in complete suppression of tumorigenicity (see Table 1).82,85 In both cases, the investigators could demonstrate the restoration of normal APC gene activity. These two reports also showed complete or partial suppression of tumorigenicity after introduction of chromosome 18 into these cells with concomitant expression of normal DCC. However, these results remain correlative as single gene transfection experiments have not been performed. Dowdy et al. have also shown that the operative tumor suppressor for the G401 cell line maps to a different region of chromosome 11 from the wt-1 gene. 45

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CONCLUSIONS

Several individual but not exclusive mechanisms have arisen to account for the actions of tumor suppressor genes. In this final section, we will consider some of these potential mechanisms. The introduction of a tumor suppressor gene into cells could downregulate the expression of a key oncogene, thus altering tumorigenic potential. Hybrids between the EJ bladder carcinoma expressing an activated c-H-ras oncogene and normal human fibroblasts showed suppressed tumorigenic potential. 22 The expression of the activated oncogene was not altered in the non-tumorigenic hybrids, suggesting that the tumor suppression occurred through a different mechanism. However, another report showed a different result when examining the control of c-myc expression in a colorectal carcinoma cell. Transfer of human chromosome 5 by microcell fusion to CC1 221 and 233 colon carcinoma cells resulted in suppression of deregulated c-myc expression accompanied by a loss of tumorigenicity in nude mice. 86 Loss of the transferred chromosome 5 resulted in reexpression of the tumorigenic phenotype and in constitutive expression of c-myc. These data suggest that a gene or genes on chromosome 5 control the tumorigenicity of these cells and the regulated expression of c-myc, although it remains unknown whether the same gene or genes controls both. These results suggest that the APC gene, located on chromosome 5, functions to regulate Myc either directly or indirectly through another gene or gene product. In a similar fashion, we have observed that suppression of metastasis in a human melanoma cell line after chromosome 6 transfer correlates with reexpression of the NM-23 tumor suppressor gene on chromosome 17.52,79 Fusion with normal cells or transfer of specific chromosomes into human tumor cell lines often causes a complete loss of proliferative capacity. One interpretation of these results credits the resetting of the program for cellular senescence for the growth inhibition. 59 However, without a reliable biochemical or molecular marker to identify the process of cellular senescence, other possibilities remain. Another possible mechanism for this phenomenon lies in the restoration of cell-cycle checkpoint control. Normal human cells undergo a block in the cell cycle in response to cellular-damaging agents, presumably to allow DNA repair. 87 Most human tumor cell lines have lost this program which may contribute to their genomic instability. 88 At least one of the known tumor suppressor genes, p53, appears to function in the control of cell cycle checkpoints. 89 Transfection of this gene into p53-deficient cell lines results in the apparent restoration of cell-cycle checkpoints as evidenced by the suppression of gene amplification. 9~ If tumor cells suffer the loss of other cell cycle checkpoint genes, their restoration by cell fusion or monochromosome transfer could bring a complete inhibition of growth. Since most tumor cells have sustained extensive chromosome damage, the reexpression of one cell cycle checkpoint gene could lead to a cessation of cellular growth. However, the cells may find the large amount of DNA damage irreparable

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and choose to undergo suicide perhaps through apoptosis. The end result, cell death, would be indistinguishable from cellular senescence. A final route for tumor suppression in vivo may involve the induction of terminal differentiation. Many of the original human intraspecific hybrids between tumor and normal cells behaved almost identically to the parental cells in culture. ~~ However, when tested in an in vivo assay, these hybrid cells showed a complete inhibition of growth. 1~ When Stanbridge and co-workers first examined the fate of HeLa x normal fibroblast hybrid cells after inoculation into nude mice, they observed the apparent induction of terminal differentiation along a mesenchymal pathway. 92 Peehl and Stanbridge reported similar results with HeLa x normal keratinocyte hybrids although these cells underwent keratinocyte terminal differentiation. 93'94 These studies led to the hypothesis that the tumor suppression occurs via restoration of the tumor cell's ability to respond to the normal differentiation signals in the animal. 95 Our recent study supports this notion as microcell transfer of chromosome 11 into a squamous cell carcinoma resulted in complete suppression of tumorigenicity only when the hybrids were inoculated at the orthotopic site. 96 The results may implicate a role for these types of tumor suppressor genes in signal transduction pathways for differentiation and/or growth inhibition. The ability to manipulate tumor cell lines in culture along with in v i v o tumorigenicity assays has allowed investigators to delve into the genetics of human cancer development. These studies have solidified the concept that loss or inactivation of genetic information plays a major role in the initiation and progressio n of human malignancies. They have also given some insights into the potential normal functions of these genes. As we continue to identify and characterize these genes, new avenues for cancer detection and treatment should arise as well as a better understanding of human development.

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32. Craig, R. W.; Sager, R. Suppression of tumorigenicity in hybrids of normal and oncogene-transformed CHEF cells. Proc. Natl. Acad. Sci. USA 1985, 82, 2062-2066. 33. Wiener, F.; Klein, G.; Harris, H. The analysis of malignancy by cell fusion VI. Hybrids between different tumour cells. J. Cell Sci. 1974, 16, 189-198. 34. Weissman, B. E.; Stanbridge, E. J. Complementation of the tumorigenic phenotype in human cell hybrids. JNC11983, 70, 667-672. 35. Pasquale, S. R.; Jones, G. R.; Doersen, C.-J.; Weissman, B.E. Tumorigenicity and oncogene expression in pediatric cancers. Cancer Res. 1988, 48, 2715-2719. 36. Choi, K.-H.; Tevethia, S. S.; Shin, S. Tumor formation by S V40-transformed human cells in nude mice: the role of SV40 T antigens. Cytogenet. Cell Genet. 1983, 36, 633--640. 37. Geiser, A. G.; Anderson, M. J.; Stanbridge, E. J. Suppression of tumorigenicity in human cell hybrids derived from cell lines expression different activated ras oncogenes. Cancer Res. 1989, 49, 1572-1577. 38. Ege, T.; Ringertz, N. R.; Hamberg, H.; Sidebottom, E. Preparation of microcells. Methods Cell Biol. 1977, 15, 339-357. 39. Fournier, R. E. K.; Ruddle, E H. Microcell-mediated transfer ofmurine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc. Natl. Acad. Sci. USA 1977, 4, 319-323. 40. Saxon, P. J.; Srivatsan, E. S.; Stanbridge, E. J. Introduction of human chromosome 11 via microce 11transfer controls tumorigenic expression of HeLa ceils. EMBO J. 1986,15, 3461-3466. 41. Misra, B. C.; Srivatsan, E. S. Localization of the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Am. J. Hum. Genet. 1989, 45, 565-577. 42. Riccardi, V. M.; Sujansky, E.; Smith, A.C., et al. Chromosomal imbalance in the Aniridia-Wilms' tumor association: 11p interstitial deletion. Pediatrics 1978, 61,604--610. 43. Koufos, A.; Grundy, P.; Morgan, K., et al. Familial Wiedemann-Beckwith syndrome and a second Wilms" tumor locus both map to I I pl 5.5. Am. J. Htlm. Genet. 1989, 44, 711-719. 44. Weissman, B. E.; Saxon, P. J.; Pasquale, S. R.; Jones, G. R.; Geiser, A. G.; Stanbridge, E. J. Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression. Science 1987, 236, 175-180. 45. Dowdy, S. E; Fasching, C. L.; Scanlon, D. J., et al. Suppression of tumorigenicity in Wilms' tumor by the pl4:pl5 region of chromosome 11. Science 1991, 254, 293-295. 46. Call, K. M.; Glaser, T.; lto, C. Y., et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor gene locus. Cell 1990, 60, 509-520. 47. Gessler, M.; Poustka, A.; Cavenee, W., et al. Homozygous deletion in Wilms' tumours of a zinc-finger gene identified by chromosome jumping. Nature (Lond.) 1990, 343, 774-778. 48. Nigro, J. M.; Baker, S. J.; Preisinger, A. C., et al. Mutations in the p53 gene occur in diverse human tumor types. Nature 1989, 342, 705-708. 49. Viskochil, D.; Buchberg, A. M.; Xu, G., et al. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell 1990, 62, 187-192. 50. Wallace, M. R.; Marchuk, D. A.; Andersen, L. B., et al. Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science 1990, 249, 181-186. 51. Hall, J. M.; Lee, M. K.; Newman, B., et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1991, 250, 1684-1690. 52. Steeg, P. S.; Bevilacqua, G.; Pozzatti, R., et al. Altered expression of NM23, a gene associated with low tumor metastatic potential, during adenovirus 2 Ela inhibition of experimental metastasis. Cancer Res. 1988, 48, 6550--6554. 53. Hayflick, L.; Moorhead, P. S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 1961, 25, 585-621. 54. Goldstein, S. Replicative senescence: The human fibroblast comes of age. Science 1990, 249, 1129-1133. 55. Smith, J. R.; Pereira-Smith, O. M. Genetic and molecular studies of cellular immortalization. Adv. Cancer Res. 1990, 54, 63-77.

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56. Littlefield, J. W. Variation, Senescence and Neoplasia in Cultured Somatic Cells. Harvard University Press, Cambridge, MA, 1976, pp. 1-163. 57. Pereira-Smith, O. M.; Smith, J. R. Phenotype of low proliferative potential is dominant in hybrids of normal human fibroblasts. Somat. Cell Genet. 1982, 8, 731-742. 58. Bunn, C. L.; Tarrant, G. M. Limited lifespan in somatic cell hybrids and cybrids. Exp. Cell Res. 1980, 127, 385-396. 59. Pereira-Smith, O. M.; Smith, J. R. Evidence for the recessive nature of cellular immortality. Science 1983, 221,964-966. 60. Muggleton-Harris, A.; De Simone, D. Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components. Somatic Cell Genet. 1980, 6, 689-698. 61. Pereira-Smith, O. M.; Smith, J. R. Expression of SV40 T antigen in finite lifespan hybrids of normal SV40 transformed fibroblasts. Somatic Cell Genet. 1981, 7, 411-421. 62. Pereira-Smith, O. M.; Smith, J. R. Functional simian virus 40 T antigen is expressed in hybrid cells having finite proliferative potential. Mol. Cell. Bio. 1987, 7, 1541-1544. 63. Pereira-Smith, O. M.; Smith, J. R. Genetic analysis of indefinite division in human cells: Identification of four complementation groups. Proc. Natl. Acad. Sci. USA 1988, 85, 6042-6046. 64. Ning, Y.; Weber, J. L.; Killary, A. M., et al. Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4. Proc. Natl. Acad. Sci. USA 1991, 88(13), 5635-5639. 65. Sugawara, O.; Oshimura, M.; Koi, M., et al. Induction of cellular senescence in immortalized cells by human chromosome 1. Science 1990, 247, 707-710. 66. Klein, C. B.; Conway, K.; Wang, X. W., et al. Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control. Science 1991, 251,796-799. 67. Wang, X. W.; Lin, X.; Klein, C. B., et al. A conserved region in human and Chinese hamster X chromosomes can induce cellular senescence of nickel-transformed Chinese hamster cell lines. Carcinogenesis 1992, 13, 555-561. 68. Loh, W. E. Jr.; Scrable, H. J.; Livanos, E., et al. Human chromosome 11 contains two different growth suppressor genes for embryonal rhabdomyosarcoma. Proc. Natl. Acad. Sci. USA 1992, 89, 1755-1759. 69. Koi, M.; Johnson, L. A.; Kalikin, L. M., et al. Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11. Science 260, 361-364. 70. Casey, G.; Plummer, S.; Hoeltge, G., et al. Functional evidence for a breast cancer growth suppressor gene on chromosome 17. Human Molec. Genet 1993, 2, 1921-1927. 71. Nowell, P. C. The clonal evolution of tumor cell populations. Science 1976, 194, 23-28. 72. Otto, E.; McCord, S.; Tlsty, T. D. Increased incidence of CAD gene amplification in tumorigenic rat lines as an indicator of genomic instability of neoplastic cells. J. Biol. Chem. 1989, 264, 3390-3396. 73. Tlsty, T. D. Normal human diploid human and rodent cells lack a detectable frequency of gene amplification. Proc. Natl. Acad. Sci. USA 1990, 87, 3132-3136. 74. Wright, J. A.; Smith, H. S.; Watt, E M., et al. DNA amplification is rare in normal human cells. Proc. Natl. Acad. Sci. USA 1990, 87, 1791-1795. 75. Tlsty, T. D.; White, A.; Sanchez, J. Suppression of gene amplification in human cell hybrids. Science 1992, 255, 1425-1427. 76. Ichikawa, T.; Ichikawa, Y.; Isaacs, J. T. Genetic factors and suppression of metastatic ability of prostatic cancer. Cancer Res. 1991, 51, 3788-3792. 77. Ichikawa, T.; Ichikawa, Y.; Dong, J., et al. Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome 11. Cancer Res. 1992, 52, 3486-3490. 78. Zajchowski, D. A.; Band, V.; Trask, D. K., et al. Suppression of tumor-forming ability and related traits in MCF-7 human breast cancer cells by fusion with immortal mammary epithelial cells. Proc. Natl. Acad. Sci. USA 1990, 87, 231 4-2318.

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79. Welch, D. R.; Chen, P. C.; Miele, M. E., et al. Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity. Oncogene 1994, 9, 255-262. 80. Friend, S. H.; Bernards, R.; Rogeli, S., et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986, 323, 643-646. 81. Banerjee, A.; Xu, H.-J.; Hu, S.-X., et al. Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13. Cancer Res. 1992, 52, 6297-6304. 82. Goyette, M. C.; Cho, K.; Fasching, C. L., et al. Progression ofcolorectal 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. 83. Baker, S. J.; Markowitz, S.; Fearon, E. R., et al. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 1990, 249, 912-915. 84. Dillar, L.; Kassel, J.; Nelson, C. E., et al. p53 functions as a cell cycle control protein in osteosarcomas. Mol. CelL Biol. 1990, 10, 5772-5781. 85. Tanaka, K.; Oshimura, M.; Kikuchi, R., et al. Suppression of tumorigenicity in human colon carcinoma cells by introduction of normal chromosome 5 or 18. Science 1991, 349, 340-342. 86. Rodriguez-Alfageme, C.; Stanbridge, E. J.; Astrin, S. M. Suppression of deregulated c-MYC expression in human colon carcinoma cells by chromosome 5 transfer. Proc. Natl. Acad. Sci. USA 1992, 89, 1482-1486. 87. Kaufmann, W. K.; Kaufman, D. G. Cell cycle control, DNA repair and initiation of carcinogenesis. FASEB J. 1993, 7, 1188-1191. 88. Hartwell, L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 1992, 71,543-546. 89. Kastan, M. B.; Onyinye, O.; Sidransky, D., et al. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991, 51, 6304-6311. 90. Livingstone, L. R.; White, A.; Sprouse, J., et al. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 1992, 70, 923-935. 91. Yin, Y.; Tainsky, M. A.; Bischoff, E Z., et al. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 1992, 70, 937-948. 92. Stanbridge, E. J.; Ceredig, R. Growth-regulatory control of human cell hybrids in nude mice. Cancer Res. 1981, 41,573-580. 93. Peehl, D. M.; Stanbridge, E. J. Characterization of human keratinocyte x HeLa somatic cell hybrids, hit. J. Cancer 1981, 27, 625-635. 94. Peehl, D. M.; Stanbridge, E. J. The role of differentiation in the suppression of tumorigenicity in human cell hybrids, hit. J. Cancer 1982, 30, 113-120. 95. Stanbridge, E. J. A genetic basis for tumor suppression. C1BA Found. Symp. 1989,142, 149-159. 96. Conway, K.; Morgan, D.; Phillips, K. K., et al. Human chromosome 11 suppresses the tumorigenicity of a human skin squamous cell carcinoma cell line. Cancer Res. 1992, 52, 6487-6495. 97. Yamada, H.; Wake, N.; Fujimoto, S.-I., et al. Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer. Oncogene 1990, 5, 1141-1147. 98. Kugoh, H. M.; Hashiba, H.; Shimizu, M., et al. Suggestive evidence for functionally distinct tumor suppressor genes on chromosomes 1 and 11 for a human fibrosarcoma cell line, HT1080. Oncogene 1990, 5, 1637-1644. 99. Tanaka, K.; Yanoshita, R.; Konishi, M., et al. Suppression of tumourigenicity in human colon carcinoma cells by introduction of normal chromosome lp36 region. Oncogene 1993, 8, 22532258. 100. Shimizu, M.; Yokota, J.; Mori, N., et al. Introduction of normal chromosome 3p modulates the tumorigenicity of a human renal cell carcinoma cell line YCR. Oncogene 1990, 5, 185-194.

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101. Satoh, H.; Lamb, P. W.; Dong, J. T., et al. Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosome 3 and l l introduced via microcell-mediated chromosome transfer. Mol. Carch~og. 1993, 7, 157-164. 102. Trent, J. M.; Stanbridge, E. J.; McBride, H. M., et al. Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6. Science 1990, 247, 568-571. 103. Koi, M.; Morita, H.; Yamada, H., et al. Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa. Moi. Carcin. 1989, 2, 12-21. 104. Oshimura, M.; Kugoh, H.; Koi, M., et al. Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines. J. Cell Biochem. 1990, 42, 135-142. 105. Ning, Y.; Shay, J. W.; Lovell, M., et al. Tumor suppression by chromosome I 1 is not due to cellular senescence. Exp. Cell Res. 1991, 192(1), 220-226. 106. Negrini, M.; Castagnoli, A.; Sabbioni, S., et al. Suppression of tumorigenesis by the breast cancer cell line MCF-7 following transfer of normal human chromosome l l. Oncogene 1992, 7, 2013-2018. 107. Bader, S. A.; Fasching, C.; Brodeur, G. M. et al. Dissociation of suppression of tumorigenicity and differentiation fir vitro effected by transfer of single human chromosomes into human neuroblastoma cells. Cell Growth and Differentiation 1991, 2, 245-255. 108. Annab, L. A.; Dong, J. Y.; Futreal, P. A., et al. Growth and transformation suppressor genes for BHK Syrian hamster cells on human chromosomes 1 and 11. Mol. Carcinog. 1992, 6, 280-288.

P21 ras.. FROM ONCOPROTEIN TO SIGNAL TRANSDUCER

Johannes L. Bos and Boudewijn M. Th. Burgering

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

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r a s Gene Mutations in H u m a n Tumors ..................... r a s Gene Mutations During Tumor Development . . . . . . . . . . . . . . . . Heterogeneity of r a s Gene Mutations . . . . . . . . . . . . . . . . . . . . . . The ras Gene Products: p21ras . . . . . . . . . . . . . . . . . . . . . . . . . p 2 1 r a s Functions in Signal Transduction. . . . . . . . . . . . . . . . . . . . . Growth Factors Can Activate p21 r a s . . . . . . . . . . . . . . . . . . . . . . . p 120GAP and the Relation of P21 r a s with Tyrosine Kinases . . . . . . . . . . Neurofibromatosis Type I Gene as GTPase Activating Protein of p21 r a s Guanine Nucleotide Exchange of p21 r a s . . . . . . . . . . . . . . . . . . . . p2 lras and Serinefrhreonine Kinases . . . . . . . . . . . . . . . . . . . . . . A c knowle dgm e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Genome Biology Volume 3, pages 163-183. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

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164 1 64 166 169 . 170 171 172 172 174 174 175 177 178

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JOHANNES L. BOS and BOUDEWIJN M. TH. BURGERING I.

INTRODUCTION

Alterations in genes involved in the regulation of growth and differentiation are considered to be the main cause of cancer. Molecular cancer research aims at identifying the genes that are altered in human tumors and elucidating the function of their encoded gene products. One of the paradigms of this research is the r a s gene family. Members of this family were first identified as genes responsible for the oncogenic properties of the Harvey and Kirsten rat sarcoma RNA tumorviruses. These viral genes, which were derived from the host and had been integrated into the viral genome, were designated c - H - r a s and c - K - r a s . By a different approach these same cellular r a s genes were also identified as genes that may play a role in human carcinogenesis. DNA isolated from tumor cells was analyzed for the presence of "cancer genes" by introducing the DNA into an established nontumorigenic fibroblast cell line, NIH/3T3. It was found that DNA derived from a variety of tumors could transform NIH/3T3 cells into tumorigenic cells. In the majority of the cases, the cellular gene responsible for this transforming capacity was found to be a member of the r a s gene family. In the human genome, three different functional r a s genes are present, the H - r a s gene and the K - r a s gene already mentioned, and the N - r a s gene, which is not found in viral genomes. Each of these genes has been isolated from human tumors as a "transforming gene". Subsequent analysis revealed that transforming r a s genes differed from non-transforming r a s genes by the presence of a point mutation at one of a few restrictive positions within the gene. Point mutations at these positions led to the conversion of a normal cellular (protoonco)gene into a transforming oncogene. 1 This chapter will focus on the relevance of mutated r a s genes for the development of tumors, on the stage in tumor development at which these mutation occurs, and on the observation that mutated r a s genes are frequently found in only a subfraction of tumor cells. In addition, the function of the p 2 1 ras gene products in signal transduction will be discussed.

I!. RASGENE MUTATIONS IN H U M A N TUMORS The original NIH/3T3 assay used to identify mutated r a s genes in human tumors as described above, is not suitable to screen large numbers of tumor samples, but it did reveal that the mutations in the different transforming r a s genes are restricted to codons 12, 13, and 61. Several biochemical approaches (for discussion see Ref. 2) have been used to identify these mutations in a large number of tumor samples 3 and the results of these analyses are summarized in Table 1. From these data several conclusions can be drawn. First, the presence of mutated r a s genes can be demonstrated in a number of different malignancies, both in solid tumors and in hematopoietic malignancies. However, in several other malignancies r a s mutations

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Table 1. Incidence of ras Gene Mutations in Human Tumors a Tumor

%ras +b

breast adenocarcinoma ovarian carcinoma cervical carcinoma

0 0 0

esophageal carcinoma glioblastomas neuroblastomas

0 0 0

stomach carcinomas lung squamous cell carcinomas

0

ras gene c

0

large cell carcinomas adenocarcinomas colon adenoma adenocarcarcinoma

0 30 50 50

K-ras

pancreatic carcinoma cholangiocarcinoma seminomas

80 60 d 40

K-ras

melanomas bladder carcinomas myeloid disorders myelodysplastic syndrome acute myeloid leukemia chronic myeloid leukemia lymphoid disorders acute lymphoid leukemia non-Hodgkin's lymphomas Hodgkin's lymphomas multiple myelomas

20 10 30 30 0 10 0 0 30 e

K-ras

K-ras K-ras N-, K-ras K-ras

N-ras N-ras

N-ras

N-, K-ras

Notes: aFromBos, 1989

b%ras+ indicates the percentage of tumors containing a mutated ras gene. Cthepreponderant mutated ras gene is indicated. dref (Levi et al., 1991) eref (Neri et al., 1989)

do not occur or occur only sporadically. This finding implies that the occurrence of mutated r a s genes is restricted to certain malignancies. This specificity is illustrated quite strikingly in the case of three different types of lung tumors. First, r a s Mutations occur rather frequently in adenocarcinoma, whereas they are seldom observed in squamous cell carcinoma and large cell carcinoma, although these three different tumors are thought to originate from the same bronchiolo-alveolar epi-

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thelial cell. Second, there is some specificity with respect to the type of r a s gene that is mutated. In solid tumors, the K - r a s gene is predominantly mutated, whereas in hematopoietic malignancies most frequently the N - r a s gene is mutated. Third, mutations in r a s genes are not restricted to malignant tumors but have also been shown to occur in benign lesions, such as polyps of the colon and myelodysplastic syndrome. It is generally accepted that mutated r a s genes contribute to the formation of those tumors where the mutated gene is present. With respect to the process of tumor formation the role of mutated r a s genes is primarily demonstrated by their ability to induce cellular transformation in vitro. Furthermore, its presence in, for instance 80% of pancreatic carcinomas can hardly be ascribed to "accidental presence", which may be the case in tumors where only occasionally a mutated r a s gene is observed. In none of the malignancies analyzed, r a s mutations have been observed in all the tumor samples. This genetic heterogeneity, however, does not result in clear phenotypic differences, although in certain cases, the presence of a mutated r a s gene may serve as a prognostic marker. For example, for patients having a curative resection of a primary adenocarcinoma of the lung, the presence of a K - r a s mutation in the tumor indicates poor prognosis. 4

II!. RAS GENE MUTATIONS DURING TUMOR DEVELOPMENT Tumor development is considered to progress through several stages. For some tumors, these different stages have been analyzed for the presence of mutated r a s genes. Studies on colorectal cancer and myelodysplastic syndrome(MDS) were in particular very informative. Colorectal cancer is considered to develop through several distinct stages. One of the early stages involves the development of hyperplasia of the colon epithelium, which is followed by the development of a benign tubular type ofadenoma or polyp. Polyps occur frequently in the colon of healthy individuals and in most cases do not progress. The villous type of polyp, a more advanced stage, is usually larger and frequently contains patches of frank adenocarcinoma tissue. These patches may progress into the fourth stage, the invasive carcinoma. Finally, the tumor will metastasize. In about 50% of the cases, mutated r a s genes can be detected in carcinoma tissue or in the villous type of adenomas. 5-7 Furthermore, in several tumors with both carcinomatous and adenomatous tissue, the r a s mutation was found in both tissue types. This finding implies that usually the mutation occurs prior to the conversion to malignant carcinoma. However, in a few mixed tumors the r a s mutation was found in the carcinomatous part of the tumor only, indicating that the onset of the mutation is not fixed to a particular stage of tumor development. In the smaller adenomas, the frequency of r a s mutations (approx. 10%) is much lower than in the larger villous adenomas. 7 This may imply that in the majority of

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the cases the mutation occurs later in development. Alternatively, mutation of the r a s gene is (partly)responsible for the development of a small fraction of the polyps, but polyps with a mutated r a s gene have a higher probability to progress into a malignancy. A mutation in one of the r a s genes is not the sole genetic defect in colorectal cancer. In particular, from the work of Vogelstein and colleagues, it is known that several other genetic defects are involved in the induction of colon tumors. These genetic defects include mutation and or deletion of the p53 gene, 8 the APC (adenomatous polyposis coli) gene 9 and the DCC (deleted in colon cancer) gene. 1~ Similar to what was found for the r a s gene, mutations in these other genes do not seem to occur at a set stage in tumor development, although alterations in the APC gene is usually an earlier event compared to mutation/deletion of the p 5 3 gene. 7 In general, an accumulation of different genetic lesions is observed during progression. The fact that some carcinomas appear to have none of the described genetic lesions, might thus indicate that other "cancer genes" await identification. MDS is a heterogeneous group of disorders characterized by abnormally low counts of one or more of the blood lineages and bone-marrow abnormalities. MDS may progress into acute myeloid leukemia (AML) in one third of the cases. Most likely, the initiating event in this disease affects an early stem cell, and the subsequent progression is marked by the accumulation of chromosome abnormalities and the gradual increase of blast cells. Arbitrarily, patients with more than 30% immature blast cells in their bone marrow are diagnosed as having AML. It was found that 30% of the MDS and AML patients carry a mutated r a s gene in their leukemic cells. 3 From studies in which serial samples from the same patient were analyzed, it was found that, like for colorectal cancer, the onset of the r a s mutation was not restricted to one particular stage in the development of the leukemia (Figure 1). 11

In one group of patients, the mutation is present early in the course of the disease and can be detected in both the affected and unaffected blood lineages, such as the peripheral blood lymphocytes, suggesting that the mutational event had occurred in a multipotent stem cell (Figure 2 row 1). During complete clinical remission, cells with a mutated r a s gene are still present and may comprise a major fraction of the bone marrow cells. In the subsequent relapse the mutation is still present in the leukemic cell clone. In a second group of patients, the mutation is not present during the initial phase of the disease, but occurs during progression (Figure 2 row 2). Initially, a mutated r a s gene is detected only in a subfraction of the bone marrow cells, but gradually the fraction of cells containing a mutant ras gene increases, more or less concomitant with the increase in immature blast cells ~Apparently, in these patients the mutated r a s gene is present in a newly evolved cell clone, which is the major cell clone during the acute phase of the disease. At clinical remission, no mutated r a s genes can be detected in the bone marrow, and also in a subsequent relapse no mutated r a s genes are present. This indicates

Figure 1. Mutated ras genes in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Mutated ras genes are found at different stages during the progression of MDS/AML. Indicated are four examples of diseases, where the presence of mutated ras genes has been analyzed during the course ofthe disease. Striped squares indicated bone marrow cells with a mutated ras gene; open circles indicate bone marrow cells without a mutated ras gene. For further explanation see text.

Figure 2.

The GDP/GTP cycle of p21 ras 168

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P21 ras

that during treatment the cell clone harboring the mutated r gene is eradicated. In some cases of MDS/AML it has been demonstrated that a mutation in a r a s gene occurs even later in the course of the disease (Figure 2 row 3 and 4). 3 Combined, these data show that a mutation in one of the r a s genes in MDS/AML can be an early or even initial event as well as an event which occurs in a later stage, during progression of the disease. In conclusion, the studies on colorectal cancer and MDS/AML showed that mutations in r a s genes are one of many possible genetic defects that underlie the development of tumors, none of which appears to be obligatory for the formation of a particular tumor type. Furthermore, the studies showed that the mutational event itself can occur at different stages during tumor development.

IV. HETEROGENEITY OF RAsGENE MUTATIONS One puzzling observation with respect to mutated r a s genes in human tumors concerns the fact that, frequently, a mutation is observed in only a subset of the tumor cells--for instance, in cholangiocarcinomas 12 and seminomas. Seminomas are germline tumors of the testis. In 40% of the tumors, a mutated r a s gene can be detected, but in most of these cases the mutation is present in only a subfraction of the cells. 13To exclude that infiltrating lymphocytes and normal stromal cell account for this observation, aneuploid nuclei of the tumor cells were separated from the diploid nuclei of the nontumor cells by flow sorting. Even after this purification of tumor cell nuclei, the mutation was found to be present in a subfraction of the tumor cells only. In some tumors, areas which do not contain mutant r a s genes were detected, whereas in other areas a mutated r a s gene was clearly present. However, both the histopathology and the DNA index of the different areas of such a tumor were identical. This indicates that the different areas represent the same primary tumor. The tumor cells having the r a s mutation may represent a newly evolved cell clone. Alternatively, the mutant r a s - c o n t a i n i n g cells may be a relict of a cell clone overgrown by a more malignant cell clone that has lost the mutant r a s gene (one can imagine that initially a mutated r a s gene is favorable for cell growth, but that later other genetic defects overcome the requirement of the mutated r a s gene, or that cells with a mutated r a s gene are recognized by specific cytotoxic T cells). Finally, cells with a mutant r a s gene may stimulate the growth of neighboring cells not having the mutation--for instance, by the production of growth factors. In melanoma, r a s gene mutations are also illustrative for events that occur during tumor development. Melanomas are highly malignant tumors which metastasize very rapidly. The incidence of r a s mutations is approximately 20%. The analysis of these tumors resulted in a striking observation with respect to heterogeneity of the tumor.14 In each of two separate tumors, two different mutated r a s alleles were found, whereas in the metastases of these same tumors only one of the mutated alleles could be detected. In one case both mutated alleles were identified in

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JOHANNES L. BOS and BOUDEWIJN M. TH. BURGERING

different metastases. This finding implies that in these cases the primary tumor consists of two different cell clones, each with a mutated r a s gene and each with metastatic potential. Apparently, the cell clones that constitute the primary tumor are phenotypically identical, but genotypically different. Since the two mutations are at a similar position in the gene, the development of the two phenotypically identical cell clones might be explained by assuming that the last genetic event in the formation of the primary tumor is a lesion in the r a s gene (e.g., a DNA adduct or a pyrimidine dimer) induced by a carcinogenic agent (e.g., UV-light). During replication a mutation in the strand opposite of the lesion occurs and, thus, one of the daughter cells will obtain a mutated r a s gene. When the lesion persists in the other daughter cell, another mutation could occur opposite the lesion in the next round of replication. This process might result in two cell clones, each with a different mutated r a s gene. Although this model is attractive, it cannot be excluded that the two cell clones are independent tumors or independent malignant subclones of a premalignant lesion.

V. THE RAS GENE PRODUCTS: P21ras The three human r a s genes encode very similar products of 188/189 amino acid residues, which, by virtue of their apparent molecular weight in SDS-polyacrylamide electrophoresis, are commonly referred to as p21 ras. The K - r a s gene, due to alternative splicing, gives rise to two different p21 ras proteins, and thus four different p21 ras proteins can be identified. The four p21 ras proteins only differ in their carboxy-terminal part. Thus far, these four different p 2 1 ras proteins have shown indistinguishable with respect to biochemical properties and function. Therefore, in the remainder of this paper we will refer to them commonly as p21 r a s , unless stated otherwise. p 2 1 ras is modified posttranslationally at its carboxyterminus. Covalent attachment of a farnesyl group at the cysteine of the carcoxyterminal CAAX sequence is followed by removal of the three carboxyterminal amino acids. Subsequently, the remaining carboxyterminal cysteine is carboxymethylated. Additionally, some of the p21 ras proteins are palmitoylated at another cysteine in the carboxy-terminal part of the protein. These fatty acid modifications serve as anchors to position p21 r a s at the innersite of the plasma membrane. 15 The most striking feature of p21 ras is its very high affinity for GTP and GDP. Furthermore, the protein possesses an intrinsic GTPase activity, which serves to hydrolyze bound GTP. Due to these characteristics p 2 1 ras can be considered a member of the family of guanine nucleotide binding proteins. This family includes, among others, heterotrimeric G-proteins, initiationand elongation-factors of protein synthesis and a large number of small GTPases. 16'17 The activity of these proteins is regulated cyclicly by association of GTP, hydrolysis of GTP to GDP, and phosphate and dissociation of GDP (Figure

2).

171

P21 ras

The dissociation of GDP is considered to be the rate-limiting step in the exchange of GDP for GTP. Recent reports indicate the existence of proteins that stimulate the dissociation of GDP from p21 ras, the guanine nucleotide exchange factors (GNRF). 18'19 This is immediately followed by binding of GTP, of which the concentration in the cell is at least 10-fold higher than GDP. Hydrolysis of GTP occurs through intrinsic GTPase activity of p21ras. This intrinsic GTPase activity is very slow, but greatly augmented by GTPase activating proteins. 2~Two of these proteins are already identified and cloned: p 120GAP and neurofibromin. Both the exchange factors and the GTPase activating proteins will be discussed in detail later. Mutated p21 r a s found in human tumors differs from the normal counterpart by substitutions of either residue 12, 13, or 61. In all these cases, the intrinsic GTPase activity of p21 ras is impaired. 1 This results in a p21 ras protein that is constitutively in the GTP-bound "on" state. In the proper cell type this will lead to constitutive stimulation of cell growth.

VI.

P21rasFUNCTIONS

IN SIGNAL T R A N S D U C T I O N

In mammalian cells, p21 ras function was studied initially by micro-injection of neutralizing antibodies, in particular the rat monoclonal antibody Y 13-259. 21These studies have shown that p21 ras is critically required for serum- and growth factorinduced DNA synthesis in a variety of cell types. The growth factors involved include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), 22 the phorbol ester TPA, prostaglandin F2t~, and phosphatidic acid. 23 In addition, p21 ras was shown to be required for serum-induced c-fos expression in NIH/3T3 cells e4 and nerve growth factor (NGF)-induced differentiation of PC-12 cells. 25 Furthermore, oncogenic transformation by several tyrosine kinase oncogenes (vsrc, v-fes) is transiently reverted after microinjection of monoclonal antibody Y 13-259, indicating that p21 ras is required for transformation by these oncogenes. Cellular transformation by cytoplasmic serine/threonine kinase oncogenes (v-raf and v-mos) appears not to depend on p21 ras.26 The function of p21 ras in signal transduction pathways has also been studied using dominant interfering mutants. 27'28The mutant p2 1ras, a s n l 7 , which displays a reduced affinity for GTP, but normal affinity for GDP, was found to be most effective. This mutant may compete with normal cellular p21 ras for GNRE 19'29 Expression of p21 ras'asnl7 inhibits cellular responses induced by activated p21 ras, that are also inhibited by neutralizing antibodies, that is, inhibition of EGF- and serum-induced DNA synthesis and early gene expression and inhibition of NGF-induced differentiation of PC 12 cells. 27'28 The examples described above and other experiments not discussed 15 show that p21 r,,s is necessary for proper signal transduction by several growth and differentiation factors. However, this does not imply that p21 ras is also directly activated

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JOHANNES L. BOS and BOUDEWIJN M. TH. BURGERING

by these stimuli. It could be that a basal level of p21 rasGTP and not an increase of the GTP-form of p21 r a s is sufficient for most of these pathways.

VIi. GROWTH FACTORS CAN ACTIVATE P21ras Convincing evidence that receptor stimulation can indeed increase the ratio of GTP/GDP bound to p21 ras and thereby presumably activates p21 ras, was provided by experiments in which T-lymphocyte activation was analyzed. Stimulation of the T-cell receptor by phytohaemagglutinin or specific monoclonal antibodies rapidly increases the percentage GTP-bound p21 ras from 7 to 60%. 30 In this type of experiment, the cells were labeled with 32p-orthophosphate in vivo and after receptor stimulation, p21 ras was collected by immunoprecipitation. Labeled GDP/GTP was eluted from p21 r a s and separated on TLC. The increase in p21 rasGTP occurred very rapidly, was maximal within 2 min and sustained for at least 15 min. This strong induction was in sharp contrast to the small increase in the amount of GTP bound to p21 ras following serum, PDGE and EGF stimulation of NIH/3T3 cells (7 to 15% GTP) 31 or Swiss/3T3 cells overexpressing normal p21H ras (0.5 to 1% GTP). 32'33 Possibly, mitogenic stimulation ofT lymphocytes requires a higher relative level of p21 rasGTP than fibroblasts. Insulin is a very weak mitogen for NIH/3T3 cells, presumably due to the low number of insulin receptors expressed by these cells. However, in a NIH/3T3 cell line expressing high numbers of human insulin receptor, insulin is mitogenic and acts as efficient as serum, or other potent mitogens. In these cells it was found that p21 r a s Was loaded with GTP rapidly upon insulin stimulation (15 to 70% GTP), but hardly upon serum stimulation. 34 Taken together, these results have provided the first direct proof that p21 r a s can become activated; that is, accumulation of p21 r a s in the GTP-bound state, upon growth factor stimulation is thus involved actively in growth factor signal transduction. Now it has been demonstrated that many growth factors can increase the amount of p2 lrasGTP. Common to all these growth factors is that tyrosine kinases appear to be involved in passing the signal to p2 lras. However, there is a difference in the level of increase induced by the different growth factors. In part, this difference is determined by the cellular context. An illustrative example is provided by protein kinase C-induced activation of p2 lras: In T lymphocytes this results in an increase of p21rasGTP up to 70%, whereas in fibroblasts no or hardly any increase in p2 lrasGTP is observed. 30 '35

VIII.

P120GAP AND THE RELATION of TYROSINE KINASES

P21 ras

WITH

Originally, p 120GAP was discovered as a cytosolic protein which augmented the GTPase activity of p21 r a s in vitro. 36 The p 120GAP coding sequence has been cloned

P21 ras

1 73

and its predicted amino acid sequence revealed few clues as to its function. 37'38 It showed the presence of SH2 (src homology) and SH3 domains. SH2 domains are found in nonreceptor tyrosine kinases (src, yes, etc.), phospholipase C-7, phosphatidylinositol-3-kinase, the crk oncogene, and several other proteins. 39 The SH2 domains are probably involved in the interaction with phosphotyrosine containing domains, either intra- or intermolecularly. 4~ Also the SH3 domain may serve as a protein recognition sequence. 42 Deletion analysis revealed that the carboxyterminal part (334 amino acids) of p l20GAP is sufficient for the GTPase-activating function and that the N-terminal part, including the SH2 and three domains, may fulfill a regulatory function. 43 An important role for p120GAP in PDGF signaling is suggested by the observation that following PDGF stimulation p l20GAP is rapidly phosphorylated on tyrosine44 and associates with the activated PDGF receptor. 45'46 In vitro, PDGF receptor autophosphorylation, but not p120GAP tyrosine phosphorylation, is necessary for association. 45'46 Furthermore, certain PDGF receptor mutants, defective in signal transduction, do not bind pl20GAP. 46 Also, after EGF -47 and CSF-1 stimulation, 48 p l20GAP becomes phosphorylated on tyrosine, but stable association with the respective receptors has not been observed. In cells transformed by tyrosine kinase oncogenes (v-src, v-abl, v-fms, v-fps), p 120GAP is phosphorylated on tyrosine and complexes with two other tyrosine-containing phosphoproteins, p 190 and p62. 47 Both proteins have been cloned recently. Protein p62 has weak homology with proteins that bind heterogeneous nuclear RNA, and indeed was found to have RNA-binding properties. 49 The function of this protein as well as the relevance for GAP functioning is still unclear. Protein p 190 has homology with a region of the Bcr-protein, N-chimaerin and RhoGAP. 5~Recently, it was shown that p 190 can serve as a GTPase activating protein for members of the Rho family, such as Rho and Rac. 51 Both Rac and Rho function in the regulation of the cytoskeleton organization. 52'53 Also for p190, the significance of binding to GAP remains unclear. A variety of observations indicates that the effector region of p21 r a s (amino acids 32 to 40)provides part of the binding site for pl20GAP on p21ras. 54'55 However, evidence that p 120GAP may be the effector of p21 ras is limited. Studies analyzing the activation ofK § channels of atrial cells in vitro suggests that a complex between p21 raS.GTP and p 120GAP inhibits the coupling of activated muscarinic receptors to a G-protein involved in the regulation of K § channel opening. 56 Moreover, it has recently been shown that a fragment of p l20GAP, containing 0nly the SH2-SH3 domains can inhibit K § channel opening independent of p21 ras. This suggests that p21 ras is involved in the unfolding of p 120GAP to allow the SH2-SH3 domains to bind to proper target proteins. 57These target proteins could be the above-mentioned proteins that associate to p 120GAP or other proteins that can bind to the SH2-SH3 region of GAP. In addition to serving as an effector, p l20GAP may downregulate the level of GTP bound to p21 r~s and as such it provides an obligate negative feedback to signal

1 74

JOHANNES L. BOS and BOUDEWIJN M. TH. BURGERING

transmission by p21 ras. Alternatively, p 120GAP may fulfill an upstream function. In this case, inhibition of p l20GAP activity can be either permissive for the activation of p21 ras by other signals or the signal that inactivates p 120GAP directly results in p2 1 ras activation. This latter possibility is suggested by the inhibition of in vitro p 120GAP activity after stimulation of the T-cell receptor and the concomitant activation of p21 ras.30 It is not excluded that p 120GAP functions both upstream and downstream of p21 ras. In conclusion, tyrosine phosphorylation of p l20GAP and association of p 120GAP to the PDGF receptor and other cellular proteins may be involved in the various possible functions of p 120GAP. These associations and phosphorylations may inactivate p l20GAP resulting in the activation of p21 ras, activate the effector function of p 120GAP, or play a role in the feedback control of p2 1 ras activation. Thus far, no clear evidence exists to support any of these possibilities.

IX. NEUROFIBROMATOSIS TYPE ! GENE AS GTPASE ACTIVATING PROTEIN OF l'21ras Recently, the gene has been identified that carries the defect leading to the von Recklinghausen form of neurofibromatosis (NF1) and the DNA sequence of the large coding region (> 2485 codons) has been determined. 58-64 Surprisingly, the protein, neurofibromin, has a region of approximately 300 residues that shows 30% similarity with pl20GAP, the p l20GAP related domain (GRD). Subsequently, it was found that the GRD segment of neurofibromin is able to induce GTPase activity of normal p21 ras, but not of oncogenically mutated p21 ras. GRD binds stronger than p 120GAP to the effector domain loop of p2 lras, but its specific activity is lower. This may indicate that at low concentrations of p2 l rasGTP, neurofibromin is as efficient as p 120GAP in inducing GTPase activity. 64 Specific antibodies to neurofibromin protein revealed a protein of 240 kDa, which is present in a large complex of proteins. 65 Although little is known about the function of neurofibromin, the significance of this protein for the regulation of p2 l rasGTP levels is indicated by the observation that in neurosarcomas which are devoid of neurofibromin, p2 lras is predominantly in the GTP-bound state. 66

X. GUANINE NUCLEOTIDE EXCHANGE OF P21ras Several proteins have been identified that stimulate the exchange of GDP bound to p21 ras for GTP in vitro. These proteins were denoted guanine nucleotide release factor (GNRF), 67 ras guanine nucleotide exchange factor (rGEF), 68 and rasexchange protein (REP). 69 rGEF is reported to be localized at the plasma membrane and appears to display a broad specificity. GNRF and REP apparently are predominantly localized in the cytosol. Recently, several genes have been cloned based on homology with GNRFs of p2 1 ras in lower eukaryotes. First, CDCMn homologous

1 75

P21 ras

to CDC25, the p21 ras GNRF of yeast, 7~ and second SOSMn, a clone homologous to the Drosophila SOS gene. 72 Whether these proteins are involved in the regulation of nucleotide exchange on p21 ras in vivo, or whether they are responsible for constitutive nucleotide exchange awaits further study. In T lymphocytes, a constitutive high level of nucleotide exchange is observed and the level of p21 rasGTP appears to be regulated predominantly by inhibition of GAP activity. 3~ In fibroblasts, however, increase in nucleotide exchange is the predominant way of regulating the level of p21 rasGTP, at least after stimulation with EGF and insulin. 19 Which of the forementioned rasGNRFs is responsible for this activation is still unknown. Recently two proteins have been identified which may be involved in signal transduction from the receptor to p21 ras. Grb2 is a 23-Kda protein containing a SH2 and a SH3 domain that binds to the EGF receptor. This protein is linked with the p21 ras pathway through its homology to the C. elegans Sem-5 protein. The Sem-5 locus is genetically linked to the p21 ras signaling pathway involved in the determination of cell fate in vulva development. 73 The Shc protein is an SH2 domain-containing protein which serves as a substrate for the EGF-, PDGF-, and insulin receptor. 74 Furthermore, Grb2 associates to a 55-Kda protein which was recently found to be Shc. 75 Whether these proteins are indeed involved in the regulation of p2 lras awaits further analysis.

XI. P21rasAND SERINE/THREONINE KINASES What is the direct biochemical consequence of p21 ras activation? Recent developments indicate that serine/threonine kinases are important proteins in mediating p21 ras-elicited signals further downstream towards the nucleus. In particular, protein kinase (PKC), c-Raf, and the family of extracellular signal-regulated kinase(s) (ERK) (see Figure 3). ERK2 is a 42-Kda serine/threonine protein kinase, also called MAP2 kinase. 76'77 Gene cloning revealed that ERK2 is a member of a family of related kinases that are regulated by extracellular signals. 78 A large variety of stimuli, including growth factors, can induce ERK2 activity. The kinase activity of ERK2 is activated by phosphorylation on both a tyrosine and a threonine residue, located within one tryptic peptide. 79 Scrape loading of active p21H ras protein into Swiss-3T3 cells induced activation of ERK2, suggesting ERK2 as a potential downstream target of p21 ras. In concordance, constitutive activation of ERK2 was observed in p21 r o s -transformed cell lines. 8~ These results show that p21 r~s can regulate ERK2 activity. Recently, using the dominant negative p21 rasasn 17 mutant, we have obtained evidence that indeed in fibroblasts p21 ras is involved in the activation of ERK2 by some growth factors, in particular insulin and PDGF, but not TPA-induced ERK2 activity. 81 Similar results were obtained for nerve growth factor and EGF in PC12 cells. 82'83How p21 ras regulates ERK2 activity is at present unknown. ERK2 activity can be modulated in vitro by an ERK2 kinase, which in turn is regulated by an

176

JOHANNES L. BOS and BOUDEWIJN M. TH. BURGERING receptor tyrosine kinase PI 3 K " s 9

i

9

Ic P i

Grb2 Shc

,,

|

I~ [p120GAPI ~

v

i !

t

('RA

i

9 9

PKC

-~

raf-1

f ERKq~

s

9

s

9

s

fos-SRF

c-jun

~'"

9

~

"4k

$6 kinase II MAPKAP kinase-2

glycogen

synthesis

Figure 3. Schematic representation of various proteins and their interactions in the p21 ras signal transduction pathway in fibroblasts. ERK2 kinase-kinase. 84 Recently, it has been demonstrated that raf- 1 could serve as the ERK2 kinase-kinase 85 (Figure 3). One of the first substrates to be discovered for ERK2 was $6 kinase II (p90rsk).77 Independent observations already suggested a role for p21 ras in the regulation of $6 kinase activity. Increased $6 protein phosphorylation has been observed in viral H-ras-transformed cells and microinjection of active oncogenic p21 r a s stimulates $6 phosphorylation. 86 ERK2/p90 rsk mediated phosphorylation of $6 protein would connect the p21ras pathway to the control of translation, although p90 rsk may not be the guinine in vivo kinase for $6 protein. 87 Control of transcription by p21 r a s may also be regulated through ERK2. In vitro, ERK2 phosphorylates the transcriptional transactivation domain of c-jun, at sites that are also phosphorylated in response to oncogenic p21 ras.88,89 Transcriptional activity of genes containing AP-1 sequence elements within their promoter is thought to be regulated in part by this phosphorylation of c-jun. Using transient expression systems the AP-1 binding site was shown to be the target for regulation of gene transcription by p21ras.90'91 Also, the serum response factor of the c-fos promoter is an in vitro substrate of ERK2. The role PKC plays in p21 ras signaling is very confusing. Depending on cell type or the parameter that is being measured, PKC has been shown to play a role in the

1 77

P21 ras

activation of p21 ras or in the downstream signaling from p21 ras. A nice example of the involvement of PKC in the activation of p21 ras is presented by T lymphocytes, where the phorbol ester TPA, presumably through activation of PKC can activate p21 ras.3O However, PKC activation is not essential for p21 r,~s activation by T-cell receptor stimulation. 92 In fibroblasts, most evidence indicate that PKC is not involved in the activation of p21ras. 93 In signaling from p21 ras to downstream targets, the picture of the involvement of PKC is not quite clear. Both PKC-dependent and PKC-independent pathways have been described. An informative example is provided by Marshall and co-workers who within one assay system (i.e., scrape loading of active p21 ras protein) observe effects that are either dependent (mitogenesis, PC hydrolysis) 94'95 or independent of functional PKC (cellular transformation, induction of c - m y c expression). 96 In addition it has been demonstrated that even the same downstream parameter (i.e., the induction of c-fos) can be induced by active p21 ras through PKC-dependent pathway(s) in one assay system (microinjection of active p21 ras protein)97 or PKC-independent pathway(s) in other assay systems (transient expression with CAT reporter genes). 98 These experiments suggest that in certain cell types, under certain conditions, PKC may receive signals from p21 ras for further processing, but the precise interaction between the two proteins is still elusive. An attractive model would be that PKC serves as a separate signal transduction pathway which is directly activated by receptors through the production of diacylglycerol. This signal transduction pathway will have a close interplay with p2 l raS-mediated signaling. From the above discussion it is clear that p21 r a s is involved in signal transduction. The signal transduction pathways have in common that they transmit signals from cell surface receptors to the nucleus, regulating proliferation and/or differentiation of the cell. A further common feature appears to be that all the cell surface receptors that are somehow linked to p21ras belong to the tyrosine kinase receptors or to receptors that are directly linked to tyrosine kinases. Therefore, the elucidation of the interaction between p21 r a s and tyrosine kinases will be of paramount importance to understand the functioning of p21 r'~s in signal transduction. The observed interaction of p l20GAP with tyrosine kinases, and the identification of putative intermediates between receptor and p21 ras (grb-2 and shc) are already major breakthroughs, but a lot of questions remain to be answered. The variety of receptors that can activate p21 r a s and the involvement of different target molecules, indicate that p21 r a s is part of a complex interplay of signal transduction pathways. With all the unsolved issues, however, there are many exciting years to come before p21 raS-mediated signaling is elucidated.

ACKNOWLEDGMENTS We thank our colleagues for discussion and critical reading of the manuscript. This work is supported in part by a grant from the Dutch Cancer Society.

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20. Trahey, M.; McCormick, E A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science 1987, 238, 542-545. 21. Stacey, D. W.; Tsai, M.-H.; Yu, C.-L.; Smith, J. K. Critical role of cellular ras proteins in proliferative signal tranduction. CSHSQB 1988, 53, 871-881. 22. Mulcahy, L. S.; Smith, M. R.; Stacey, D. W. Requirements for ras protooncogene function during serum-stimulated growth of NIH 3T3 cells. Nature 1985, 313, 241-243. 23. Yu, C.-L.; Tsai, M.-H.; Stacey, D. W. Cellular ras activity and phospholipid metabolism. Cell 1988, 52, 63-71. 24. Stacey, D. W.; Watson, T.; Kung, H.-E; Curran, T. Microinjection of transforming ras protein induces c-fos expression. Mol. Ceil. Biol. 1987, 7, 523-527. 25. Hagag, N.; Lacal, J. C.; Graber, M.; Aaronson, S.; Viola, M. V. Microinjection of ras induces a rapid rise in intracellular pH. Mol. Cell, Biol. 1987, 7, 1984-1988. 26. Smith, M. R.; DeGudicibus, S. J.; Stacey, D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature 1986, 320, 540-543. 27. Cai, H.; Szeber6nyi, J.; Cooper, G. M. Effect of a dominant inhibitory Ha-ras mutation on mitotic signal transduction in NIH 3T3 cells. Mol. Cell. Biol. 1990, 10, 5314-5323. 28. Szeber6nyi, J.; Cai, H.; Cooper, G. M. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PCl2 cells. Mol. Cell. Biol. 1990, 10, 5324-5332. 29. Farnsworth, C. L.; Feig, L. A. Dominant inhibitory mutations in the Mg2+-binding site of ras H prevent its activation by GTP. Mol. Cell. Biol. 1991, 11, 4822-4829. 30. Downward, J.; Graves, J. D.; Warne, P. H.; Rayter, S.; Cantrell, D. A. Stimulation of p21 ras upon T-cell activation. Nature 1990, 346, 719-723. 31. Gibbs, J. B.; Marshall, M. S.; Scolnick, E. M.; Dixon, R. A. F.; Vogel, U. S. Modulation of guanine nucleotides bound to ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP). J. Biol. Chem. 1990, 265, 20437-20442. 32. Satoh, T.; Endo, M.; Nakafuku, M.; Nakamura, S.; Kaziro, Y. Platelet-derived growth factor stimulates formation of active p21 ras-GTP complex in Swiss mouse 3T3 cells. Proc. Natl. Acad. Sci. USA 1990, 87, 5993-5997. 33. Satoh, T.; Endo, M.; Nakafuku, M.; Akiyama, T.; Yamamoto, T.; Kaziro, Y. Accumulation of p21 ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 1990, 87, 7926-7929. 34. Burgering, B. M. T.; Medema, R. H.; Maassen, J. A.; Van de Wetering, M. L.; Van der Eb, A. J.; McCormick, E; Bos, J. L. Insulin stimulation of gene expression mediated by p21 r a s activation. EMBO J. 1991, 10, 1103-1109. 35. Medema, R. H.; Wubbolts, R.; Bos, J. L. Two dominant inhibitory mutants of p21 ras interfere with insulin-induced gene expression. Mol. Cell Biol. 1991, 11, 5963-5967. 36. Trahey, M.; Milley, R. J.; Cole, G. E.; Innis, M. I.; Paterson, H.; Marshall, C. J.; Hall, A.; McCormick, F. Biochemical and biological properties of the human N-ras p21 protein. Mol. Cell. Biol. 1987, 7, 541-544. 37. Trahey, M.; Wong, G.; Halenbeck, R.; Rubinfeld, B.; Martin, G.A.; Ladner, M.; Long, C. M.; Crosier, W. J.; Watt, K.; Koths, K.; McCormick, E Molecular cloning of two types of GAP complementary DNA from human placenta. Science 1988, 242, 1697-1700. 38. Vogel, U. S.; Dixon, R. A. E; Schaber, M. D.; Diehl, R. E.; Marshall, M. S.; Scolnick, E. M.; Sigal, I. S.; Gibbs, J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature 1988, 335, 90-93. 39. Cantley, L, C.; Auger, K. R.; Carpenter, C.; Duckworth, B.; Graziani, A.; Kapeller, R.; Soltoff, S. Oncogenes and signal transduction. Cell 1991, 64, 281-302. 40. Anderson, D.; Koch, C. A.; Grey, L.; Ellis, C.; Moran, M. E; Pawson, T. Binding of SH2 domains of phospholipase Cgl, GAP, and src to activated growth factor receptors. Science 1990, 250, 979-982.

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41. Pawson, T. Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. Oncogene 1988, 3, 491-495. 42. Cicchetti, P.; Mayer, B. J.; Thiel, G.; Baltimore, D. Identification of a protein that binds to the SH3 region of abl and is similar to bcr and GAP rho. Science 1992, 257, 803-806. 43. Marshall, M. S.; Hill, W. S.; Assunta, S. N.; Vogel, U. S.; Schaber, M. D.; Scolnick, E. M.; Dixon, R. A. E; Sigal, I. S.; Gibbs, J. B. A c-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989, 8, ll05-1110. 44. Molloy, C. J.; Bottaro, D. P.; Fleming, T. P.; Marshall, M. S.; Gibbs, J. B.; Aaronson, S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature 1989, 342, 711-714. 45. Kaplan, D. R.; Morrison, D. K.; Wong, G.; McCormick, E; Williams, L. T. PDGF b-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell 1990, 61, 125-133. 46. Kazlauskas, A.; Ellis, C.; Pawson, T.; Cooper, J. A. Binding of GAPto activated PDGF receptors. Science 1990, 247, 1578-1581. 47. Ellis, C.; Moran, M.; McCormick, F.; Pawson, T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature 1990, 343, 377-381. 48. Reedijk, M.; Liu, X.; Pawson, T. Interactions of phosphatidyl kinase, GTPase activating protein (GAP) and GAP-associated proteins with the colony-stimulating factor 1 receptor. Mol. Cell. Biol. 1990, 10, 5601-5608. 49. Wong, G.; MUller, O.; Clark, R.; Conroy, L.; Moran, M. F.; Polakis, P.; McCormick, E Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell 1992, 69, 551-558. 50. Settleman, J.; Narashimhan, V.; Foster, L. C.; Weinberg, R. A. Molecular cloning of cDNAs encoding the GAP-associated protein p 190: implications for a signaling pathway from ras to the nucleus. Cell 1992, 69, 539-549. 51. Settleman, J.; Albright, C. E; Foster, L. C.; Weinberg, R. A. Association between GTPase activators for Rho and Ras. Nature 1992, 359, 153-154. 52. Ridley, A. J.; Hall, A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992, 70, 389-400. 53. Ridley, A. J.; Paterson, H. E; Johnston, C. L.; Diekman, D.; Hall, A. The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling. 1992, 54. Adari, H.; Lowy, D. R.; Willumsen, B. M.; Der, C. J.; McCormick, F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 r a s effector binding domain. Science 1988, 240, 518-521. 55. Cales, C.; Hancock, J. E; Marshall, C. J.; Hall, A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature 1988, 332, 548-551. 56. Yatani, A.; Okabe, K.; Polakis, E; Halenbeck, R.; McCormick, E; Brown, A. M. Ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels. Cell 1990, 61,769-776. 57. Martin, G. A.; Yatani, A.; Clark, R.; Conroy, L.; Polakis, P.; Brown, A. M.; McCormick, E GAP domains responsible for ras p2 l-dependent inhibition of muscarinic atrial K+ channel currents. Science 1992, 255, 192-194. 58. Viskochil, D.; Buchberg, A. M.i Xu, G.; Cawthon, R. M.; Stevens, J." Wolff, R. K.; Culver, M." Carey, J. C.; Copeland, N. G.; Jenkins, N. A.; White, R.; O'Connell, P. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell 1990, 62, 187-192. 59. Xu, G.; O'Connell, P.; Viskochil, D.; Cawthon, R.; Robertson, M.; Culver, M.; Dunn, D.; Stevens, J.; Gesteland, R. ;White, R.; Weiss, R. The neurofibromatosis type I gene encodes a protein related to GAE Cell 1990, 62, 599-608. 60. Wallace, M. R.; Marchuk, D. A.; Andersen, L. B.; Letcher, R.; Odeh, H. M.; Saulino, A. M.; Fountain, J. W.; Brereton, A.; Nicholson, J.; Mitchell, A. L.; Brownstein, B. H.; Collins, E S.

P21 ras

61.

62.

63.

64.

65. 66.

67. 68. 69. 70.

71. 72.

73.

74.

75.

76.

77.

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Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NFI patients. Science 1990, 249, 181-186. Xu, G.; Lin, B.; Tanaka, K.; Dunn, D.; Wood, D.; Gesteland, R.; White, R.; Weiss, R.; Tamaonoi, F. The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae. Cell 1990, 63, 835-841. Cawthon, R. M.; Weiss, R.; Xu, G.; Viskochil, D.; Culver, M.; Stevens, J.; Robertson, M.; Dunn, D.; Gesteland, R.; O'Connell, P.; White, R. A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell 1990, 62, 193-201. Ballester, R.; Marchuk, D.; Boguski, M.; Saulino, A.; Letcher, R.; Wigler, M.; Collins, F. The NFI locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell 1990, 63, 851-859. Martin, G. A.; Viskochil, D.; Bollag, G.; McCabe, P. C.; Crosier, W. J.; Haubruck, H.; Conroy, L.; Clark, R.; O'Connell, P.; Cawthon, R. M.; Innes, M. A.; McCormick, F. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell 1990, 63, 843-849. DeClue, J. E.; Cohen, B.; Lowy, D. R. Identification and characterization of the neurofibromatosis type 1 protein product. Proc. Natl. Acad. Sci. 1991, 88, 9914-9918. Basu, T. N.; Gutmann, D. H.; Fletcher, J. A.; Glover, T. W.; Collins, F. S.; Downward, J. Aberrant regulation of ras proteins in malignant tumor cells from type 1 neurofibromatosis patients. Nature 1992, 356, 713-715. Wolfman, A.; Macara, I. G. A cytosolic protein catalyzes the release of GDP from p21 ras. Science 1990, 248, 67-69. West, M.; Kung, H.; Kamata, T. A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Len. 1990, 259, 245-248. Downward, J.; Riehl, R.; Wu, L.; Weinberg, R. A. Identification of a nucleotide exchange-promoting activity for p21 ras. Proc. Natl. Acad. Sci. USA 1990, 87, 5998-6002. Martegani, E.; Vanoni, M.; Zippel, R.; Coccetti, P.; Brambilla, R.; Ferrari, C.; Sturani, E.; Alberghina, L. Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator. EMBO J. 1992, 11, 2151-2157. Shou, C.; Farnsworth, C. L.; Neel, B. G.; Feig, L. A. Molecular cloning of cDNAs encoding a guanine-releasing factor for ras p21. Nature 1992, 358, 351-354. Bowtell, D.; Fu, P.; Simon, M.; Senior, P. Identification of murine homoloques of the Drosophila Son of Sevenless gene: potential activators of ras. Proc. Natl. Acad. Sci. USA 1992, 89, 6511-6515. Lowenstein, E. J.; Daly, R. J.; Batzer, A. G.; Li, W.; Margolis, B.; Lammwers, R.; Ullrich, A.; Skolnik, D.; Schlessinger, J. The SH2 and SH3 domain-containing protein grb2 links receptor tyrosine kinases to ras signaling. Cell 1992, 70, 431-442. Pelicci, G.; Lanfrancone, L.; Grignani, E; McGlade, J.; Cavallo, E; Forni, G.; Nicoletti, I.; Grignani, E; Pawson, T.; Pelicci, P. G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell 1992, 70, 93-104. Rozalis-Adcock, M.; McGlade, J.; Mbamalu, G.; Pelicci, G.; Daly, R.; Li, W.; Balzer, A.; Thomas, S.; Brugge, J.; Pellici, P. G.; Schlessinger, J.; Pawson, T. Association of the shc and grb2/sem5 SH2-containing proteins is implicated in activation of the ras pathway by tyrosine kinases. Nature 1992, 360, 689-692. Ray, L. B.; Sturgill, T. W. Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adiposites that phosphorylates microtubule-associated protein 2 in vitro. Proc. Natl. Acad. Sci. USA 1987, 84, 1502-1506. Cobb, M. H.; Boulton, T. G.; Robbins, D. J. Extracellular signal regulated-kinases: ERKs in progress. Cell Regulation 1991, 2, 965-978.

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78. Boulton, T. G.; Yancopoulos, G. D.', Gregory, J. S.; Slaughter, C.; Moomaw, C.; Hsu, J.; Cobb, M. H. An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 1990, 249, 64-67. 79. Anderson, N. G.; Maller, J. L.; Tonks, N. K.; Sturgill, T. W. Requirement for integration of two distinct phosphorylation pathways for activation of MAP kinase. Nature 1990, 343, 651-653. 80. Leevers, S. J.; Marshall, C. J. Activation of extracellular signal-regulated kinase, ERK2 by p21 ras oncoprotein. EMBO J. 1992, 11,569-574. 81. De Vries-Smits, A. M. M.; Burgering, B. M. T.; Leevers, S. J.; Marshall, C. J.; Bos, J. L. Involvement of p21 r a s in activation of extracellular signal-regulated kinase 2. Nature 1992, 357, 602-604. 82. Wood, K. W.; Sarnecki, C.; Roberts, T. M.; Blenis, J. Ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, raf- 1, and RSK. Cell 1992, 68, 1041-1050. 83. Thomas, S. M.; DeMarco, M.; D'Arcangelo, G.; Halegoua, S.; Brugge, J. S. Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases. Cell 1992, 48, 525-534. 84. G6mez, N.; Cohen, P. Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature 1991, 353, 170-173. 85. Kariakis, J. M.; App, H.; Zhang, X.; Banerjee, P.; Brautigan, D. L.; Rapp, U. R.; Avruch, J. Raf- 1 activates MAP kinase-kinase. Nature 1992, 388, 417-421. 86. Barrett, C. B.; Schroetke, R. M.; Van der Hoorn, F.; Nordeen, S. K.; Maller, J. L. Ha-ras val12"~r59 activates $6 kinase and p34 cdc2 kinase in Xenopus oocytes: evidence for c-mosXE-dependent and -independent pathways. MoL Cell. Biol. 1990, 10, 310-315. 87. Chung, J.; Kuo, C. J.; Crabtree, G. R.; Blenis, J. Rapamycine-FKBP specifically blocks growthdependent activation of and signaling by the 70kd $6 protein kinases. Cell 1992, 69, 1227-1236. 88. Bin6truy, B.; Smeal, T.; Karin, M. Ha-ras augments c-jun activity and stimulates phosphorylation of its activation domain. Nature 1991, 351, 122-127. 89. Pulverer, B. J.; Kryakis, J. M.; Avruch, J.; Nikolakaki, E.; Woodgett, J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature 1991, 353, 670-674. 90. Wasylyk, C.; lmler, J. L.; Perez-Mutul, J.; Wasylyk, B. The c-Ha-ras oncogene and a tumor promoter activate the polyoma virus enhancer. Cell 1987, 48, 525-534. 91. Sch6nthal, A. Requirement forfos gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters. Cell 1988, 54, 325-334. 92. Izquierdo, M.; Downward, J.; Graves, J. D.; Cantrell, D. A. Role of protein kinase C in T-cell antigen receptor regulation of p21 ras. evidence that two p21 r a s regulatory pathways coexist in T cells. Mol. CeiL Biol. 1992, 12, 3305-3312. 93. Medema, R. M.; de Vries-Smits, A. M. M.; van der Zon, G. C. M.; Maassen, J. A.; Bos, J. L. Ras-activation by insulin and EGf through enhanced exchange of guanine nucleotides on p21 ras 1992, 94. Morris, J. D. H.; Price, B.; Lloyd, A. C.; Self, A. J.; Marshall, C. J.; Hall, A. Scrape-loading of Swiss 3T3 cells with ras protein rapidly activates protein kinase C in the absence of phosphoinositide hydrolysis. Oncogene 1989, 4, 27-31. 95. Price, B. D.; Morris, J. D. H.; Marshall, C. J.; Hall, A. Stimulation of phosphatidylcholine, diacylglycerol release, and arachidonic acid production by oncogenic ras is a consequence of protein kinase C activation. J. Biol. Chem 1989, 264, 16638-16643. 96. Lloyd, A. C.; Paterson, H. E; Morris, J. D. H.; Hall, A.; Marshall, C. J. p21H-ras-induced morphological transformation and increases in c-myc expression are dependent of functional protein kinase C. EMBO J. 1989, 8, 1099-1104. 97. Gauthier-Rouvi~re, C." Fernandez, A.; Lamb, N. J. C. ras-lnduced c-fos expression and proliferation in living rat fibroblasts involves C-kinase activation and the serum response element pathway. EMBO J. 1990, 9, 171-180.

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98. Fukumato, Y.; Kaibuchi, K.; Oku, N.; Hori, Y.; Takai, Y. Activation of the c-fos serum response element by the activated c-Ha-ras protein in a manner independent of protein kinase C and cAMP-dependent protein kinase. J. Biol. Chem. 1990, 265, 774-780. 99. Neri, A.; Murphy, J. P.; Cro, L.; Ferrero, D.; Tarella, C.; Baldini, L.; Dalla-Favera, R. Ras oncogene mutation in multiple myeloma. J. Exp. Med. 1989, 170, 1715-1725.

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CHROMOSOMAL BASIS O!HEMATOLOGIC MALIGNANCIES

Ram S. Verma

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

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Consistent Chromosomal Abnormalities

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Chronic Myelogenous Leukemia (CML)

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Acute Leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V. VI. VII. VIII.

A.

Acute Lymphocytic Leukemia (ALL)

B.

Acute Nonlymphocytic Leukemia (ANLL)

Lymphoproliferative Disorders

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B-Cell Non-Hodgkin's Lymphoma . . . . . . . . . . . . . . . .

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T-Cell N o n - H o d g k i n ' s L y m p h o m a

C h r o m o s o m e P a t t e r n in H u m a n T u m o r s . . . . . . . . . . . . . . . . . . . . .

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Neoplasms of Chromosome Breakage Syndromes

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Conclusion

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References

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Advances in Genome Biology Volume 3A, pages 185-210. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

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RAM S. VERMA I.

INTRODUCTION

Eighty years ago Boveri I hypothesized that the transformation of a normal cell to a neoplastic cell was due to an abnormal chromatin content. Later, Winge 2 proposed that a neoplastic cell was the result of a single genetic event and a variable chromatin content would be considered the consequences of the origin of malignancy. Furthermore, it was speculated that malignant cells originate from a "single" cell which has lost the property of a normal function. 3 However, the chromosomal basis for human neoplasia could not be recognized until 1958, primarily due to technical difficulties in the analysis of chromosomes. Due to the advent in chromosome banding techniques, many neoplasms have been associated with gross chromosomal abnormalities during the past two decades. 4 A number of reports concluded that breakpoints are limited to specific regions which contained genes for neoplastic transformation. 5'6 Exhaustive attempts have resulted in a renewed focus on chromosome rearrangements as a causative factor in cancer progression. 71 shall present only a brief account of a genomic diversity in human neoplasia since much of the detailed information pertaining to chromosome rearrangement has been reviewed earlier. 8-2~

I!. CONSISTENT C H R O M O S O M A L ABNORMALITIES Major advances in cancer cytogenetics took place in the early 1970s with the introduction of both improved banding techniques and tissue culture methods. 21 The surprising relationship of unique chromosomal abnormalities with specific neoplasia during neoplastic development has been a prime concern. 22 Almost all human cancers have aberrant genomic constitution whose significance has been explained using chromosome bands. 23 Our unfolding knowledge of the human gene map, taken together with information about which chromosomes are preferentially altered during neoplastic development, have led to the cloning of genes which cause cancer 24 (Table 1). Consequently, a definite relationship between genetic anomalies and specific types of proliferative processes has been established. 25 The gain of genetic material has been implicated with gene amplification while complex chromosomal abnormalities suggest different mechanism of genetic changes which may be responsible for the development of malignancies. 26 Literature pertaining to chromosomal changes in neoplastic diseases has been quite extensive. However, in an attempt to elucidate the basic principles concerning the role of chromosomes in hematologic malignancies, I have selected a few examples whose etiologic factors are related to the target cells in proliferation of human neoplasia. It became quite necessary to present concise information on these neoplasms in a tabulated form which shall serve as a quick reference source.

187

Chromosomes and Cancer

Table 1. Cloned Familial Cancer Genes* Entity

Re ti nob last oma Wilms' tumor Li-Fraumeni syndrome Familial adenomatous polyposis Neurofibromatosis type 1 Neurofibromatosis type 2 vonHippel-Lindau syndrome Multiple endocrine neoplasia type 2A

Gene Symbol

Chromosomal Location

rb I wt l tp53 apc nfl

13q 14

nf2 vhl men2a

11p 13

17p 13 5q21 17ql 1 22q 12 3p25 10q 11

Note: *AfterKnudson18

!!1. CHRONIC MYELOGENOUS LEUKEMIA (CML) Chronic myelogenous leukemia (CML) was first described in 1845 by Bennett, 27 but was not fully understood until the early 1900s. 28 CML is the most common hematologic disorder among the myeloproliferative diseases. It is one of the most important subtypes of leukemias because the first consistent chromosomal abnormalities in any malignant diseases were detected in CML. 29 The discovery of the balanced reciprocal translocation involving chromosomes 9 and 22 [t(9;22) (q34;ql 1)] in CML 3~ became a fundamental basis of consistent chromosomal abnormality in other neoplasia. It was further demonstrated that the same chromosomes 9 and 22 were involved in every cell, confirming that the cancer has a colonal origin which was based on earlier studies by enzyme markers. Only 95% of these cases have t(9;22) standard translocation while remaining cases have a variety of so-called variant translocations. Some variant translocations are highly complex in nature and the readers are referred to various reviews. 31-34 The molecular structure of Ph-chromosome has provided a most important turning point in understanding the disease. The location of c-abl gene on chromosome 9 (band q34) which translocates to chromosome 22 have demonstrated that the breakpoints on chromosome 22 are clustered within a 5.8 kb sequence of DNA, designated the breakpoint cluster region (BCR). 35 The translocation produces a unique mRNA transcript from the chimeric gene that is formed by fusion of the 5' portion of the bcr gene with the 3' portion of abl. The results enhanced tyrosine kinase activity. 36'37 A variety of mechanisms, which activate c-abl gene during leukemogenesis, have been proposed 38 including lacking of complete bcr/abl gene in CML. A number of animal models have been proposed to understand the complex pattern of bcr/abl gene fusion and progression of disease. 39'4~

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RAM S. VERMA

IV. ACUTE LEUKEMIAS The acute leukemias are a highly heterogeneous group of malignant proliferations and are generally classified as either acute lymphocytic leukemia (ALL) or acute nonlymphocytic leukemia (ANLL). The classification ofleukemias was originally based upon the French-American-British (FAB) system, 41 whose criteria were the morphological and immunological characteristics of the cells. Later subsets were recognized based on cytogenetic changes. ALL has been divided into three FAB subtypes: (1) L 1 (small cells, homogeneous morphology); (2) L2 (large cells, heterogeneous morphology); and (3) L3 (large cells, homogeneous morphology-Burkitt type). ANLL has seven FAB subtypes that have distinctive hematological and clinical features: (1) M1A [acute myeloblastic leukemia (AML) without differentiation]; (2) M2 [acute myeloblastic leukemia (AML) with differentiation]; (3) M3 [acute promyelocytic leukemia (APL)]; (4)M4 [acute myelomonocytic leukemia (AMMoL)]; (5) M5a [acute monocytic leukemia (AMoL), poorly differentiated]; M5b [acute monocytic leukemia (AML), well differentiated]; (6) M6 [erythroleukemia (EL)]; and (7) M7 [acute megakaryocytic leukemia]. The nonrandom major chromosomal abnormalities frequently seen in ALL are listed in Table 2, while the consistent abnormalities found in ANLL are listed in Table 2. Consistent and Primary Structural Chromosome Abnormalities in Acute Lymphocytic Leukemia (ALL)a

Rearrangement

Typical Morphology

t(1;11)(p32;23) t( 1; 19)(q23;pl 3) t(2;8)(p12;q24) t(4;1 l)(p21 ;q23) del(6q) t(8;14)(q24;ql 1) t(8; 14)(q24;q32) t(8;22)(q24;ql 1) del/t(9p) t(9;22)(q34;ql 1) t(10; 14)(q24;ql 1) t( 11 ;14)(pl 3;ql 1) t( 11 ;14)(q23;q32) t(11 ;19)(q23;p13) del/t(12p) Near haploid

Note: aAfterCin and Sandberg42

L1 L1 L3 L1, L2 LI, L2 L1, L2 L3 L3 L 1, L2 L1, L2 L1, L2 L1, L2 L1, L2 L1, L2 L 1, L2 L 1, L2

Typical ImmunophenoO,pe Pre-B ALL Pre-B ALL B ALL Early B-precursor ALL, mixed phenotype Common ALL TALL B ALL B ALL T ALL or early T-precursor ALL Early B-precursor, common, or pre-B ALL T ALL T ALL non-B, non-T ALL B ALL, mixed phenotype Common ALL B ALL

Chromosomes and Cancer

189

Table 3. Consistent and Primary Structural Chromosome Abnormalities in Acute Nonlymphocytic Leukemia (ANLL)

M 1: M2: M3: M4: M5: M6: M7:

-7; -17; +8; +21; 5q-; -5; Ph; 7q-, del or t with 3. t(8;21);-7; +8; 5q-; 7q-; -5; del or inv(3); t(6;9), Ph t(15;17), i(17q), t(l;17) del ort or inv(16); +8; -7; del or t(llq); t(6;9); 5q-/-5; 7q-; +4; Ph del ort(llq); +8; t(9;ll) -7; +8; 5q-; -5; 7q-; -3 or t with 3; dup(l) inv or del (3); ring; +8; +21

Table 3. Anumber of investigators have subtyped the ANLL based on chromosomal findings. Diversity of chromosomal abnormalities implicate the existence of various genetic mechanisms associated with different subtypes of leukemia 42 (Table 4). Consequently, a multistep evolution of malignancy has been proposed. However, in ANLL, with translocations and inversions, fewer steps are required. It has been suggested that ANLL patients with normal karyotypes have a better prognosis than those with abnormalities, though survival varied from patient to patient. The type of chromosomal abnormalities has been associated with survival rate. 43 It is quite interesting to note that chromosomal gain and loss in ALL is nonrandom. Chromosome 16 has not been observed in gain and chromosome 1 is not lost so far in ANLL. Chromosome 8 is most frequently gained, while loss of chromosome 7 is the most common anomaly.

A. Acute Lymphocytic Leukemia (ALL) WBC count and immunological markers are the most important prognostic factors in childhood acute lymphoblastic leukemia (ALL). However, cytogenetic findings can be correlated with other prognostic factors because various chromosomal abnormalities have been associated with distinct immunological phenotypes of ALL. So far, several hundred patients have been evaluated and hyperdiploidy was the most common abnormality. 53'-54 However, abnormalities involving chromosome t(4;ll), t(8;14), and the Ph-chromosome have drawn much attention in recent years.

The 8; 14 Translocation The third international workshop confirmed the definite association of t(8;14) and L3 morphology (FAB) of ALL with B-cell characteristics. However, a similar

Table 4. Neoplasias with a Shared Single Recurrent Chromosomal Defect a Chromosomal Defect

del lp t( 1;3)(p36;q21 ) inv(3)(q21q27) t(4;11)(q21 ;q23) del(5)(q 13q31) del(6)(q21q25) t(6,9)(p21.2;q34) del(7)(q31.2q36) +8 t(8;14) (q24.1 q32.3)

t(8;21)(q22.1 ;q22.3) t(9; 11)(p22 ;q23.3) t(9,22)(q34.1 ;ql 1.21)

t( 11;14)(q13.3;q32.3)

t(ll ;22)(q23;ql 1.2)

+12 isol2p del 13(q14)

Disease

Neuroblastoma Melanoma Myelodysplasia Acute nonlymphocytic leukemia (M4) Myelodysplasia Acute nonlymphocutic leukemia (MI,M2) Acute lymphocytic leukemia (LI,L2) Acute myelomonocytic leukemia Myelodysplasia Acute nonlymphocytic leukemia (M 1,M2,M4,M5,M6) Diffuse large-cell lymphoma Acute lymphocytic leukemia (L2) Acute nonlymphocytic leukemia (M1,M2) Myelodysplasia Acute nonlymphocytic leukemia (M 1,M2, M4,M5,M6) Myelodysplasia Acute nonlymphocytic leukemia (MI,M2, M4,M5,M6) Burkitt's lymphoma Acute lymphocytic leukemia (L3) Small noncleaved non-Burkitt's lymphoma Immunoblastic lymphoma (B cell) Acute myelogenous leukemia (M2,M4) Acute monocytic leukemia (M5) Acute myelomonocytic leukemia (M4) Acute nonlymphocytic leukemia (M2) Chronic myelogenous leukemia Acute myelogenous leukemia (M1,M2) Acute lymphocytic leukemia (L I,L2) Chronic lymphocytic leukemia, B cell Small-cell lymphocytic lymphoma (B cell) Diffuse large-cell lymphoma (B cell) Ewing's sarcoma Neuropithelioma Askin's tumor Chronic lymphocytic leukemia (B cell) Small-cell lymphocytic lymphoma (B cell) Seminoma Teratoma Retinoblastoma Constitutional retinoblastoma Osteosarcoma (continued)

190

191

Chromosomes and Cancer

Table 4. (continued) Chromosomal Defect

inv( 14q 11.2q32) or t(14;14)(ql 1.2;q32.3)

t(14;18) (q32.3 ;q21.3)

inv(16)(p13.1q22.1)

Disease

Chronic lymphocytic leukemia (T cell) Small lymphocytic lymphoma (T cell) Sezary syndrome Mycosis fungoides Follicular small cleaved cell lymphoma Follicular mixed cell lymphoma Follicular large-cell lymphoma Acute monocytic leukemia (M5b) Acute myelomonocytic leukemia (M4) Acute nonlymphocytic leukemia (M2)

Note: aAfterYunis2~

translocation has been observed in a high proportion of Burkitt's tumors (BL) of both African and non-African origin, 55 indicating BL and ALL are probably different manifestations of the same disease. 56 A variant translocation involving chromosomes 2 and 8 [i.e. t(2;8) (pll-12;q24) and t(8;22)(q24;qll)] have been reported in Burkitt's lymphoma and B-cell ALL. 57 Complex translocations have also been found but the breakpoints were localized within band 8q24.

The 4;11 Translocation About 8-10% of patients with ALL have a consistent translocation involving the long arms of chromosomes 4 and l l[t(4;ll)(q21;q23)]. The patients have poor prognosis owing to high leukocyte counts and have a very high tumor burden. The leukemic cells can be L 1, L2, and L3 subtype. The immunological markers were non-T, non-B, and T-cell ALL. The patients who have poor outcome have complete remission rate of 67% and the median survival was 7 months. It is suggested that cells by t(4; 11) are not lymphoid but rather early myeloid forms. However, Parkin and his colleagues disagreed. 58 The breakpoints on chromosome 11 is the same (11 q23) as noted in children with AMoL-M5a. Light and heavy immunoglobulin chain gene arrangements have been demonstrated in patients with t(4; 11).59'6~

Near Haploid in ALL Near haploidy in ALL is a rare occurrence. 61 The chromosome number usually ranged from 26 to 36 and the gain was chromosomes + 1, +6, + 10, + 18, + 19, +22, +X, +Y. ALL with near haploid chromosomes may be a unique subgroup of ALL with a prognosis that is poor compared with that for other types of non-T and non-B ALL.

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Hyperdiploidy in ALL Hyperdiploidy in patients with ALL has been recognized. 62 The chromosome numbers range from 50 to 60 with gain of a few chromosomes. Patients can be non-T and non-B ALL and the L 1 and L2 types are equally common. Patients with hyperdiploidy have better prognosis and may have other structural abnormalities.

The t(9;22) Translocation in ALL The Philadelphia (Ph) chromosome which results due to t(9;22) commonly associated with CML is also found in 6% of children and 17% adult patients with ALL. Variant translocations observed in CML are also seen in ALL. A normal cell line are usually observed in ALL with Ph-positive karyotypes although rarely seen in CML patients. It is suggested that ALL with Ph-positive karyotype is of pre-B-cell or B-cell lineage while a few cases were found to have B-cell and myeloid markers. 63'64 Molecular studies have yielded similar genes (ABL and BCR) which are involved in the pathogenesis of CML and ALL. Nevertheless, ALL patients with Ph-positive karyotypes suggested that the disease has two subgroups. 65 In one group, the identical breaks were found in the BCR region producing a 8.5-Kb message with a 210-kDa fusion protein while in the second it was absent due to lack of 8.5-Kb mRNA. 66 Nevertheless, the breaks occur in 5' (upstream) of the BCR giving rise to smaller fusion protein (185-190 kDa) owing to a smaller chimeric mRNA (6.5-7.4 Kb). 67 These findings have clearly shed some light concerning clinical differences between these entities. 68

The 1; 19 Translocation Williams and colleagues 69 reported a consistent recurring chromosomal abnormality involving chromosomes 1 and 19 [i.e., t(1;19)(q23;p13] in patients with B-cell ALL. Later, these findings were confirmed in children who have had low white blood counts with pre-B ALL and a t(1; 19) translocation. 7~These cytogenetic findings helped to distinguish a subgroup of patients with pre-B cell ALL of poor prognosis. 71

T-Ceil Acute Lymphoblastic Leukemia A distinct chromosomal abnormality has been noted in T-cell acute lymphoblastic leukemia. 72 Notably, the involvement of chromosome 14 (band q l l) and chromosome 7 (7q34-36). However, there are other variable yet distinct chromosomal abnormalities that are observed, which are also observed in lymphomas of T-cell origin.

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B. Acute Nonlymphocytic Leukemia (ANLL)

The 8;21 Translocation in Acute Myeloblastic Leukemia (AML-M2) The precise identification of the t(8;21) was first described by Rowley. 44 At least 10% of the cases had a balanced translocation involving chromosomes 8 and 21 [t(8;21)(q22;q22)]. The abnormality is although restricted to AML-M2, the similar translocation has been reported in acute myelomonocytic leukemia (M4). Complex translocations involving chromosomes 8 and 21 are also seen. Furthermore, the t(8;21) is often accompanied by the loss of a sex chromosome which denotes a broader prognosis. The survival of patients with AML-M2 having t(8;21 ) was much longer when compared with patients who had other chromosomal abnormalities.

The 15;17 Translocation and Acute Promyelocytic Leukemia (APL-M3) The structural rearrangement involving chromosomes 15 and 17 in acute promyelocytic leukemia (APL-M3) was first recognized by Rowley and colleagues. 45 With improved culturing and banding procedures the exact breakpoints are now being resolved [i.e., t(15;17) (q22;qll-12)]. 46 APL is comprised of two subtypes hypergranular (M3) and the less common microgranular variant (M3V). APL is a heterogeneous disease and presentation of the t(15;17) has been very helpful in diagnosing and clarifying the disease.

Structural Aberrations of Chromosome 16 in Acute Myelomonocytic Leukemia (AMMoL) Recently, cytogenetic association has been established in acute myelomonocytic leukemia with abnormal eosinophils (AMMoL-M4Eo) having deleted chromosome 16; del(16)(q22). 47 Le Beau et al. 48 have reported a related entity with an inversion of the long arm of chromosome 16, inv(16)(q13q22), and also del(16)(q22). The breakpoints in the long arm for the del(16) and for the inv(16) were the same with an increase in the number of eosinophils with morphological and cytochemical abnormalities. Another distinct subtype of AMMoL has been referred to as M4Eo with abnormal eosinophils and abnormalities of chromosome 16. Le Beau 49 has suggested that breakpoints at both 16p 13 and 16q22 are necessary to develop M4Eo leukemia. Patients with either inv(16) or +(16;16) have good response to treatment.

The 6;9 Translocation in ANLL Patients with an increase in basophils in the marrow which appear to be morphologically normal were found to have a consistent translocation involving chromosomes 6 and 9 [t(6;9)(p23;q34)]. 5~This is a relatively rare abnormality comprising

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RAM S. VERMA

only about 2% of patients with ANLL. Patients with a t(6;9) respond poorly to intensive remission induced therapy.

Aberrations of 11q in Acute Monoblastic Leukemia (AMoL) Berger and colleagues 51 found unexpectedly higher incidence of abnormalities of the long arm of chromosome 10 in AMoL (M5). Later Rowley suggested a strong association between 11 q abnormalities and the poorly differentiated form of acute monoblastic leukemia (M5a). This abnormality occurs most frequently in children with monoblastic leukemia. The breakpoints in 11 q involving band 11 q23 are more frequent but other chromosomes are also involved. 52

V. LYMPHOPROLIFERATIVE DISORDERS Consistent chromosomal abnormalities in non-Hodgkin's lymphoma (NHL) have been reported and, more importantly, these nonrandom chromosomal abnormalities have been correlated with histology and immunological phenotype. 73'74Using high resolution chromosome analysis, over 97% of the patients were found to have some sort of chromosomal abnormalities. For example, the presence of a t(14;18)(q32.3;q21.3) translocation of follicular lymphoma was the most significant finding. 75'76 A deletion of the long arm of chromosome 6 (6q) or duplication of the long arm of chromosome 18 (18q) was an exclusive finding in follicular large cell lymphoma. Patients with a t(8; 14)(q24;q32) have either small non-clear cells or diffuse large cell lymphoma. The most compelling observation was the involvement of band 14q32 in neoplasm of B-cell lineage while T-cell neoplasms were characterized having 14q 11, 7q34-36, or 7p 15 abnormalities. 77

A. B-Cell Non-Hodgkin's Lymphoma Burkitt's Lymphoma Burkitt's lymphoma (BL) is the one for which a viral etiology has been more seriously considered than for any other neoplastic diseases. A consistent chromosomal abnormality was first described by Manolov and Manolova 78 in the five of six BL patients in the malignant cells and in seven of nine cultured cell lines.79 They noted an additional band at the end of the long arm of one chromosome 14 (i.e. 14q+). Later, Zech et al. 8~ reported that it was a reciprocal translocation both in African American types and had identical breakpoints on 8q and 14q [i.e., t(8;14)(q24;q32)]. The c-myc oncogene located at band 8q24.1, becomes deregulated when rearranged. The 5' end fuses with the constant gene of the immunoglobin heavy chain (IG) at band 14q32.381 in at least 75% of BL patients. In the remaining

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25% of these cases, two other variant translocations involving chromosomes 2 or 22 may occur [i.e., t(2;8) (p 11.1 q24.1) or t(8;22) (q24.1 ;q I 1.2)]. The myc oncogene remained on chromosome 8 in such variant translocations but rearranged with the ~c-or k-immunoglobin light-chain genes of chromosomes 2 or 22, respectively. The chromosome region distal (3') to the myc oncogene is involved in these translocations. 82 It is a DNA binding protein which is a product of the myc gene that is responsible for the unrestricted proliferation of the B cells. 83'84 The diagnosis and classification of lymphomas have been an arduous task but recent availability of molecular techniques have been extremely helpful.

Other B-Ceil Non-Hodgkin'sLymphoma There are several other specific chromosomal abnormalities which are noted in non-Hodgkin lymphoma. In follicular lymphoma, which constitutes about 40% of all n o n - H o d g k i n l y m p h o m a , 87% of the reported cases have a t(14;18)(q32.3;q21.3) translocation. A deletion of 6q or a duplication of 18q was found almost exclusively in follicular large cell lymphoma. The well-differentiated small cell lymphoma or small lymphocytic lymphoma (SLL) and chronic lymphocytic leukemia (CLL) are a heterogeneous group of disorders. The nonrandom chromosomal abnormality observed in these disorders include: Trisomy 12, t(ll;14)(q13.1;q32.3), del(ll)(q14.2;q23), t(12;14)(q13.1;q32.3), t( 14; 14)(q 11.2;q32.3 ), and inv( 14)(q 11.2;q32.3 ). Molecular characterization of the recurring chromosomal abnormalities in t(ll;14)(ql 3.3;q32.3) have assisted in localizing the bcll gene within the IGH gene at band 14q32, 85 and the bcl2 gene was identified at 18q21.3 in t(14;18)(q32.3;q21.3) in follicular lymphoma (Table 5). 86

B. T-Cell Non-Hodgkin's Lymphoma Leukemias and lymphomas of T-cell origin, have consistent recurring chromosomal abnormalities. 88-9~The most common rearrangements in T-cell neoplasms involve chromosome 14, band 14ql 1, and chromosome 7, band 7q 34-36 and 7p 15. T-cell receptor gene loci are apparently involved in such translocations T-cell receptor o:-chain and 8-chain genes (TCRA and TCRD) are mapped to 14q I 1 while the 13-chain and y-chain genes are located at bands 7q34-35 and 7p 15, respectively. The breakpoints occur within these genes which may activate the cellular proliferation (Table 6). 87 The chromosomal abnormalities in other lymphoproliferative disorders are not fully characterized due to poor culture growth. 91 However, in B-cell chronic lymphocytic leukemia (CB-CLL), trisomy 12 is the most common abnormality. 92 It has been suggested that prognosis is poorer in patients who have other than

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Table 5. Chromosomal Abnormalities in Lymphoma Immunophenotype

Abnormalities

Burkitt's lymphoma t(8; 14)(q24.1;q32.3) t(8;22)(q24.1;ql 1.2) t(2;8)(pl 1.1;q24.1) Non-Hodgkins lymphoma +3 +5 6p+7 14q+ +18 CLL and diffuse small-cell lymphoma t(l I ;14)(ql3.1 ;q32.3) del(l l)(q14.2q23) t( 12;14)(ql 3.1 ;q32.3) t( 14;14)(q11.2;q32.3) inv(l 4)(qi ".2q32.3) +12 6qFollicular lymphoma t( 14;18)(q32;q21) +7 + 12 or +7 and + 12 both 13q2p+ T cell 14qI 1

trisomy 12 karyotype. 93 Chromosome abnormality involving 14ql 1 have been reported in chronic T-cell leukemia (T-CLL). 94

Polycythemia Vera (PV) The chromosomal abnormalities in polycythemia vera (PV) are nonrandom though PV represents a diagnostically difficult and prognostically diverse group of disorders. For some unknown reasons, the chromosomal abnormalities have been found three times more often in treated patients than in those untreated. However, in PV, cytogenetic abnormalities do not necessarily predict leukemia but an evolutionary change in karyotype during the course of disease has important prognostic value. Eighty-three percent of patients with PV had a modal number of 46 to 47

Table 6. Cytogenetic-lmmunophenotypic Correlations in Malignant Lymphoid Diseasesa Phenot)pe

Rearrangement

Involved Genesb

Acute lymphoblastic leukemia Pre-B B(Sig+)

B or

B-myeloid

Other

T

t(1 ;9)(q23;p13) t(8;14)(q24;q32) t(2;8)(pl 1-12;q24) t(8;22)(q24;ql 1) dic(9; 12)(pl 1;p12) t(9;22)(q34;ql 1) t(4;11)(q21 ;q23) hyperdiploidy (50-60 chromosomes) del(9p),t(9p) del( 12p),t(12p) t(11; 14)(pl 3;q 11) t( 11 ; 14)(p 15 ;q 11) t(8;14)(q24;ql 1) inv(14)(ql lq32) inv(14)(ql lq32) t( 10; 14)(q24;ql 1) t(1;14)(p32;ql 1) t(7;9)(q34-36;q34) t(7;9)(q34-36;q32) t(7;7)(pl 5;ql 1) t(14; 14)(ql 1;q32) t(7;14)(q34-36;ql 1) t(7;14)(pl 5;ql 1) t(7;19)(q34-36;pl 3)

pbxl myc myc

e2a igh igk igl

abl

bcr

myc tcra tcra tcl5 tcrb

myc

tcl2 tall tcra igh

tcrd tcrd

tcl3 tcrd

tcrd

tcrb

tcl4

tcrb

lyll

tcrg

Non-Hodgkin's lymphoma B(Sig+)

T

t(8;14)(q24;q32) t(2;8)(pl 1-12;q24) t(8;22)(q24;ql 1) t( 14;18)(q32 ;q21 ) t(11 ; 14)(ql 3;q32) see T-cell ALL

myc myc

igh igk igl igh bcll

myc bcl2 igh

Chronic lymphocytic leukemia B

t(11; 14)(ql 3 ;q32) t(14;19)(q32;q13) t(2;14)(p13;q32)

igh

bcll bcl3 igh

igh

myc igh

tcra

t(14q)

T

+12 t(8; 14)(q24;ql 1) inv(14)(ql 1;q32) inv(14)(ql 1;q32)

197

tcra tcra

(continued)

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RAM S. VERMA

Table 6. (continued) Phenotype

Rearrangement

h~volved Genes b

Multiple myeloma B

t( 11 ;14)(q13;q32)

bcll

igh

t(14q) Adult T-cell leukemia

t(14;14)(ql 1;q32) inv(14)(ql I ;q32) +3

Notes: aAfterM.M. LeBeau87 hpbxl, pre-B cell leukemia gene 1; e2a, immunoglobulin enhancer binding protein gene; myc, cellular protooncogene, homolog of the transforming sequence of the avian myelocytomatosis virus; igh, immunoglobulin heavychain gene; igk, immunoglobulin kappa light-chain gene; igl, immunoglobulin lambda light-chain gene; abl, cellular protooncogene, homolog of the transforming sequence of the Abelson murine leukemia virus; bcr, cellular gene

containing the breakpoint cluster region, encodes a protein with a tyrosine kinase activity; tcl2, cellular protooncogene, T-cell leukemia/lymphoma gene 2; tcrd, T-cell receptor 8-chain gene; tall, T-cell acute leukemia gene 1; tcra, T-cell receptor or-chain gene; tcl3, cellular protooncogene, T-cell leukemia/lymphoma gene 3; tci5, cellular protooncogene, T-cell leukemia/lymphoma gene 5; tcrb, T-cell receptor I]-chain gene; tcrg, T-cell receptor ](-chain gene; lyll, lymphoid leukemia gene 1; bcl2, cellular protooncogene, B-cell leukemia/iymphoma gene 2; bcll, cellular protooncogene, B-cell leukemia/lymphoma gene 1; bcl3, cellular protooncogene, B-cell leukemia/lymphoma gene 3.

chromosomes, while hypoploidy accounted for only 10%. 95 The nonrandom distribution of chromosomal changes is particularly evident in the additional gain of both chromosomes 8 and 9 which are seldom observed in other neoplastic diseases and may be unique to PV. 96 The most frequently rearranged chromosomes are #18 and #20, while 20q- was seen in at least 32% of PV patients with aneuploidy. 97 The rearrangement of chromosome 20 is less frequent in an advanced stage. Furthermore, structural rearrangement of chromosome 12 which is rare in the stable stage of the disease is most frequent in the advanced phase, while 5q- may be a specific abnormality frequently associated with the terminal stage.

Refractory Anemia and Preleukemia There is a considerable disagreement concerning the classification of refractory anemia (RA) as they belong to a heterogenous group. About 50% of patients have a normal karyotype. The most frequent abnormalities are monosomy 5, 7, and trisomy 8. The deletion of long arm of chromosome 5 is quite variable [del (5)(q 1322q33)] and as many as 75% of the cases have such interstitial deletions. 98- 100 The presence of chromosomal abnormalities in patients with cytopenia is a sign of poor prognosis.

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VI. CHROMOSOME PATTERN IN HUMAN TUMORS Owing to technical difficulties, the chromosomal basis of solid tumors have not been fully explored. Most of the chromosomal findings are based on materials from advanced tumors, often metastatic lesions or effusion material which has resulted in difficulties in differentiating between primary and secondary chromosomal changes in solid tumors (Table 7). Furthermore, the chromosomal abnormalities found in solid tumors are far more heterogeneous as compared to leukemias. 1~ In general, the chromosomal abnormalities in solid tumors can be classified into three classes" (1) translocation, insertion and inversion, (2) interstitial deletion and chromosome monosomy, and (3) amplification (double-minute chromosomes and homogeneously staining region) (Tables 7-9). The amplifications and deletions involving large segments can disrupt blocks of genes resulting in deregulation of cells. Certain embryonic tumors have nonrandom chromosomal abnormalities while others predisposes to specific tumors. Specific chromosome deletion have been used in cloning oncogenes or tumor suppressor genes. A number of tumors have been found to have a specific chromosome deletion. One of them, retinoblastoma, is a highly malignant congenital tumor that characteristically arises multicentrically in one or both retina. 1~ Approximately 6% of all retinoblastoma cases are familial and of these 50% are bilateral. The remaining sporadic cases have more than one primary tumor. Retinoblastoma is one of the tumors whose inheritance is autosomal dominant with about 90% penetrance. The hereditary tendency is governed by a locus which is mapped to the long arm of chromosome 13 [del (13) (q14)]. Hereditary tumors are bilaterally affected while non-hereditary types are generally unilateral which has led to the hypothesis that embryonic tumors arise as the results of inactivation of both tumor suppressor gene. 1~ In other words, a single normal allele is quite sufficient for the maintenance of normal cell growth while second mutation is required to initiate malignant cell proliferation. 1~ There are a number of mechanisms by which the loss of one specific allele has been documented. The loss of heterozygosity by means of restriction fragment length polymorphisms (RFLPs) has been a hallmark finding confirming the involvement of recessive inactivation of genes during cellular transformation. ~~ Wilms' tumor (WT) is another classic example of a single-minute chromosome band deletion of one of the two chromosome 11 's (p 13-p 14.1), but this abnormality in itself is not sufficient for development of the tumor. 1~ However, single-band deletion in one of the chromosome 1l's have been implicated as strong predisposition towards neoplasm. 1~ Like retinoblastoma, a second mutation is necessary for the development of Wilms' tumor; a deletion alone is regarded as a primary event, suggesting that retinoblastoma and Wilms' tumor may share a similar genetic mechanism. 1~ However, other additional loci have been implicated in the etiology of Wilms' tumor.110

Table 7. Chromosomal Abnormalities in Solid Tumors a Tumor

Chromosomes Changes

Benign Meningioma and acoustic neuroma Mixed tumors of salivary glands Lipoma Colonic adenoma Leiomyoma Cortical adenoma of Kidney Carcinoma Bladder

-22 or 22qt(3;8)(p21 ;q12) t(9; 12)(p13-22;q 13-15) t with 12q 14 12q- and/or +7 12q- and/or +8 t( 12; 14)(q14-15;q22-24) 7q+7, +8, +17,-Y i(5p) +7 -9/9qlipdel(10)(q24) del(3)(p14p23)

Prostate Lung (SCLO) Colon

+T +8/) +12 17(~11)b 17p~" _18b del (3)(pl lp21) lq b 6qb

Kidney Uterus Ovary

t(6; 14)(q21 ;q24) Trisomy lq +10

Endometrium Sarcomas

Liposarcoma (myxoid) Synovial Sarcoma Rhabdomyosarcoma (alveolar) Extraskeletal myxoid chrondrosarcoma Leiomyosarcoma Embryonal and other Testicular (germ-cell tumors) Retinoblastoma

t( 12; 16)(ql 3;pl 1) t(X;18)(p 11.2;q11.2) t(2,13)(q37;q14) t(9;22)(q31;q12.2) _22b i(12p) del( 13)(p14)c i(6p) del(l 1)(pl 3)b del( 1)(p32-p36) (continued)

Wilms' tumor Neuroblastoma 200

Table 2'. Chromosomal Abnormalities in Solid Tumors a Tumor

Chromosomes Changes

Embryonal and other Malignant melanoma

del(6)(ql lq27) b i(6p) a del( 1)(p 11p22) b t(1; 19)(ql 2;ql 3) del(3)(pl3 p23)

Mesothelioma Ewing sarcoma and peripheral neuroepithelioma Glioma

t(11 ;22)(q24;q 12) _22 b

Notes: aAfterCin and Sandberg.1~

t'Not yet proved to be primary. "Associated with a constitutionalchromosomechange.

Table 8. Deletion and Loss of Heterozygosity in Solid Tumors a Tumor Ref

RB Colorectal carcinoma WT Bladder adenocarcinoma Breast adenocarcinoma

Glioma

Leiomyosarcoma (intestine) Leiomyoma (uterus) Lipoma Lung adenocarcinoma Lung small cell carcinoma Mesothelioma Mesthelioma (pleura) Malignant fibrous histiocytoma Melanoma

Chromosomal Deletion in Tumor

Cloned 13q 14 17p 18q llp13 (Noted) 1q21-23 Monosomy-9 lpl 1-13 3pl 1-13 3ql 1-13 lp32-36 6p 15-q27 7q22-q34 8p21-23 9p24-p13 1p 12-12 6p21 7q21-31 13q 12-13 3pl 3-23 3p 13-23 3p21-25 I p I 1- 13 lql 1 lpl 1-22 6q I 1-27

201

Allele Loss

13q 5q; 17p; 18q lip 9q; 11p; 17 lp;lq;3p;l lp; 13q;16q;17p; 17q;18q 17

NT b NT NT 3p;l 3q; 17p 3p; 13q; 17p NT NT NT lp (continued)

Table 8. (continued) T.mor Ref

Chromosomal Deletion in Tumor

Allele Loss

Menigioma

Monosomy-22 22q12-13 1p32-36 3p 13-21 6q15-23 7q22 10q24 13p 13-21 I q21-23

22q 12-qter

Ne uroblastoma Ovarian adenocarcinoma Prostatic adenocarcinoma Renal cell carcinoma Uterine adenocarcinoma

1p 3p;6q; 11p; 17q 10; 16 3p 3p

Notes: aAfterSolomonet al. 13

bNT-nottested.

Table 9. Translocations in Solid Tumors a Tumor

Translocation

Breast adenocarcinoma Glioma Ewing's sarcoma Leiomyoma (uterus) Lipoma

Liposarcoma (myxoid) Melanoma

Myxoid chondrosarcoma Malignant histiocytosis Ovarian adenocarcinoma Pleomorphic adenoma

Renal cell carcinoma Rhabdomyosarcoma (alveolar) Synovial sarcoma Note: aAfterSolomonet al.~3

202

t(1)(q21-23) t(19)(q13) t(11 ;22)(q24;q 12) t( 12; 14)(ql 3-15 ;q23-24) t(3; 12)(q27-28;q 13-15) t(6)(p22-23) t(12)(q13-15) t( 12; 16)(ql 3;pl 1) t(1)(qll-ql2) t( 1;6)(q 11-12;ql 5-21 ) t(l,19)(q21 ;p13) t(6)(pl 1-ql 1) t(7)(qll) t(9;22)(q22;ql 1.2) t(2;5)(p23;q35) t(6;14)(q21 ;q24) t(3;8)(p21;q12) t(9;12)(pl 3-22;ql 3-15) t(12)(q13-15) t(3;8)(p21 ;q24) t(2;13)(q35-37;q14) t(X;l 8)(pl 1;ql 1)

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203

Neuroblastoma is a tumor found in children whose origin is neural crest cells. 111 The disease is both hereditary and nonhereditary. The deletion of the short arm of one of the chromosomes 1 band p32.2 is the most consistent abnormality and a duplication of the long ann of chromosome 1 or an isochromosome 17q which are regarded as secondary changes that arise during the evolution of malignancy. Homogeneous staining regions (HSRs) and double minutes (DMs) have been noted in the more advanced stages. Furthermore, it has been suggested that the N-myc gene is responsible for amplification of HSRs and DMs. VII.

NEOPLASMS OF CHROMOSOME BREAKAGE SYNDROMES

In recent years, tremendous strides have been made towards understanding the molecular mechanisms of chromosome breakage in individuals who are predisposed to develop neoplasia. 113There are a number of neoplasms where chromosomal instability have been noted. TM Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome are the most common disorders. Xeroderma pigmentosum, Werner's syndrome, and Kostmann's agranulocytosis can be considered as a part of chromosome breakage syndrome. Fanconi's anemia which is an autosomal recessive disorder manifests 10 to 100% of the cells with chromosome breakage. Various types of chromosomal abnormalities are noted in these individuals which are not only limited to lymphocytes but occurred in fibroblasts as well. Individuals with Fanconi's anemia are very sensitive to certain clastogenic agents. The rate of sister chromatid exchange (SCE) is high ll5 and a faulty DNA repair mechanism has been postulated. It has been suggested that Fanconi's anemia predisposed to leukemia. Bloom syndrome is another autosomal recessive disorder with an increased predisposition toward an early onset of cancer, having increased frequency of symmetrical quadriradials with an increased rate of SCE. 116Bloom syndrome, with its highly increased incidence of cancer, constitutes a cardinal example of a gene defect predisposing to neoplasm. 117 An increased SCE frequency and decreased capacity for cell division have been observed in cells exposed to mitomycin C (MMC) and other clastogens and mutagens. Ataxia telangiectasia (AT) is also an autosomal recessive disorder, ll8 A significantly high rate of spontaneous chromosome breakage is found when lymphocytes are exposed to X-ray irradiation. 119 The incidence of sister chromatid exchange is normal, but tandem translocations involving both chromosome 14 with breakpoints in band 14ql 1-12 have been demonstrated. 12~ Furthermore, translocations affecting chromosomes 7 and 14 are found much more often in AT-lymphocytes. The nature and causation of chromosomal abnormalities in AT is unknown, but genetic instability and a defective immune system may be leading factors.

204

RAM S. VERMA

Xeroderma pigmentosum (XP) is a recessively transmitted trait and individuals with the disorder show hypersensitivity to sunlight. 121 However, UV irradiated cells of patients with XP undergo an abnormally low amount of "repair replication" of damaged DNA which results in a significantly high rate of chromosome breakage. 122 The incidence of SCE is normal in cells from patients with XP, but cells are hypersensitive to SCE when irradiated by UV light. Highly unusual chromosomal changes were observed both in fibroblasts and lymphocytes in persons with Werner syndrome (WS). 123 Stable clonal translocations were the most common abnormalities. 124 Although chromosomes 2, 3, 10, and X were the most common, the involvement of individual chromosomes in translocation was random. Furthermore, increased chromosomal breakage has been noted in individuals with the WS trait. 125'126 Sarcomas are the most common neoplasm in Werner syndrome.

VIii.

CONCLUSION

For the last three decades, the chromosomal basis of human neoplasia has been a primary concern in understanding the genetic basis. The occurrence of nonrandom chromosomal abnormalities and their association with particular types of cancer have led to a prerequisite for cytogenetic evaluation prior to treatment. It was the chromosomal abnormalities which led to identify a number of genes which are causing cancer. Recent revolution in molecular techniques have opened new avenues in elucidating the molecular mechanisms of oncogenesis while the chromosomal basis of cancer is losing ground. The DNA, RNA, and protein isolated from tumors is gaining popularity in the search for the causes for transformation of normal to malignant cells. Certainly, neoplastic transformation is a complex, multistep process and a number of strategies have been used to characterize and clone the cancer-causing genes. The chromosomal and molecular basis are currently being coupled for evaluating human neoplasias and over 50 oncogenes have been identified. The loss of heterozygosity by various mechanisms have led to a better understanding of tumor suppressor genes. Consequently, remarkable progress has been made towards understanding the function of a gene in a normal cell and an oncogene in a diseased state. Gene amplification in certain tumors have contributed much towards understanding of rational approach to neoplastic transformation.

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THE MOLECULAR GENETICS OF CHROMOSOMAL TRANSLOCATIONS IN LYMPHOID MALIGNANCY

Frank G. Haluska and Giandomenico Russo

I. II.

III.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Features of the Molecular Genetics of Lymphoid neoplasms . . . . . A. Genes Involoved in Translocations . . . . . . . . . . . . . . . . . . . . . B. Mechanisms of Deregulation . . . . . . . . . . . . . . . . . . . . . . . . C. Mechanisms of Chromosome Translocation . . . . . . . . . . . . . . . . Chromosome Rearrangements in B-Cell Malignancies . . . . . . . . . . . . . A Translocations Involving m y c . . . . . . . . . . . . . . . . . . . . . . . . B. The t(8; 12) Translocation: btg I and myc . . . . . . . . . . . . . . . . . . C. The t(11;14) Translocation: b c l l , p r a d l , and Cyclins . . . . . . . . . . . D. bcl2: The t(14;l 8) Translocation . . . . . . . . . . . . . . . . . . . . . . E. The t(l 4;19) Translocation and bcl3 . . . . . . . . . . . . . . . . . . . . F. bci6 and Diffuse Large Cell Lymphoma . . . . . . . . . . . . . ..... G. Fusion Transcripts and Proteins: The Philadelphia Chromosome . . . . . . H. The t(1 ;19) Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Genome Biology Volume 3, pages 211-231. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

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IV. Common Characteristics of T-Cell Leukemia . . . . . . . . . . . . . . . . . . A. The t(8;14)(q24;qll) Translocation and the Myc Locus . . . . . . . . . . B. The lp32 Locus: tall/scl/tcl5 . . . . . . . . . . . . . . . . . . . . . . . . C. The lyll Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Rhombotin 1 and 2 Loci . . . . . . . . . . . . . . . . . . . . . . . . . . E. The Chromosome 10 h o x - l l / t c l 3 Gene . . . . . . . . . . . . . . . . . . . F. The t a n l Translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . G. The tcll Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. I N T R O D U C T I O N No approach to the understanding of the molecular basis of acute and chronic leukemias has been more productive than that of molecular cytogenetics. This approachmthe utilization of the techniques of molecular cloning to isolate and characterize regions of various chromosomes involved in chromosome translocations, inversions, and deletions--has been successfully employed to implicate known oncogenes in such rearrangements, to identify new oncogenes, and to demonstrate the involvement of genes previously not known to contribute to malignancy. Its utility has been proven for hematologic neoplasms of myeloid and lymphoid lineages, and more recently for solid tumors as well. Thus the scope of molecular cytogenetics has come to encompass much of molecular oncology. This chapter will concentrate on the molecular biology of the lymphoid malignancies as elucidated by molecular cytogenetic analyses. The lymphoid malignancies include the lymphomas and the acute and chronic lymphocytic leukemias. The non-Hodgkin's lymphomas (NHL) alone account for over 30,000 new malignancies per year in the United States. Taken together with adult and childhood acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), lymphoid neoplasms constitute nearly 40,000 newly diagnosed neoplasms yearly. Yet aside from their epidemiologic significance, the lymphoid malignancies are also important in that they have served to provide the molecular foundation of cancer cytogenetics. With the notable exception of chronic myeloid leukemia, the elucidation of the molecular genetics of lymphoid malignancies has preceded that of most myeloid neoplasms and solid tumors. This is largely a consequence of the chromosomal juxtaposition of the immunoglobulin (Ig) and T-cell receptor (TCR) loci to various loci, and of the early widespread availability of probes for these genes. Thus the detailed characterization of the Ig and TCR genes enabled rapid cloning ofbreakpoints from translocations involving these loci before other regions of the genome were mapped densely enough for cloning experiments. In addition, hematologic tumors have generally been easier to analyze cytogenetically than

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solid tumors, the latter usually carrying more complex karyotypes. As a result, many of the methods and principles now more broadly applied in molecular cytogenetic analyses were first worked out for lymphoid tumors. In this review we will first discuss general features of the molecular cytogenetics of B- and T-cell neoplasms. We will then discuss individual translocations and the insights derived from their analysis.

Ii. GENERAL FEATURES OF THE MOLECULAR GENETICS OF LYMPHOID NEOPLASMS It has long been understood that virtually all malignant tumors carry abnormal karyotypes. 1 The identification of karyotypic abnormalities in hematologic neoplasms, 2-4 as well as in solid tumors, and the realization that hematologic tumors were consistently characterized by nonrandom chromosomal translocations, was a crucial insight. 5 These observations led to the supposition that the junctions between translocated chromosomes carried genes central to the pathogenesis of the tumors carrying the aberration, and provided the rationale for the molecular analyses of the chromosome breakpoints. Several principles have emerged from these analyses. First, the chromosome translocations involve genes generally involved in cellular growth control. Second, the translocation disrupts the normal control of expression or function of these genes. This may occur in several ways. The gene may be juxtaposed to control elements that result in its being expressed at inappropriate times or abnormal levels. The translocation may alter the gene's own control elements so that they no longer function correctly. Or translocation may result in a fusion protein having a functional capacity not found in either of the original fusion partners. Third, translocations may result from the aberrant operation of cellular devices which cause physiologic recombination. In B cells and T cells, the variable (V), diversity (D), and joining (J) segment (V-D-J) recombinase system has been so implicated.

A. Genes Involved in Translocations In many instances the genes located at translocation breakpoints code for proteins which play a role in the cellular signal-transduction cascade. 6 Nuclear, cytosolic, membrane, receptor, and extracellular portions of the cascade have each been shown to be dysregulated in various translocations. Tables 1 and 2 list the translocations in lymphoid malignancy that have been characterized on a molecular level, and thegenes involved in the translocations. The genes discussed briefly here will be addressed in more detail in the sections detailing the particular translocations involving them.

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FRANK G. HALUSKA and GIANDOMENICO RUSSO Table 1. Chromosomal Translocations Observed in B-Cell Malignancies and Loci Involved

Translocation t(8;14)(q24;q32) t(2;8)(pl 1;q24) t(8;22)(q24;ql 1)

Affected Loci lgH I gK I gL

MYC

Gene Class bHLH

Disease Burkitt's Lymphoma, undifferentiated lymphoma

t(ll;14)(q13;q32) IgH

BCLI/PRAD1 cyclinD1

B-CLL, centrocyticNHL

t(14;18)(q32;q21) IgH

BCL2

abate apoptosis

follicular NHL

t(14;19)(q32;q13) IgH

BCL3

CDCI0

B-CLL

t(3;14)(q27;q32)

lgH

BCL6

zinc finger

diffuse large cell NHL

t(5; 14)(q31;q32)

IgH

IL-3

growth factor

ALL

t(8;12)(q24;q22) MYC BTG1

bHLH, early response

B-CLL

t(9;22)(q34;ql 1)

tyrosine kinase

CML, ALL

enhancer binding, homeobox

pre-B-All

ABL BCR

t(l ;19)(q23;p13) E2A

PBXI

Nuclear transcription factors of several different types have been localized to translocation breakpoints. 7'8 Transcription factors including: the helix-loop-helix (HLH) class, such as myc, E2A, lyll, and tcl5; the zinc finger class, such as bcl6; the homeobox domain class, such as pbx and hoxll/tcl3; and NF-KB have all been shown to be involved by translocations. Genes encoding proteins that, while not directly binding to DNA, interact with DNA binding factors also play roles in translocations. Two recently identified genes, Rhomboitin 1 and 2 (RBTNI, RBTN2) contain LIM domains. These are cysteine-rich motifs thought to mediate protein dimerization, and although these proteins lack DNA binding domains, they may dimerize with DNA-binding transcription factors, thus effecting regulation. Membrane and cytosolic elements of the signal cascade can also be perturbed by translocation. The Philadelphia chromosome fusion protein joins the bcr gene (BCR), a ras GAP, to the ABL tyrosine kinase. In addition, growth factors themselves have been implicated, as for example IL-3. Cell cycle control elements have also been shown to be involved in chromosome translocations. The bcll/pradl gene encodes cyclin D 1. The bcl3 gene carries the CD 10 motif, which is found elsewhere in yeast cell cycle control genes, and the human homolog of the Drosophila gene notch, involved in development and differentiation, is tan1. Finally, the control of programmed cell death is disrupted by translocation at the bcl2 locus. The Bcl2 protein blocks apoptosis, and its overexpression by translocation appears to prevent normal programmed cell death.

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Table 2. Chromosomal Translocations Observed in T-Cell

Malignancies and Loci Involved

Translocation

Affected Loci

Gene Class

Disease

bHLH

T-ALL

unknown

ATL, T-prolymphocytic leukemia, ataxia te langiectasia

t(8;14)(q24;q32)

TCR~8

MYC

t(7;14)(q35;q32) t(14;14)(ql 1;q32) inv( 14; 14)(q 11;q32)

TCR~

TCLI TCRct/8

t(7;19)(q35;p13)

TCRI3

LYL1

bHLH

T-ALL

t(1;14)(p32;ql 1)

TCRct/8 TALl/SCL/TCL5

bHLH

T-ALL

t(7;9)(q35;q34)

TCR~

TAL2

bHLH

T-ALL

t(ll;14)(pl5;qll)

TCR~I

RBTN1/TTGI

LIM domain

T-ALL

t(ll;14)(pl3;qll) t(7;1 l)(q35;p 11)

TCR~

RBTN2/TTG2

LIM domain

T-ALL

t(10;14)(q24;ql 1) t(7;10)(q35;q24)

TCR~[3 HOX11/TCL3 TCRI3

homeobox

T-ALL

t(7;9)(q34;q34.3)

TCR~

Notch homolog

T-ALL

TAN1

B. Mechanisms of Deregulation Activation of oncogenes by chromosome aberrations occurs by alteration of expression by the juxtaposition of inappropriate control elements, by alteration of the target oncogene, or by gene fusion. 9 In lymphoid tumors by far the most common mechanism of oncogene activation is the juxtaposition ofimmunoglobulin or TCR loci to the oncogene. The expression of the immunoglobulin and TCR genes is strictly controlled in an ontogenetic and tissue-specific manner. A portion of this control is exerted through the Ig and TCR enhancers, l~ The hallmark of the action of enhancers is twofold; they have the capacity to stimulate transcription independent of their position and orientation relative to the activated element, and at variable distances from it. Thus they are ideal candidate-deactivating elements for chromosome rearrangements whose architecture relative to the translocated oncogene may vary. For translocations involving myc, it is clear that expression of the oncogene is deregulated by the juxtaposition of Ig and TCR loci. The myc gene is expressed constitutively in a manner similar to otherwise normal, proliferating cells. 12'13 Initially, in the best studied cases of Burkitt's lymphoma, it was shown that only some translocations joined the upstream heavy-chain enhancer (E~) to myc. 14,15 However, it seems likely that additional transcriptional activation elements have yet to be described. For example, widespread transcription of VH segments is

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observed early in B-cell development; 16this phenomenon may be mediated through undiscovered long-range acting elements. Thus for Ig and TCR translocations not involving defined enhancers, one might speculate that as yet undefined cis-acting elements may be operative. However, alteration of onc0gene control elements has also been implicated in deregulation. Changes in the 5' region of myc have been shown to affect mRNA stability, 17 and to release a block to transcriptional elongation. 18 Thus alteration of such sequences, or truncation of putative silencer (negative control) elements, may contribute to inappropriate onc0gene expression when the gene is translocated into the Ig or TCR loci. Finally, fusion proteins may result from chromosome rearrangement. Although this mechanism of oncoprotein generation is more prominent in myeloid and solid tumor chromosome rearrangements, it occurs in lymphoid tumors as well. The hallmark translocation of chronic myelogenous leukemia (CML), the Philadelphia chromosome (Ph'), occurs in a subset of approximately 20% of cases of ALL as well. This translocation fuses abl and bcr to generate a fusion transcript. Several other lymphoid translocations similarly result in the production of fusion proteins, including the pre-B ALL t(l" 19), and lymphoid variants oftranslocations involving chromosome 1lq23. These will be discussed in more detail below.

C. Mechanisms of Chromosome Translocation The generation of antibody and TCR diversity occurs by a process of somatic recombination that takes place as the respective lymphocyte matures. The Ig and TCR loci are organized so that regions of the proteins that take part in antigen recognition are comprised of gene segments that join combinatorially. The V-D-J segments of the Ig and TCR genes thus are physiologically recombined by the recombinase system during lymphocyte ontogeny. The recombinase enzymes Rag- 1 and Rag-2 have been identified and cloned; these enzymes recognize specific sequences in the flanking regions of V-D-J segments, and catalyze their recombination during lymphocyte maturation. 19 In addition, the Ig loci undergo isotype switching following lymphocyte exposure to antigen. This process too occurs as a consequence of enzymatic recognition of specific switching sequences upstream of heavy-chain constant segments in the IgH locus. It is now clear that mistakes in this process may give rise to chromosome translocation. Translocations and inversions involving the Ig and TCR loci have been abundantly demonstrated to occur at regions of physiologic rearrangement. The initial translocations from Burkitt's lymphoma cells involving IgH and myc were found to occur at sites of IgH isotype switching. 15'2~Subsequently, translocations into Ig JH segments were demonstrated, and sequence analyses demonstrated that the sequences from the translocation breakpoints shared homology with recombinase recognition sequences. 21'22This was subsequently demonstrated to be

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true in TCR translocations as well. 23 It has been pointed out that the features suggesting recombinase involvement are variable at chromosome rearrangement breakpoints, in that sequence homologies are only variably preserved. 24 However, the placement of sites of chromosome breakage near Ig and TCR segments that undergo recombination in the normal cell is widespread. Although the precise molecular mechanisms underlying these observations remain to be elucidated, the validity of the process is generally accepted. For some rearrangements the mechanism of recombination is less clear. For example, translocations involving 11q23 do not involve Ig or TCR loci. The association of these malignancies with prior epidophyllotoxin (topoisomerase inhibitor) therapy suggests that topoisomerase may play a role. This conjecture has yet to be examined. For other rearrangements in lymphoid tumors, and for many other myeloid and solid tumor translocations, inversions, and deletions, mechanisms of chromosome rearrangement are lacking.

i!i. C H R O M O S O M E REARRANGEMENTS IN B-CELL MALIGNANCIES The catalog of structural chromosome aberrations observed in B-cell neoplasms is extensive. Although many of these lesions remain uncharacterized, the most prevalent rearrangements, almost all of them translocations, have been cloned and analyzed. A brief compendium of these is given in Table 1. We will discuss the most important cases.

A. TranslocationsInvolving myc Initially the best characterized of the lymphoid chromosome abnormalities were the translocations involving myc. As early as 1972, it was demonstrated that Burkitt's lymphomas carried, in up to 80% of cases, a marker chromosome derivative of chromosome 14. Later analysis revealed this 14q+ chromosome to be the product of the t(8; 14)(q24;q32) translocation. Cases of Burkitt's lymphoma not carrying t(8; 14) translocations were ultimately shown to carry t(8;22)(q24;ql 1) or t(2;8)(pl I ;q24) variant translocations. These cytogentic observations were placed in a molecular context when it was demonstrated that the common site of involvement of the three rearrangements, band 8q24, also was the site to which the myc protooncogene mapped. 25'26 Overexpression of myc from translocated chromosomes was immediately demonstrated, 12 and somatic cell hybrid studies demonstrated that inappropriate myc expression was a consequence of its becoming subject to expression in a lymphoid background. The IgH enhancer was also implicated. The demonstration of rearrangement of the myc gene by Southern analysis, and the concomitant mapping of the Ig heavy, ~, and ~cgenes to the regions of chromosome 8, 22, and 2 involved in the translocations eventually facilitated

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cloning of these breakpoints. These experiments also served as the foundation for later studies which also implicated myc in translocations into the TCR loci (see below). Examination of the t(8;14) and variant translocations revealed that the relative orientation of the activating Ig locus and myc was variable; 9 myc was upstream of IgH constant segments, but downstream of ~: and X constant regions. Additional analysis of the t(8;14) translocations revealed two subtypes of Burkitt's translocation. Most sporadic tumors, those occurring throughout the world, and with an approximately 30% association with EBV, carry translocations which disrupt the 5' portion of myc, and which usually involve switching regions on the chromosome 14 side of the breakpoints. In contrast, endemic, African tumors, essentially uniformly occurring in EBV+ patients, demonstrate breakage far upstream from a structurally intact myc. These translocations take place at V-D-J segments. These data suggest that the translocations occur at different times in lymphocyte ontogeny in these related, but probably different, malignancies. 22'27 The precise role of myc in inducing malignancy as a consequence of its juxtaposition to Ig loci is still under active investigation, myc is a transcription factor having DNA-binding, HLH, and leucine zipper motifs. It normally dimerizes with Max, but Myc homodimers may abnormally bind DNA as a consequence of amino-terminal truncation. The results of its deregulation of alteration in the Burkitt's lymphomas, as well as in other B- and T-cell tranlocations in which it is involved, as still being elucidated.

B. The t(8;12) Translocation: btgl and myc myc has also been recently shown to be involved in a translocation specific to CLL. The t(8; 12) (q24;q22) translocation is occasionally seen in CLL. Molecular analysis of a case of a secondary t(8;12) occurring with a t(ll;14) demonstrated a myc rearrangement in the 3' portion of the gene. 28 Juxtaposed to the 3' region of myc is a gene named btgl. This gene apparently is a negative regulator of cell proliferation, and is homologous to an early growth factor response gene pc3. 29 At this time it is not clear if dysregulation of myc, btgl, or both is central to the pathogenesis of this tumor.

C. The t(11;14) Translocation: ball, pradl, and Cyclins The t(ll;14)(q13;q32) translocation occurring in CLL was one of the initial chromosome translocations to be cloned through the analysis of rearranged Ig genes, and analysis of sequences near the breakpoints of the translocated chromosomes led to the proposed implication of the V-D-J recombinase enzymes. 30 However, despite clustering of translocation breakpoints on chromosome 11, no transcription unit was initially revealed at this locus. The locus was putatively labeled bcll. Subsequent studies revealed that this translocation, although seen in

Chromosomal Translocations in Lymphoid Malignancy

219

CLL at only low frequencies, is characteristic of an NHL subtype known as centrocytic or intermediate lymphocytic lymphoma. 31'32 The bcll gene was actually identified through the analysis of a translocation found in a parathyroid adenoma. 33 Subsequently this gene, pradl, was implicated in the centrocytic lymphoma translocations as well and proposed as a candidate for bcll. 34"35 Pradl has since been demonstrated to be cyclin D1. 36 This gene is normally not expressed in lymphoid cells, but appears to be inappropriately turned on in centrocytic lymphomas carrying the translocation. 34'35'37Disruption of normal expression of this gene likely leads to abnormal cell cycle progression and malignancy. It is instructive to note that parathyroid adenoma, CLL, and centrocytic lymphoma are relatively indolent neoplasms. It is probable that the full attainment of the malignant phenotype requires additional genetic changes aside from loss of tightly controlled cyclin D 1 expression.

D. bcl2: The t(14;18) Translocation The translocation that led to the identification of the bcl2 gene is the t(14;18)(q32;q21). The most common B-cell malignancy is nodular or follicular lymphoma, a slowly progressive subtype of NHL. Over 85% of these tumors carry the t(14; 18). 38 These translocations may occur in isolation in follicular lymphomas, but they may also precede the development of translocations such as the t(8;14) which produce a more malignant, acute phenotype. The bcl2 gene was first isolated by cloning IgH rearrangements. 39--41The translocations occur upstream oflgH J segments, and appear to occur by an aberrant V-D-J recombinase activity. 21 Like other translocations taking place by this mechanism, they thus likely occur early in B-cell differentiation, at a point at which the immunoglobulin genes functionally rearrange. Translocations group into major and minor breakpoint clusters, and lead to overexpression of the bcl2 gene. 42'43 The tight clustering of t(14;18) breakpoints has led to the development of sensitive PCR-based assays to detect the translocations. These have been utilized to assay for the presence of minimal residual disease after therapy. They have also been used to examine normal individuals, and thus it is noteworthy that t( 14; 18) translocations have been detected in normal tonsils. 44 Moreover, cases have been described carrying different bcl2 translocations. 45 Thus the possibility that bcl2 only indirectly contributes to malignancy must be assessed. The bcl2 gene encodes a protein that localizes to the inner mitochondrial membrane. 46 Overexpression of the protein abrogates apoptosis, prolonging cell lifetimes. 46 '47 The gene and its expression have been studied in several systems; transgenic murine strains have been constructed; 48 the function of the bcl2 homologue in Caenorhabditis and its role in the determination of cell fate has been examined. In short, the identification of the role of bcl2 in malignancy has led to the rapid expansion in the investigation of the biology of apoptosis that is largely outside the scope of this review. However, recently one aspect of the biology of

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bcl2 which may have special bearing on lymphoid malignancy has begun to emerge. This is the relationship between bcl2 and the Epstein-Barr virus (EBV). EBV infection is widely associated with lymphoid malignancy in Burkitt's lymphoma, in NHL in immunosuppressed patients, and in Hodgkin's disease. The EBV latent membrane protein (LMP) is a virally-encoded 60-kDa transmembrane protein. 49 LMP may play a part in the control of bcl2 expression. Transfection of this protein into human B cells protects them from apoptosis by augmentation of bcl2 expression. 5~ This suggests a parallel in the induction of bcl2 expression by EBV infection and its dysregulation by translocation. Both t(14; 18)and EBV infection have been abundantly demonstrated to precede the development of discrete second changes, including t(8;14) translocations activating MYC. It is possible that an initial genetic change, consisting of inappropriate bcl2 expression, EBV infection, or an as yet uncharacterized alteration, prolongs lymphocyte lifetimes and allows for the accrual of secondary changes leading to a more aggressive phenotype. In summary, it is likely that the most relevant aspect of bcl2 function to lymphomagenesis is that, by delaying or preventing apoptosis, its inappropriate expression may enlarge populations of cells susceptible to progressive malignant changes. This may occur by diverse pathways.

E. The t(14;19) Translocation and bcl3 The bcl3 gene is found next to the t(14; 19)(q32;ql 3.1) translocation found in a subset of CLL patients. The breakpoint was cloned from Ig switch rearrangements. 52'53 The transcript adjacent to the breakpoint encodes a protein that carries seven tandem repeats of the cdc 10 motif; this motif is found in proteins functioning in cell cycle control and lineage determination, including the Drosophila Notch gene. (A notch h0molog, tan1, is involved in TCR translocations, and is discussed below.) The bcl3 gene is overexpressed in leukemic cells carrying the translocation; 53 further details regarding its role in transformation remain obscure.

F. bcl6 and Diffuse Large Cell Lymphoma Recently translocations specific to diffuse large cell NHL have been identified. The t(3;14) (q27;q32) has been cloned. 54'55 Band 3q27 carries a gene, bcl6, that encodes a 79-kDa zinc-finger transcription factor. 56 Truncation of the 5' noncoding regulatory sequences is found in approximately 33% of large cell lymphoma cases. 56 This gene may come to play a central role in the pathogenesis of this common B-cell malignancy.

G. Fusion Transcripts and Proteins: The Philadelphia Chromosome All of the previously discussed translocations result in the activation of oncogenes by the juxtaposition of ectopic activating regions, usually from the Ig locus,

Chromosomal Translocations in Lymphoid Malignancy

221

with genes that function in cellular growth and differentiation control. However, the second mechanism of activation is the production of a fusion transcript, and a fusion protein as a result, that carries transforming function. The prototypical fusion translocation is that of the Philadelphia (Ph') chromosome, t(9;22). This is the specific chromosome abnormality that characterizes the stem cell defect of CML. The translocation joins the abl oncogene on chromosome 9 to the bcr gene on 22. In CML, an 8.5-kb fusion mRNA results from the translocation. This transcript carries bcr sequences 5' of the abl second exon, which normally accepts either the abl exon Ia or lb. The resultant transcript encodes a 210-kDa protein tyrosine kinase. The detailed analysis of the function of this fusion protein in CML is outside of the scope of this discussion. However, it is important to note that the Ph' chromosome is an important finding in adult ALL as well as in CML. In fact, about 20% of adultALL case carry the translocation; it is an adverse prognostic feature of the disease. The translocations of the t(9;22) in ALL have been cloned and analyzed. It has been found that these translocations differ from the CML type. 57 In ALL, the breakpoint in the abl gene is the same; that is, it lies upstream of the exon II. But BCR breakpoints fall immediately after the first large intron. As a consequence, the ALL fusion transcript is 7 kb; the resulting protein is 190 kDa in size. 58 This protein is a more active kinase than the p210. 59 Note that the situation described for the Ph' is different from that described for the myc translocations. Translocations involving myc confer a tissue type specificity as a result of the specificity of the activating loci, i.e. Ig or TCR. The actual activated oncogene may be the same in different cases. However, in CML, subtle variations in the structure of the fusion protein confer different histopathologies.

H. The t(1;19) Fusion About 25% of childhood pre-B-ALL carries the t(1; 19). Cloning of this breakpoint has shown that the translocation shares some features with the Ph' translocation. 6~ The translocation generates a fusion transcript between the E2A gene on chromosome 19, and a homeobox gene pbxl on chromosome 1. 61'62 Breakpoints cluster within the intron separating E2A exons 13 and 14, and within a single large intron in pbx; 63 as in the Ph' chromosome, different translocation breakpoints generate a common fusion transcript. The E2A gene encodes the transcription factors El2 and E47. These factors are members of the H-L-H class of proteins. They bind to the E-box of the ~clight chain enhancer. The chimeric protein carries the amino portion of E2A, on which the activation domain resides, and the carboxyl DNA-binding and homeobox domains of pbxl. The proteins produced vary slightly: a p77 and p85 are observed. Either protein can transform NIH 3T3 cells. 64 The mechanisms through which this occurs are being investigated.

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FRANK G. HALUSKA and GIANDOMENICO RUSSO

COMMON

CHARACTERISTICS OF T-CELL LEUKEMIA

Approximately 50% of T-cell leukemias carry chromosomal translocations at the cytogenetic level. Most of these molecular lesions have now been analyzed at the molecular level as well (Table 2). As described earlier, common patterns of pathophysiology have emerged from their study. Most of the cloned translocations have been shown to join TCR loci to target genes, resulting in the activation of the latter. The target genes are participants in the cellular pathways of signal transduction and division and proliferation control and, in fact, often encode transcription factors. These genes are often developmentally regulated, and encode proteins with a potential to dimerize with other factors, thus maximizing their capacity for interaction with other control elements. The mechanism of translocation also is conserved between T cells and B cells. TCR translocations apparently originate as a consequence of errors in the V-D-J joining process that occurs in T-cell maturation just as it does in B cells, utilizing the same enzymes. And as has been shown for many B-cell translocations, chromosome breakpoints usually involve 5' ends of the D or J segments of the TCR loci. Sequence analyses of rearranged and normal partner chromosomes involved in translocations or inversions has demonstrated recombinase signal sequences to be present. These findings strongly suggest a common origin of B- and T-cell translocations in V-D-J joining mistakes that occur during early stages of T-cell differentiation. In the following sections we will discuss some of the most frequent translocations observed in T-cell malignancies and the possible roles of the genes involved in the translocations.

A. The t(8;14)(q24;q11) Translocation and the myc Locus The most frequent abnormalities observed in T-lymphocyte neoplasia involve chromosome 14qll, the site of the TCR ct/8 locus. The t(8;14)(q24)(ql 1) juxtaposes the TCR 3' of the myc gene. 23'65 This is analogous to those rearrangements found in the Burkitt's lymphoma variant t(2;8) and t(8,22) translocations. In the latter cases, the Ig genes translocate 3' of myc as well. This reflects the common orientation on their respective chromosomes of the TCR ~/8 and Ig light-chain genes: centromere-V-J-C-telomere. In contrast, the IgH locus is oriented centromere C-J-V-telomere, and so that when myc translocates into IgH, the immunoglobulin genes lie 5' of myc. It should be noted that, lacking alterations in myc sequences, the position-independence of the translocations suggests the operation of enhancer or enhancer-like elements operating in cis to deregulate myc expression. As in B cells, translocation deregulates myc expression. The Myc protein interacts with the Max basic H-L-H protein, 66'67 and translocation may disrupt the normal equilibrium that obtains and disturbs the normal binding of Myc with Max, altering transcriptional control. It must be emphasized that the cell-type of the resulting

Chromosomal Translocations in Lymphoid Malignancy

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malignancy does not follow as a simple consequence of myc's deregulation itself. Rather, the occurrence of the translocation by a differentiation-dependent mechanism (V-D-J joining) appears to lead to abnormal myc expression in the background of the B or T lymphocyte carrying the translocation. Thus the phenotype of the particular malignant cell is a direct result of the mechanism and timing of the chromosome translocation.

B. The lp32 Locus: tall/scl/tcl5 Rearrangements of chromosome lp32 occur in T-cell leukemias, but also in malignant melanoma and in neuroblastoma. 68'69 Several t (1;14) breakpoints have been cloned and analyzed from the human leukemia stem cell In DU528 and from patient T-ALL samples. 7~ All of these translocations cluster within approximately 10-15 kb on chromosome 1 and involve D~ or J~5segments on chromosome 14. Translocation alters the 5' noncoding region of a gene named tall (for T-cell acute leukemia- 1), SCL (for stem cell leukemia), of tcl5 (for T-cell leukemia/lymphoma-5). tall encodes a 42-kDa phosphoprotein of the bHLH transcription factor class. The protein's HLH domain is highly homologous to the lyl-1 and tal-2 genes, and localizes to the nucleus. 73-75 Analysis of the tall product has revealed that its HLH domain mediates specific interaction with two other ubiquitously expressed HLH proteins: E47 and El2. 76 The resultant heterodimers specifically recognize the E-box DNA motif found in the eukaryotic transcriptional enhancer. For this reason Hsu 76 and colleagues have suggested that the tall protein can behave as a transcriptional regulatory factor influencing cell-type determination during hemaotpoietic development in a manner similar to that of MyoD, which forms heterodimers with ubiquitous proteins to control myogenesis. More recently it has been shown that the tall gene plays a pivotal role in erythroid differentiation. Its promoter binds the erythroid transcription factor GATA-1 and its overexpression in erythroid cell lines correlates with increased erythroid differentiation in the absence of added inducer. On the basis of these findings Aplan et al. 77 have also suggested that while tall in the erythroid background can promote differentiation, inappropriate expression of tall in the T-lymphocyte background can lead to interactions with factors that are normally not coexpressed with tall. These interactions may contribute to leukemogenesis. Interestingly, deregulation of the 5' control elements of tall may also take place because of deletion. 78 In from 12 to 26% of T-ALL, approximately 90 kb of tall upstream DNA is deleted. This is designated tal d. The 5' portion of the gene is replaced by another gene named the SCL interrupting locus (sil). The apposition of the sil elements 5' of tall apparently deregulate the latter-like translocation.

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C. The lyll tocus The T-cell leukemia cell line SupT7 carries a t(7; 19) translocation. The cloning of this translocation by isolation of the rearranged TCRI3 gene led to the identification of lyll. 79 The translocation breaks the lyll gene in its first intron, truncating exon 1, and placing TCR~ and lyll in opposite transcriptional orientations. The lyll gene is expressed in most murine myeloid, erythroid and B-cell lines, and only at low levels in T-cell lines. 8~ In SupT7, only the translocated allele is expressed. Lyll codes for a bHLH protein with strong homology to the tall gene. Its precise role in differentiation and proliferation control is under active investigation.

D. Rhombotin 1 and 2 Loci The rhombotin (rbtn) genes map to chromosomes 1 lp15 and 1lp13 respectively; they have been characterized through their involvement in translocations with TCRt~/8 at 14q 11. rbtnl (also named Ttg-1) was cloned from the T-ALL cell line RPMI 8402. 82 Analysis of the translocation breakpoint demonstrates that the 5' noncoding region of rbtnl is placed 5' of a D8 segment by a V-D-J joining mechanism 83 and that an upstream promoter is truncated 84 resulting in overexpression of the gene. rbm2 was originally cloned from a pediatric T-ALL case carrying the t(11; 14)(p 13;q 11).85 Approximately 50% ofbreakpoints involving this gene cluster within a 2-kb region, and the remainder a scattered over 25 kb. The gene is also overexpressed consequent to translocation. The two rbtn genes both carry duplicated cysteine rich motifs termed LIM domains 86 found in transcription factors, rbtnl lacks a DNA-binding domain, but yet by virtue of the LIM domain, it may still function in transcriptional control. The LIM domain itself may play two roles in transcription regulation: it may act through modification of redox states of homeobox or LIM domain factors, as it is homologous to ferredoxins; or, by competitive dimerization, it may modulate the activity of other transcription factors. The RBTN 1 protein is an 18-kDa protein that is highly expressed in neural tissue. The gene is also expressed in mouse thymus and several other tissues. 84 RBNT2 is 48% homologous to RBNT1; it too is expressed in the nervous system, as well as in spleen, liver, and kidney. Two groups 87'88 have constructed transgenic mouse strains. These mice develop T-cell tumors when they carry rbml and rbtn2 transgenes that are expressed in thymus-derived T cells. For rbtnl, tumor incidence is proportional to the level of transgene expression. In this case, tumorigenesis preferentially affects a minority population of thymocytes representing immature CD4- CD8+ stage cells. 88 These data support the hypothesis that these genes lead to T-lymphocyte malignancy through their aberrant expression in early T cells.

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E. The Chromosome 10 hox-11/tc13 Gene In about 7% of T-cell ALL cases, malignant cells carry the t(10; 14) (q24;ql 1) translocation. 89 Variant translocations consist of t(7; 10)(q35;q24). Of note, 10q24 translocations also occur in pre-B-ALL. 9~ The characterized breaks involve either D8 or J~ segments at 14q I 1 or Jl~ at 7q35. An approximately 15-kb region of chromosome 10 is involved; 9~ on this chromosome, translocations with chromosome 7 cluster 12 to 13 kb centromeric to the chromosome 14 breakpoints. Probes telomeric to the breakpoint site have been able to detect a transcript of 2.1 kb, the hox-ll gene. hox-11 is expressed in normal liver cells, but not in normal hemaotpoietic cells. It is overexpressed in T-cell lines carrying the t(7;10) and t(10;14). 91'92 Characterization of hox-ll revealed its similarity to the homeobox class of genes, with a homeodomain similar to that of the murine hlx gene, having a markedly glycine-rich variable region and acidic carboxyl terminus. 92 It also shares homology with the Antennapedia gene of Drosophila; its role in leukemogenesis may again be to play an as yet undefined part in inappropriate transcriptional activation.

F. The tan1 Translocation Chromosome translocation involving the tan 1 (translocation-associated notch- 1) gene was first examined in the cell line SupT1. 93 More recently, molecular data have been gathered from three additional T-ALL patients. 94 The rearrangements involve TCR J[~ sequences and chromosome 9q34.3 within 100 bp of an intron of the locus named tanl. Transcripts originating near the breakpoint have been identified in many normal fetal and adult tissues. The tan1 transcript is highly homologous to the Drosophila gene notch, a neurogenic gene required for the correct segregation of epidermal from neuronal cell precursors during embryogenesis. The N terminal half of Tanl contains a domain of 36 tandemly repeated units, epidermal growth factor (EGF) cysteine repeats, found in EGF and in other cell-surface and secreted proteins. Translocations interrupt the tanl gene, breaking it roughly in half. The portion encoding most of the extracellular domain is separated to the der(7), and the remainder left on the der(9). The role of the tan1 gene in the pathogenesis ofT-cell leukemia remains to be fully explained. Ellisen et al. 94 have proposed, on the basis of structural data and homology, several mechanisms. The removal of the extracellular domain of the tanl gene could play a role in altered cell adhesion. Alternatively, the gene may be involved in signal transduction. A portion of it is homologous to CDC10, a yeast cell cycle gene that is involved in cytoplasmic protein-protein interactions and nuclear protein-protein and protein-DNA interactions.

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Chromosome 14q32.1 is also frequently involved in chromosome rearrangements in T-cell leukemias (TCL). These rearrangements take the form of inv(14)(q 11 ;q32) inversions, t(l 4; 14)(q 11;q32) translocations, or t(7; 14)(q35;q32) translocations. They are observed in 80% of T-prolymphocytic leukemias, and in the majority of chronic and acute T-cell leukemias arising in patients with the immunodeficiency syndrome ataxia-telangiectasia (AT), in which lymphoproliferative disease represents a major complication of the condition. Recently similar chromosomal alterations have been reported in 28% of adult T-cell leukemias (ATL) arising in the setting of HTLV- 1 infection. 67 Molecular analyses of these breakpoints have shown the involvement of J~ (on 14qll )or J~ (7q35)elements and segments of chromosome 14q32.1. This region, designated tcll, 95 is approximately 10 megabases from the IgH gene. Several breakpoints have been cloned. 96 However, until recently, except for two closely spaced inversion breakpoints, breakpoints generally have not been physically linked. Furthermore, no transcription unit has been found in the region surrounding the sites of rearrangement. Some of the rearrangements are complex, involving inversion and duplication. 97'98 Virgilio et al. 96 have reported physical mapping of a 300-kb region surrounding tcll, which encompasses most characterized rearrangements. A CpG island centromeric in the region, and two transcripts, have been found. Added in proof'. Recently, the tcll gene has been identified; it encodes a 1.3 kb transcript, and a 14-kDa lymphoid-specific protein without known homology. 99 The relationship of these findings to the pathogenesis of T-cell leukemia remains to be examined.

V.

CONCLUSION

We have provided a brief overview of the molecular basis of the specific structural chromosome aberrations that characterize lymphoid malignancies. The molecular cytogenetics of these tumors are relatively advanced, for a number of reasons. For a long period of time, hematologic cells were more readily available than cells from solid tumors, and their cytogenetic examination was more easily performed. These technical factors, coupled with the proclivity for the physiologically rearranging Ig and TCR loci to translocate, and the early availability of molecular probes from these loci, have subjected the lymphoid translocations to intense scrutiny. The field has been abundantly productive. Principles of molecular oncogenesis have been established for the hematologic malignancies that are now being more widely applied to solid tumorigenesis. And the genes identified at translocation junctions are already being shown to have important functions in the normal control of proliferation and differentiation.

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In medical terms, the molecular tools that have been generated are also proving to have significant diagnostic and prognostic applications. Perhaps the most important by-product of this research is yet to come, as a fuller understanding of the molecular steps to oncogenesis eventually lead to rational approaches to therapy as well.

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.1 A l P R E S S

Advances in Genome Biology --

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Edited by Ram S. Verma, Division of Genetics, Long Island College Hospital Volume 1, Unfolding the Genome 1992, 425 pp. ISBN 1-55938-349-6

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CONTENTS: Genetic Techniques for Mapping and Sequencing the Genome, Ram S. Verma. Cloning Defined Regions of the Genome, Bernard Horsthemke. In Situ Hybridization, Paul Szabo. Southern Blotting, K. Stam. Northern Blotting, M.A.Q. SiddiquL Slot Blot Technique: Principles and Application, Geeta Vasanthkumar. Dot Blot Technique, Roger Lebo. Polymerase Chain Reaction Technique: Principles and Application, Stephen M. Carleton. Pulse-Field Gel Electrophoresis: Detection of Large DNA Molecules, Robert M. Gimmill. Detection of Single Base Charge in Nucleic Acid, Richard G.H. Cotton. Identification of Chromosome Specific Satellite DNA from the Centromere by Biotinylated DNA Probes, Matteo Adinolfi. Construction and Usage of Linkage Libraries, Alan R. KimmeL Subject Index. Volume 2, Morbid Anatomy of the Genome 1993, 377 pp. ISBN 1-55938-583-9

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CONTENTS: Diagnosis of Human Genetic Disease, Jorg Schmidtke. Mitochondrial DNA and Disease, A.E. Harding. The Cystic Fibrosis Gene, Michael Wagner. Maffan Syndrome: A. Molecular Biology, Brendan Lee. Maffan Syndrome: B. Molecular Pathogenesis, Leena Peltonen. Molecular Genetics of the Fragile X, Grant R. Sutherland. Molecular Genetics of Huntington Disease, C. Pritchard. Gene for Von Recklinghausen Neurofibromatosis Type 1., Dalai M. Jadaye. Molecular Biology and Duchenne and Becker Muscular Dystrophies, Jamel Chelly. Molecular Genetics of Thalassemia, Stephen A. Liebhaber. Application of Molecular Genetics for Identity, Zvi G. Loewy. Current Status and Future Directions in Human Gene Therapy, Paul Tolstoshev. Prelude: Reverse Genetics, Ram S. Verma. Subject Index.

ADVANCES IN GENOME BIOLOGY Volume 3B

1995

GENETICS OF HUMAN NEOPLASIA

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UNFOLDING THE GENOME

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Volume 3A. GENETICS OF HUMAN NEOPLASIA Volume 3B. GENETICS OF HUMAN NEOPLASIA Volume 4.

GENETICSOF SEX DETERMINATION

Volume 5.

GENESANDGENOMES

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ADVANCES IN GENOME BIOLOGY GENETICS OF HUMAN

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91995

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CONTENTS (Volume 3B) xi

LIST OF CONTRIBUTORS PREFACE

Ram S. Verma

XV

TRANSCRIPTION AND CANCER

Phillip M. Cox

233

LOSS OF CONSTITUTIONAL HETEROZYGOSITY IN HUMAN CANCER" A PRACTICAL APPROACH

Jan Zedenius, GClnther Weber, and Catharina Larsson

THE ROLE OF THE BCR/ABL ONCOGENE IN HUMAN LEUKEMIA

Peter A. Benn

ADVENTURES IN MYOOLOGY

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

415 429

INDEX

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CONTENTS (Volume 3A) LIST OF CONTRIBUTORS

xi

PREFACE

Ram S. Verma

XV

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

17

55

Bernard E. Weissman and Kathleen Conway

143

P21 ras: FROM ONCOPROTEIN TO SIGNAL TRANSDUCER Johannes L. Bos and Boudewijn M. Th. Burgering

163

CHROMOSOMAL BASIS OF HEMATOLOGIC MALIGNANCIES

Ram S. Verma

THE MOLECULAR GENETICS OF CHROMOSOMAL TRANSLOCATIONS IN LYMPHOID MALIGNANCY

Frank G. Haluska and Giandomenico Russo

185

211

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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

Kathy 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

GCmther Weber

Department of Clinical Genetics Karolinska Hospital Stockholm, Sweden

Bernard E. Weissman

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

Jan Zedenius

Department of Clinical Genetics Karolinska Hospital Stockholm, Sweden

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

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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

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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

TRANSCRi PTION AN D CANCER

Phillip M. Cox

I. II.

III.

IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eukaryotic Transcriptional Regulation . . . . . . . . . . . . . . . . . . . . . A. The General Transcription Machinery . . . . . . . . . . . . . . . . . . . B. Control of Eukaryotic Transcription . . . . . . . . . . . . . . . . . . . . Transcription Factors as Oncogenes . . . . . . . . . . . . . . . . . . . . . . . A. bZip-Family Oncoproteins . . . . . . . . . . . . . . . . . . . . . . . . . B. Basic Helix-Loop-Helix Oncogenes . . . . . . . . . . . . . . . . . . . . C. Zinc-Binding Transcription Factors as Cancer Genes . . . . . . . . . . . D. Homeobox Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. reland its Relations . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. myb and ets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. The s k i O n c o g e n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Tumor Suppressor Genes and Transcription . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

233 234 234 237 245 246 249 251 253 254 255 257 258 259 261 261

INTRODUCTION

W h i l e e v e r y cell o f a m u l t i c e l l u l a r o r g a n i s m c o n t a i n s e s s e n t i a l l y the s a m e c o m p l e m e n t o f D N A in its g e n o m e , the various o r g a n s o f its b o d y p e r f o r m v a s t l y d i f f e r e n t Advances in Genome Biology Volume 3B, pages 233-278. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8 233

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PHILLIP M. COX

functions. These different functions require the production of particular sets of proteins by the cells which necessitates expression of some genes and the silence of others. In addition, the levels of proteins expressed need to be altered in response to external stimuli, as cells progress through the cell cycle or as they develop from their embryonic precursors to their differentiated state. Transcription is the process of copying small regions of the genomic DNA in the nucleus into mobile RNA species which, after processing, are translocated to the cytoplasm where the information they carry is translated into a polypeptide chain by ribosomes. Regulation of transcription is central to the control of the vast majority of cellular functions and, in particular, differentiation and the response of cells to external signals. Cancer, or more generally, neoplasms, are clones of cells that have the ability to grow and divide independent of external controls and that, on the whole, are less differentiated than the cells of the organ in which they arose. Since transcriptional regulation is of such importance to both growth responses and differentiation, it is clear that deregulation of transcription must play an important part in the development of neoplasms. Evidence is accumulating rapidly to support this notion, both from the study of the functions of viral oncogenes and their cellular counterparts and also from the genetic analysis of human tumors, most notably the leukemias.

II.

EUKARYOTIC TRANSCRIPTIONAL REGULATION

In eukaryotic organisms, RNA transcription from the DNA template requires both the general transcriptional machinery, including RNA polymerase and basal transcription factors, and proteins (trans-acting factors) which specifically recognize short DNA sequences (cis-acting elements) usually in the noncoding region of the gene. ~2 These sequences may be immediately upstream of the transcription start site where they constitute the promoter necessary for accurate and efficient initiation of transcription. Alternatively, they may lie hundreds or thousands of bases upstream or even within introns, exons, or downstream of the gene, forming enhancer elements capable of modulating promoter function. 3

A. The General Transcription Machinery R NA Polymerase II In contrast to prokaryotes, which have a single RNA polymerase, eukaryotic cells contain three enzymes responsible for transcribing distinct groups of genes from the DNA template into RNA: (1) RNA polymerase I synthesizes the precursors of the large ribosomal (r)RNAs; (2) RNA polymerase II transcribes protein coding

Transcription and Cancer

2 35

genes and some small nuclear (sn)RNAs; and (3) RNA polymerase III produces transfer (t)RNAs and the 5S rRNA. RNA polymerase II (POLII) is a multisubunit protein complex of Mr ca. 500-600 kDa comprising two large polypeptides and 8-10 smaller components. Its exact composition in vivo has recently been elucidated and exhibits some variation depending upon the organism and promoter being studied. 4'5 The largest subunit, of Mr 220-240 kDa is highly conserved in structure throughout eukaryotes and shows considerable homology to the comparable subunits of RNA polymerases I and III and also to the 13' polypeptide of the prokaryotic enzyme. 6-1~ The second largest subunit, Mr 140-150 kDa, also shows a high degree of evolutionary conservation in eukaryotes, and it shares many structural features with the 13-subunit of its prokaryotic counterpart. 11'12 The smaller components can be divided into three groups: first, those polypeptides used by all three RNA polymerases, and therefore presumably part of the fundamental core of the enzyme; second, those restricted to POLII but essential for its function; and third, those only found under some circumstances and at least partially dispensable. Although probably conserved among eukaryotes, these smaller polypeptides show only limited similarity to those in the prokaryotic enzyme. 4 Through a combination of genetic and biochemical studies, both large subunits have been found to contact the DNA template and the nascent RNA chain. 13-16 In addition, the second subunit binds substrate nucleotides and appears to be involved in fashioning phosphodiester bonds, possibly with the assistance of one of the smaller polypeptides, 17 while a region on the largest subunit, to which the mycotoxin ~-amanitin binds, is essential for RNA elongation. 18'19The carboxy-terminal domain (CTD) of the largest subunit has multiple phosphorylation sites, which are unphosphorylated during the initiation of transcription when the exposed hydroxyl groups may interact with acidic regions of other transcription factors involved in regulating the rate of initiation at some promoters. 2~ Phosphorylation, possibly by a general initiation factor (TFIIH), is essential for elongation to become established and may be involved in releasing the enzyme from the initiation complex. 2x-x6

Basal Transcription Factors In contrast to the bacterial RNA polymerase, which recognizes a specific DNA sequence a short distance upstream of the transcription start site, eukaryotic POLII depends upon a number of general transcription factors to locate the correct position for initiation. These were originally identified as activities in fractions of nuclear extracts separated by chromatography on phosphocellulose and necessary for reconstitution of a transcription system in vitro. 27 Originally three activities were defined: the transcription factors for RNA polymerase II (TFII) A, B, and D in the order of elution from the column, 27 with TFIIB subsequently being separated into

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PHILLIP M. COX

TFIIB and TFIIE. 28 The most extensively studied of these factors is TFIID, which interacts directly with an A-T-rich sequence in the promoter, the TATA box, 29'30 whose position (usually-25 t o - 3 0 relative to the transcription start site in mammalian genes) is critical in determining the exact point of initiation. 31 eDNA clones encoding yeast and mammalian TATA box-binding protein (TBP) have been isolated. 32-37The protein encoded by the mammalian cDNA is considerably smaller than TFIID purified from nuclear extracts, which has been shown subsequently to be a multisubunit complex composed, in Drosophila, of six other polypeptides known as TBP-associated factors (TAF's). 38 It is possible that several forms of TFIID coexist, comprising alternative combinations of subunits with slightly different binding specificities and distinct functions, 39 and allowing regulation of transcription via the TATA box. This may explain the earlier observations that some viral transactivators show specificity for the TATA boxes of particular genes 4~ and that some TATA boxes are only functional in a particular promoter context. 41 It has been shown recently that TBP is also required for initiation on POLII promoters lacking a TATA-box42 and, in addition, for transcription by RNA polymerases I and III, 43-46 where it appears to be associated with a different set of TAF's. Binding of TFIID to the TATA-box is probably stabilized by interaction with a heterotrimeric complex, TFIIA, 3~ and the resulting stable protein-DNA complex commits the template to transcription. 47'48 Upon this, a preinitiation complex is assembled by the addition of TFIIB, which probably interacts directly with TBP, 49 POLII, which is esconed by TFIIE and finally TFIIE, 3~ TFIIH, and TFIIJ. 26 The resulting massive structure is ready to initiate transcription and remains stable in the absence of nucleotidcs. 5~ Initiation requires the hydrolysis of ATP or dATP, possibly by one of the TFIIE subunits, 52'53 and is associated with phosphorylation of the CTD of POLII by TFIIH 25,26 and a major conformational change in the preinitiation complex. 29'3~The released energy may be employed in unwinding the template around the initiation site to give access for the polymerase, creating a structure analogous to the prokaryotic "open complex", possibly with the assistance of TFIIE/F, which may possess helicase activity. 54 Once the polymerase is released from the initiation complex and has transcribed a small number of nucleotides, it becomes part of an extremely stable elongation complex, 5~ which probably includes at least some components of TFIIE as well as elongation factors. 56"57 This transcribes the whole of the gene, including the noncoding introns, and overshoots the end of the coding sequence, eventually terminating a considerable distance downstream. Meanwhile, TFIID and possibly some of the other parts of the preinitiation complex remain attached to the TATA-box facilitating the initiation of subsequent rounds of transcription. 29"55 POLII and the general factors are necessary for the transcription of most, if not all, protein coding genes, with the rate and frequency of initiation being determined by additional trans-acting proteins which interact with specific cis-acting DNA elements in the promoter and enhancer regions. Thus, the combination of proteins

Transcription and Cancer

237

controlling the transcription of an individual gene in a particular cell type depends (1) upon which cis-acting elements are present in the gene in question, and (2) upon which trans-acting factors are expressed and active within the nucleus.

B. Control of Eukaryotic Transcription Structural Features of Transcription Factors As a general rule, transcription factor proteins are organized as a series of either spatially discrete or occasionally overlapping peptide domains which mediate the various functions, such as binding to a specific DNA sequence, the interactions with other proteins responsible for dimerization, transcriptional activation, or repression and inactivation. As an increasing number of genes for transcription factors has been isolated, it has become clear that eukaryotes have evolved a fairly limited repertoire of structures capable of mediating these activities; thus transcription factors can be grouped together on the basis of shared structural features. Homology is particularly marked in the domains responsible for binding to DNA and for the formation of homo- or heteromeric complexes. In these regions, the amino acid sequence may show a high degree of conservation between proteins which are, otherwise, largely dissimilar. However, even single amino acid differences in these domains can change the DNA-binding specificity of a protein, either directly, or by altering the repertoire of other proteins with which it can form complexes. 58-65 Two structures, the basic-helix-loop-helix (bHLH) and the leucine zipper (LZ), involved in dimer/multimer formation, have been extensively characterized and have some features in common. The bHLH motif was originally identified as a region required for dimerization and DNA-binding in three apparently functionally distinct groups of proteins with known or suspected transcriptional activity: the products of the E2A gene, El 2 and E47, involved in, among other things, stimulation of B-lymphocyte-specific immunoglobulin gene expression; the musclespecific factor, MyoD; daughterless, a Drosophila developmental gene product; and the Myc oncoproteins. 66 The same motif has since been identified in a wide variety of other proteins with diverse functions ranging from phosphatase gene regulation in yeast to peripheral nervous system development in Drosophila. 67"68 The bHLH domain comprises two predicted s-helices, separated by a peptide loop of variable length, and a 13-amino acid region containing a number of conserved basic residues, the basic domain, which lies immediately to the N-terminal side of the helices. The helices are believed to have hydrophobic amino acids positioned along one face (i.e., they are hydrophobic amphipathic helices) which mediate homo- or heterodimerization. Dimerization is required for DNA-binding by positioning the basic domains of the subunits such that they can bind to the cognate DNA sequence. Although the hydrophobic amino acids are essential for dimerization and are shared by all bHLH proteins, it is clear that other amino acids in the

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PHILLIP M. COX

m-helices dictate specificity of dimer formation, since choice of a dimerization partner is highly specific. The leucine zipper is an alternative dimerization motif, comprising a single proposed hydrophobic amphipathic m-helix with leucines on the hydrophobic face, through which related proteins interact. 69 As with the bHLH proteins, this helix may be positioned a specific short distance C-terminal to a basic domain which mediates DNA-binding, together forming the bZip domain. 7~ This motif characterizes a growing family of transcription factors, several of which are capable of combining as stable and functionally active homo- and heterodimers. 69"71'72 A separate group of proteins--the Myc proteins, and transcription factors AP-4 and USF---contains both a bHLH motif and a leucine zipper, 73 both of which are required for protein function. The LZ is positioned C-terminal to the bHLH, and probably cooperates with it in the formation of homo- or heteromeric complexes. Similarly, the LZ may be found in association with other DNA-binding motifs such as the POU-domain (see below). TM Two other frequently recurring motifs, the zinc finger (ZF) and the homeobox, have been shown to be directly responsible for sequence specific DNA-binding by separate families of transcription factors. The ZF motif was first described in the transcription factor TFIIIA, where it occurs nine times, 75'76 and up to 13 potential zinc chelation sites can be present in a single protein, as in a Y chromosome-derived protein originally believed to be the testis determining factor involved in sex determination. 77 It results from at least four appropriately spaced cysteine or cysteine and histidine residues forming a complex with a zinc ion. It was suggested that this would lead to the formation of a projecting "finger" by the amino acids between the two pairs of residues involved in the zinc complex which would interact with the DNA. X-ray crystallography has confirmed this structure in TFIIIA. 78 Although the requirement for zinc, shown by the loss of DNA-binding activity in the presence of ion chelating agents, has been demonstrated for several factors; 79 all zinc-dependent DNA-binding proteins are not structurally homologous. For example, while the "fingers" of Krtippel-like proteins interact with DNA, it is the amino acids between the two zinc chelation centers of the steroid receptors which make contact with the double helix. 59-61 In archetypal ZF proteins, such as Krtippel, the fingers are probably all functionally equivalent" in others they appear to subserve different functions. For example, Green and Chambon 8~ showed that in members of the steroid hormone receptor family, which all have two chelation centers, one is needed for DNA-binding specificity, while the other stabilizes the protein-DNA complex, possibly by participating in protein-protein interactions with the other half of a dimeric receptor. 8~'82 The homeobox was originally identified as a highly conserved 180-base pair (bp) DNA element in the homeotic selector genes 83'84 involved in segmentation of Drosophila. 85'86 Subsequently, this structure has been found in a variety of transcriptionally active proteins from many phyla, many of which are involved in the processes of differentiation and development. The homeobox has been shown to

Transcription and Cancer

239

mediate DNA binding by these proteins and to be responsible for sequence specificity. 59'87'88Predictions of the structure of the homeobox, based on its amino acid sequence, suggested it would form three stretches ofo~-helix separated by short flexible spacers, 89'9~ a motif very similar to the helix-turn-helix (HTH) first described in bacterial transcriptional repressors. 91 X-ray crystallography of the homeodomain of the Drosophila engrailed protein cocrystallized with DNA has provided final evidence of direct DNA-homeodomain interaction comparable with, although not identical to, that of the prokaryotic HTH. Helices 1 and 2 at the N-terminal end of the domain lie perpendicular to the DNA backbone and make few contacts, while helix 3 is inserted in the major groove of the DNA double helix, forming extensive contacts with the bases and sugar-phosphate backbone of the cognate sequence. In addition, amino acids N-terminal to helix 1 make further contacts in the minor groove. 92 While for many proteins the homeodomain alone is sufficient for sequencespecific DNA binding, other related proteins require the presence of additional structures. One such group includes of the proteins Pit-1, Oct 1 and 2, and Unc 86 (POU), the homeodomains of which are more closely related to one another than they are to an archetypal homeodomain, such as that of Antennapedia(Antp). In particular, all share the amino acid sequence V-RVWFCN in helix 3 with V-RV-Cbeing POU-specific. 93 In contrast to the archetypal Antp and engrailed-like homeodomains, which alone are sufficient for sequence-specific DNA binding, 87'94 the POU family possesses a second highly conserved domain, the POU-specific box, which contributes significantly to specificity of DNA binding and which is required to generate high-affinity DNA contacts. 95'96 This POU-specific region comprises approximately 75 amino acids and is linked to the N-terminal end of the homeodomain by a short flexible spacer. Within the POU-specific box, two extremely well-conserved regions, subdomains A and B, are found, separated by a short segment of variable sequence and length. The four original POU proteins exhibit combined identity of 17/26 and 18/34 amino acids, respectively, over these two domains 93'97 and, in addition, three subserve similar functions being involved in the determination of cell fate. The regions of transcription factors involved in transcriptional activation or repression are less well-conserved, although certain recurrent themes can be recognized. In particular, activation domains may contain a preponderance of amino acids with acidic side chains, or alternatively a single amino acid, such as proline or glutamine, may be especially abundant. 98 The relationship between the structure of these regions and their ability to modulate the rate of initiation of transcription by the RNA polymerase is not well understood. 99 In general it is believed that activating domains function by promoting the assembly of the preinitiation complex. Consistent with this, the acidic activating domains have been shown, under different conditions, to form a stable interaction with either TFIID or TFIIB 100,101;it has been suggested that the unphosphorylated "tailpiece" of POL II may be another target. In contrast, there is evidence that

240

PHILLIP M. COX

glutamine- and proline-rich activation domains interact with TFIID via intermediary coactivator proteins. 38'!~176 The structural elements described above are not the only ones capable of forming dimers, of binding to DNA in a sequence specific manner, or of activating transcription. Other families are emerging whose conserved domains do not fit readily in any of the above models, but their structures are being solved and in time their mode of action will doubtless be determined.

Tissue-Specific Gene Expression In a simple system, tissue-specific expression of a set of genes with related functions could be achieved by the production in the tissue of an active transcription factor capable of interacting with a cis-acting element shared by those genes. An example of this is found in the somatotroph cells of the adult anterior pituitary gland, which exclusively produce a factor, Pit-1/GHF-1, regulating expression of a number of pituitary-specific genes through a conserved DNA element. 1~176 Pit- 1 appears to provide somatotroph specificity; however, expression of the various genes it regulates is subject to additional controls determining the level at which the genes are expressed, l~ ~0 In general, the situation is not so simple since the same cis-acting element may be present in the regulatory region of genes which are not coordinately expressed, ill Furthermore, several factors capable of recognizing that particular sequence may be present in the same cell. 1~2-~15To achieve tissue specificity, the sequence context in which the basic cis-acting element occurs is varied in different genes; the relative affinity of the factors for the core recognition site may be influenced by flanking DNA sequences, while their activity may be significantly modulated by proteinprotein interactions with other sequence-specific DNA-binding proteins interacting with elements elsewhere in the same promoter/enhancer. 116.116a A major refinement of this relatively simple system has been achieved in eukaryotic organisms by the evolution of families of transcription factors which bind to their cognate sequence as homodimers or as heterodimers with other family members. By varying the combinations, different responses may be evoked from the same cis-acting element, and the range of elements recognized by a small number of factors can be extended, thus helping to facilitate the specific and flexible control of transcription required for normal cellular function. 66"71'1iv

Inducible Gene Expression While the mechanisms outlined above enable tissue-specific transcription of constitutively expressed genes, many genes are only activated in response to external stimuli. When a cell perceives such a stimulus, one of a number of signal

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transduction pathways may be activated, resulting in alteration in the level of transcription of responsive genes. 3 This may be achieved via the production of new transcription factors. For example, virus infection and exposure to IFNs t~ and leads to induction of the synthesis of the transcription factors IRF- 1 and -2, which bind to the promoters of IFN-inducible genes and to those of IFNs ct and 13 themselves. 118-12~ Alternatively, the DNA binding or transcriptional activity of factors already present within the cell may be modulated, either by covalent modification or as a result of interaction with other proteins or intracellular ligands. Covalent modification may involve phosphorylation or glycosylation and, in addition, some factors are affected by changes in the redox state of their environment. Phosphorylation, the best characterized of these mechanisms, ~21 may regulate many functions of transcription factors including" nuclear localization; 122,~23DNAbinding, which may be either inhibited 124--126or stimulated; 127-128 transactivation, also either inhibited ~29 or stimulated; ~3~ and transrepression. ~33 For some transcription factors the cellular kinases responsible have been determined, while for others they remain unknown. In addition, as exemplified by c-jun, phosphorylation of different sites on the same factor can regulate distinct functions. In the case of c-jun, glucose synthetase kinase 3 and casein kinase II-mediated phosphorylation of one set of sites, which are phosphorylated in quiescent cells, 126 inhibits DNA binding, 125 while phosphorylation of serine residue 63 or 73 stimulates transactivation in response to oncogenes. 131,132 An alternative means of influencing transcription has been developed by families of factors which need to dimerize in order to bind DNA. By forming heterodimers with related proteins which share the dimerization motif, but which are unable to bind to DNA, potentially active factors can be sequestered and thus prevented from interacting with their cognate sequence. 134-137The first such factor to be identified was IKB, an inhibitor of the factor NF-~cB, but examples of this class of proteins have been found for the bZip (IP-I),138 bHLH (Id), 135 and POU-domain (I-POU) 139 families. Direct, ligand-mediated activation is a feature of the members of the steroid hormone receptor s~perfamily, all of which are probably transcription factors. In the absence of hormone, some of the receptors are sequestered in the cytoplasm by the heat-shock protein, hsp90. When the specific ligand enters the cell it occupies its binding site on the receptor protein causing a conformational change and release from sequestration, thereby allowing translocation to the nucleus, dimerization, and performance of its transcriptional function. 14~ A further level of regulation is achieved by the modulation of the binding or function of sequence-specific transcription factors by proteins which do not bind specifically to DNA in isolation but which may interact with or modify the transcriptional complex, inducing or repressing transcription. Such proteins in-

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PHILLIP M. COX

clude: the coactivators which perform a bridging function between some groups of transcription factors and the general transcription machinery; suppressor genes such as rbl, the retinoblastoma gene product; and a number of transactivating proteins encoded by DNA viruses such as El a from adenovirus, VMW65 from Herpes simplex, and the SV40 large T antigen. In addition, as noted already, several groups of transcription factors are subject to phosphorylation by various cellular kinases. By combining all of these mechanisms, expression of the vast array of genes in the eukaryotic genome can be regulated by a much smaller number of transcription factors.

Differentiation and Development Besides being necessary for the normal function of mature cells, transcriptional regulation is central to the development of a normal body pattern and to the normal differentiation of embryonic cells into adult tissues. Genetic analysis in Drosophila of mutations leading to major structural abnormalities in the embryo or adult has identified genes whose products play a pivotal role in morphogenesis. Some 20 genes are involved in determining the dorsoventral axis of the early embryo, 142 while at least another 50 are required for segmentation of the embryonic body and the definition of structures arising from those segments. The genes involved in body segmentation encode proteins with features of transcription factors, which at specific times during the earliest stages of development, are expressed in a series of circumferential domains defining successively finer subdivisions of the embryonic body. 86 They show a hierarchy of regulatory interactions such that the first to be expressed, namely the gap genes, regulate the next class, the pair rule genes, and these in turn control the segment polarity genes. In addition, the members of each group modulate one another leading to the production of sharply defined segments. Unlike the gap and pair rule genes whose expression is transient, the segment polarity genes and the homeotic selector genes which they regulate, are persistently active, defining both the position and final fate of each cell. 86'!43'144 Besides being controlled by genes higher up this regulatory cascade, the homeotic selector genes modulate one another and are subject to positive feedback. Thus, once activated their expression persists, providing the basis for programming of embryonic cells with their fate in the adult fly. The cloning of several homeotic selector genes and subsequent structural analysis led to the identification of the homeobox DNA-binding domain 83 that is common to the products of virtually all genes of this class. Proteins with close structural similarity in man and mouse also show distinct domains of expression, and the homologues of the homeotic selector genes appear to define

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regional boundaries in the early development of the mammalian nervous system andmesoderm.]45-147

Transcription factors are also of great importance in determining cellular fate by directing precursor cells along particular lines of differentiation. In the developing nervous system of the fruit fly and the nematode, C. elegans, a variety of such proteins have been shown to be required for differentiation of particular neurons, 148'149while in mammals mutation of the gene encoding the POU protein Pit- 1 disrupts the development of three of the five cell types in the anterior pituitary gland. 15~

Transcriptional Deregulation and Neoplasia Transcriptional regulation is clearly fundamental to the normal response of cells to growth stimuli and for cellular differentiation. Disturbance of this regulation might therefore be expected to disrupt these processes and thus lead to the

Table 1A. Oncogenic Transcription Factors in Transforming Retroviruses Family

Virus (Tumor)

Oncogene

Protooncogene

Mode of Activation

bZip ASV17 (sarcoma) v-jun FBJMuSV (OS) v-fos AS42 (FS) v-maf

c-jun (AP-1) c-fos (AP-I) c-Maf

deletion of repressor region deregulated expression NK

bHLH MC29 (ML)

v-myc

c-myc

deregulated expression

AEV (EL)

v-erbA

T3 receptor

C' deletion generates constitutive repressor

ReVT (RE)

v-rel

c-rel

interferes with NF-K:B family interactions

AMV (EL)

v-myb

c-myb

deletion of CKll-regulated repressor domain

E26 (EL)

v-ets

c-ets l

gag-myb-ets fusion protein

ALV

v-ski

c-ski- 1

ZF (Steroid)

Rel

Myb

E26 (EL) Ets Ski

Note:

?

OS-osteosarcoma;FS-fibrosarcoma;ML-myeloidleukemia; EL-erythroleukemia;RE-reticuloendotheliosis; NK-not known; T3-thyroidhormone.

244

PHILLIP M. COX Table TB. Transforming Transcription Factors Activated by Retroviral Integration

Family ZF (KriJppel)

Virus

Factor

Ecotropic retrovirus Friend MuLV

Evi-1

Moloney MuLVin Ela-Myc mice

Bmi- 1

lntracisternal Aparticle (transposable element)

Hox 2.4

FeLV Spleen focus forming virus

Fli- 1 Spi- 1

(Other)

Homeobox

Ets

uncoordinated growth and failure of differentiation seen in cancer cells. Deregulation might occur in a variety of ways; for example, transcription factors normally only expressed in response to external growth signals could be produced inappropriately or their usually short-lived mRNA or protein could be stabilized by mutation. Alternatively, mutation or deletion in the protein-coding sequence of such a transcription factor gene might result in a protein which was spontaneously active or which could not be suppressed. Since the amino acids forming the DNA-binding domain of a transcription factor determine the precise cis-acting element it recognizes and thus the range of genes it regulates, a single point mutation in this region could lead to activation of the wrong set of genes. At the same time, exchange of DNA-binding domains between unrelated factors as a result of genetic rearrangement could lead to activation by the chimeric factor of the correct target genes in response to the wrong stimuli. Abnormalities of proteins of the inhibitor class that sequester transcription factors in the cytoplasm could also have major effects, as of course could abnormalities elsewhere in the growth signaling mechanism which would be transmitted to the transcriptional machinery. Mutations could also affect transcription factors that normally mediate differentiation or that repress cell growth leading to their inactivation, or, alternatively, the genes for such factors might be disrupted or deleted. In the ensuing sections, it will become clear that many of the mechanisms postulated above are responsible for the transforming activity of some retroviral oncogenes (Tables 1A and I B) and that they may also be important in the development of human tumors (Table 2).

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2 45

Table 2. Oncogenic Transcription Factors and Human Tumors Family

Oncogene

bZip

c-fos

Tumors

Mechanism

Osteosarcoma Ca colon pre-B-ALL

overexpression/ gene amplification chimeric factor [t(17;19)]

N-myc L-myc E2

BL (various) Neuroblastoma Ca bronchus pre-B-ALL

?max tal-l(TCL5)

9 T-ALL

lyl- 1

T-ALL

activation [t(14; 18)] gene amplification gene amplification gene amplification chimeric factors [t(17;19) and t(l;19)] ? [t(14)] activation [t(l;14)]/ internal deletion activation [t(7;l 9)]

wt

Wilms' tumor

gli- 1

Glioblastoma

inactivation (deletion or point mutation) gene amplification

RARA

Promyelocytic leukemia

chimeric factor It( 15;17)]

pml

Promyelocytic leukemia T-ALL

chimeric factor It( 15;17)] activation [t(ll;14)]

pbx hox- 11

pre-B-ALL T-ALL

chimeric factor [t(l;19)] activation [t( 10; ! 4 )]

lyt-lO

B-cell lymphoma

bcl-3

B-CLL

activation (truncated) [t(10;14)] activated

p53 Rbl

Many Various

Point mutation/deletion Inactivated by deletion

HLF bHLH

c-myc

ZF (Krtippel)

(Steroid)

(Other)

rhom l(ttg)) rhom2 ) Homeobox

Rel

Other

Note: Ca-carcinoma;ALL-acute lymphoblastic leukemia; BL-Burkitt's lymphoma; CLLchronic lymphocyticleukemia.

i11. TRANSCRIPTION FACTORS AS ONCOGENES Since 1987, when, for the first time, an oncogene was shown to be a transcription factor, evidence has rapidly accrued that abnormalities of proteins with a direct role in transcriptional regulation can cause cellular transformation. This has come in

PHILLIP M. COX

246

part from the study of retroviral oncogenes and their relationship to cellular protooncogenes, and also from the cloning of genes involved in nonrandom chromosome rearrangements found in a variety of human tumors. Oncogenes, originally identified as the transforming genes of highly oncogenic retroviruses, 151 were later recognized as modified forms of normal cellular genes so-called protooncogenes, the products of which are involved in the control of cell growth and proliferation. Inappropriate expression of these genes results in transformation of cells in culture. Although many protooncogene products are growth factors, surface membrane receptors and cytoplasmic or membrane-bound proteins (for review see, Ref. 15 2), a number were localized to the nucleus and it was proposed that they provided the final stage in the growth signaling pathway. 153 Comparison of the functions of the normal protooncogene product with its transforming retroviral counterpart has provided some valuable insights into the ways in which cellular proteins can be rendered oncogenic. However, to date, the large majority of retrovirus-induced neoplasms have been found in animals and birds and few abnormalities of these genes have been found in human tumors. In many human tumors certain chromosomal rearrangements occur at a much higher frequency than would be expected by chance, suggesting that the genetic lesions caused by such nonrandom events are important for development of the neoplasm. Cloning of the genes adjacent to the breakpoints of such rearrangements has demonstrated in a number of cases the involvement of a transcription factor or factors. Currently, some 30 proteins with known or presumed transcriptional activity, including members of all the major structural families, have been implicated in cellular transformation.

A. bZip-Family Oncoproteins In 1987, Maki et al. 154 were first to demonstrate a direct relationship between oncogenes and transcription factors. They demonstrated that the product of v-jun, the transforming gene of the chicken sarcoma virus ASV 17, contained a region of considerable amino acid sequence similarity with the yeast transcription factor GCN4 across from its DNA-binding domain. 155 When the DNA-binding domain of GCN4 was replaced with the homologous region of v-Jun, the chimeric protein continued to function as a transcription factor, 156 implying that the v-Jun protein would also bind DNA. The DNA sequence motif recognized by GCN4 - - (ATGA(C/G)TCAT) 157 (A = adenosine, C = cytidine, G = guanosine, T = thymidine) - - is very similar to the cognate sequence, TGACT(C/A)A, of AP-l, a transcription factor (or family of factors) involved in mediating transcriptional activation in response to stimulation of protein kinase C. 158'159 Monoclonal antibodies raised against different regions of v-Jun were shown to precipitate AP-l, but not other transcription factors, from

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nuclear extracts 16~ and subsequently the peptide sequence of a 47-kDa protein in purified AP-1 preparations was shown to be identical to c-Jun. 161 When compared with c-Jun, v-Jun shows two amino acid changes in the DNAbinding domain, an internal deletion of 27 amino acids towards its N-terminus, and its mRNA lacks a large 3' untranslated region. Normal c-Jun shows only weak transforming ability in a focus forming assay, but creation of the N-terminal deletion present in v-Jun increases the number of foci formed 10-fold. Removal of the 3' untranslated sequences also has some effect, probably as a result of mRNA stabilization, while the point mutations appear to have no role in transformation. 162 Although v-Jun and c-Jun show similar affinity for AP-1 DNA-binding sites, v-Jun is a considerably stronger transcriptional activator in HeLa cells. This is a direct result of the internal deletion since when this region, designated ~5,is removed from c-Jun its activating ability is also enhanced, suggesting that 8 acts as a repressor domain. In other cell types, however, the repressor function of 8 is not evident. This appears to be due to the absence of an additional cell-type specific factor (the 8 repressor) which mediates the repression by interacting with ~.163 Since loss of cell-type specific repressor function and transforming ability of v-Jun reside in the same region, it is likely only cells carrying the 8 repressor will prove transformable by v-Jun. The transforming gene of the Finkel-Biskis-Jinkins murine sarcoma virus (FBJMuSV) is v-fos. 164 Its cellular counterpart, c-fos, like c-jun, is an immediate early gene, whose product is a nuclear phosphoprotein expressed transiently in many cell types in response to mitogenic and other stimuli. 165 It was widely believed to be a transcription factor; however, sequence-specific DNA-binding could not be demonstrated. However, immune precipitation experiments showed that c-Fos was present in complexes binding to the AP-I sequence. 166 Furthermore, a number of other proteins were coprecipitated as a result of interaction with Fos, the most abundant of which was c-Jun. 167"168 Like Jun, Fos has a region of homology with the yeast factor GCN4, and analysis of the likely protein structure of this conserved domain led to the recognition that each contained a leucine zipper (LZ), first identified in C-EBP 7~ which facilitates the protein-protein interaction between Fos and Jun necessary for DNA binding. 69'70 Unlike Jun, which can form homodimers with, albeit relatively weak, DNA-binding and transactivating ability, Fos is unable to homodimerize. However, Fos-Jun heterodimers are stable, bind to AP- 1 sites with high affinity, and are potent activators of transcription. 71 In its C-terminal 49 amino acids, v-Fos differs from c-Fos as a result of a deletion which alters the reading frame of the viral gene. ~64 However, this has no significant effect upon the ability to form specific DNA-binding heterodimers with Jun, or to activate transcription from TPA-responsive elements; 168'169 both will transform fibroblasts. ~7~Indeed, mutations in the LZ of v-Fos which prevent it from heterodimerizing also destroy its transforming ability, lvl Therefore, it appears that it is loss of normal regulation of Fos protein expression and not the altered C-terminus,

248

PHILLIP M. COX

which is responsible for the transforming ability of v-Fos. Overexpression of Fos mRNA and protein has been detected in a number of tumors, although it is not clear whether this is a primary event or the result of growth stimulation, and amplification of the c-fos gene is also occasionally detected. 172'173 The bZip domain present in Jun and Fos has been identified in a large family of proteins, including others which bind to AP-I sites and also those recognizing ATF/CREB sites which mediate cAMP responses, 174-178 and many have been shown to be capable of selectively forming heterodimers. 69'71,72 It is also clear that some AP-I and ATF/CREB-binding factors can heterodimerize, thus there is a considerable repertoire of heterodimers able to bind to the same, or closely related, DNA sequences under different conditions. A further level of complexity in the function of AP-1 and possibly other bZip proteins was discovered when it was shown that there is a close relationship with members of the steroid receptor superfamily, 179which are also transcription factors. A small number of genes have cis-acting elements capable of binding both steroid receptor and AP-1, either separately or simultaneously, with the transcriptional response being dependent upon which factor(s) are present. 18~ In addition, AP- 1 and possibly ATF can repress promoters containing glucocorticoid or retinoic acid receptor-binding sites without an AP- 1 site, and vice versa. 182-184This effect is the result of interaction between the zinc fingers of the steroid receptor and the zipper of the bZip protein, most probably causing mutual interference with DNA binding. Since both v-Fos and v-Jun have an intact LZ it is possible that some of their effects could be produced in this way. Despite the large size of the bZip family, only one other member has been found to be a viral oncogene, v-maf, which causes naturally occurring fibrosarcomas in fowl, has both a leucine zipper motif, 185 and an adjacent domain of basic amino acids which fits the consensus sequence of the bZip proteins. 7~ In addition, v-maf has stretches of uninterrupted glycine and histidine residues, possibly representing regulatory domains. Another gene for a bZip protein, HLF, is disrupted by a t(17:19) translocation described in two patients with acute lymphoblastic leukemia (ALL) showing a primitive B-cell phenotype. 186 The part of the gene encoding its bZip domain becomes fused to the gene E2A on chromosome 19. E2A codes for two HLH proteins, E 12 and E47, which recognize a common enhancer element and are active in B cells. A chimeric protein is produced, with the HLF bZip domain fused to the E12/E47 activation domain and expressed under the E2A promoter. The normal HLF protein is most closely related to TEF, a bZip protein found in embryonic thyrotroph cells of the anterior pituitary gland, and DBP, which regulates albumin gene expression in the adult liver. HLF is expressed in adult liver and kidney, but not in lymphoid cells" thus the chimeric protein may interfere with normal bZip or bHLH protein-mediated transcriptional regulation of differentiation in B cells.

Transcription and Cancer

249

B. Basic Helix-Loop-Helix Oncogenes The oncogene v-myc, carried by the avian retrovirus MC29, was one of the earliest viral transforming genes to be identified. The protein products of its cellular counterpart, c-myc, and the related L- and N-myc genes, have features of immediate early genes, being expressed in the nucleus 187'188 and showing a rapid response to growth stimuli. 189Inappropriate expression of different members of the myc family is found in a number of human tumors, either from an apparently normal myc gene, or as a result of chromosomal translocation, such as the t(14; 18) found in Burkitt's lymphoma, or gene amplification, which is commonly present in neuroblastoma, lung, breast, and cervical carcinoma. 19~ In addition, likefos, myc can complement ras oncogenes in transforming cells. 194 Although nonspecific binding to DNA was demonstrated, 187 little more was known about the function of these proteins until a region of amino acid sequence homology was recognized between the myc gene products and a number of known and putative transcription factors involved in differentiation and tissue-specific gene expression. In these transcription factors, this domain, the bHLH motif, mediates the formation of homo- and heterodimers between related proteins and is essential for sequence-specific DNA binding. 66'195'196 Thus by inference myc was also likely to have a role in transcription. To the C-terminal side of the bHLH domain Myc, in common with the transcription factors AP-4 and USF, also has a leucine zipper domain, similar to that present in Fos and Jun. This has also been shown to be necessary for the formation of Myc multimers, but which also may enable interaction with other proteins. 197 Given the homology with other members of the bHLH family, it was clear that Myc should bind DNA. Two complementary approaches resulted in identification of the Myc recognition sequence. First, by selection and PCR amplification of DNA interacting with Myc protein from a pool of random sequences a consensus target site, CACGTG, was identified. 198Second, on the basis of homology across the basic regions of Myc and the yeast transcription factor PHO4, which binds the sequence CACGTG, it was demonstrated that a chimeric protein comprising the Myc basic domain substituted for that of PHO4 would bind the CACGTG motif in vitro. 62 These experiments, together with those from a number of other laboratories showed that Myc was potentially a sequence-specific DNA-binding protein. However, since DNA binding by Myc was extremely inefficient, requiring large amounts of protein to produce binding, it was suggested that Myc might function as a heterodimer. A dimerization partner for c-Myc was isolated from a ~.gtI 1 expression library, by screening with the dimerization domain of c-Myc. 199 This protein, Max, exists in two forms differing by the presence of an additional 9 amino acids N-terminal to the basic domain in the larger form. It can homodimerize and heterodimerizes with c- L- and N-Myc, both in vitro and in vivo, and both homo- and heterodimers recognize the CACGTG consensus. 2~176176 Max cannot heterodimerize with other

250

PHILLIP M. COX

bHLH/LZ proteins such as AP-4199,202, but recently a second partner for Max, Mad, has been identified (in press). The structure of Max is simple, comprising 160 amino acids, half of which constitute the bHLH and LZ regions responsible for dimerization, flanked by a short N-terminal domain and a C-terminal region which includes a small acidic domain. In contrast to c-Myc, Max expression is seen in quiescent as well as dividing cells and thus it may act to repress transcription from Myc-responsive genes in the absence of c-Myc protein. The ability ofMyc:Max heterodimers to transactivate gene expression is dependent upon the N-terminus of Myc, while Max homodimers repress transcription from the same genes 2~ and DNAbinding of Max homodimers, but not Myc:Max heterodimers, is prevented by phosphorylation of Max by casein kinase II. 204 Transcriptional activation is clearly important for the transforming activity of Myc proteins since the long N-terminal domain responsible for transactivation 2~ is required for cell transformation, 2~ and the relative potency of the activation domains of c- and L-Myc appears to correlate with their transforming ability in rat embryo cells. 2~ It is also possible that Max may be directly implicated in human neoplasia, since the max gene has been localized to chromosome 14 bands q22-24, a region subject to deletion in B-cell chronic lymphocytic leukemia and close to the breakpoint of the 12; 14 translocation found in uterine leiomyomas. 2~176 Three additional bHLH proteins have been implicated in different forms of acute leukemia. One of these, the product of the E2 gene, participates in separate translocations in pre-B type acute lymphoblastic leukemia with a bZip protein (see above) and with a homeobox gene (see below). The others, Tal-1 (SCL/TCL5) and Lyl-1, are closely related and both are expressed in hematopoietic cells. The tal-I gene was identified by cloning of the breakpoint on chromosome 1 caused by the translocation, t(l'14) (p33;qll.2) in a primitive acute leukemia capable of both myeloid and lymphoid differentiation. The normal tal-I gene is expressed by hematopoietic stem cells and myeloid and T-lymphocyte precursors, 21~ and may also be important in erythropoiesis. 2~ Its encoded protein can interact with other bHLH proteins forming heterodimers which specifically recognize the E-box DNA motif, CANNTG, found in a number of enhancers. The translocation results in an aberrant mRNA, including at its 3' end part of the T-cell receptor (TCR) ~i-chain gene. A small group of T-cell acute lymphoblastic leukemias (T-ALL) carry the translocation t(l" 14) (p32;ql 1) in which tal-I is also involved, 212 and up to 25% of T-ALLs carry a small internal deletion in this gene. 213Furthermore a rearrangement of tal-I has been detected in a human melanoma cell line. 214 The T-ALL translocation results in truncation of the tal-1 gene and fusion to the 5' part of the tcr8 gene, and thus the function of the protein may be abnormal and it may be expressed under the control of an inappropriate promoter. The second protein, designated Lyl- 1, is expressed in a truncated form in another subset of T-ALLs, as the result of a t(7:19) translocation which juxtaposes its gene to that of the TCR ]3-chain.215 The normal Lyl-1 protein is expressed in lymphoid cells; however, its function is not known.

Transcription and Cancer

251

Further study of these proteins should give valuable insight into the role of transcription in normal hematopoietic development and in lymphoid neoplasia.

C. Zinc-Binding Transcription Factors as Cancer Genes The archetypal zinc-dependent DNA-binding domain is the zinc finger, present in TFIIIA and the Drosophila protein Krtippel. However, other families of transcription factors use the chelation of zinc ions to form different structures. 82 For example, the steroid receptors bind DNA through a predicted o~-helix adjacent to the zinc chelation site, while in several fungal transcription factors a third structure has been defined. Other less well-characterized groups also exist for which the nature of the DNA-binding structure has not been determined precisely. Members of the various groups have been shown to be either dominant positive, dominant negative, or recessive oncogenes.

The Wilms" Tumor Gene and Other KriippeI-Like Proteins Wilms' tumor (WT) or nephroblastoma is an embryonal tumor of the kidney, which occurs sporadically in young children, but usually in a few cases it is hereditary or is associated with other congenital abnormalities including aniridia, defective genito-urinary development, and mental retardation (the WAGR syndrome). Gross cytogenetic deletions and rearrangements affecting chromosome 1l(band p13) are a common finding in the WAGR syndrome and in occasional sporadic Wilms' tumors, a finding which raised the possibility that this region contained a recessive oncogene (tumor suppressor gene). By cloning and sequencing the breakpoints of these rearrangements a gene was defined which is consistently disrupted by these changes. This proposed wt gene encodes a protein (WTI) with four "zinc fingers" of Krtippel-type near to its C-terminus, suggesting that it is a transcription factor. 216'217 Its N-terminus contains proline- and glutamine-rich domains, reminiscent of the activation domains of some other transcription factors. 218'219On the basis of amino acid conservation, WT1 is most closely related to EGR-I, 22~ an early growth response gene product which recognizes the DNA sequence element 5'-CGCCCCCGC-3' and acts as a transcriptional activator. 221'222 WT1 also recognizes this element but functions instead as a repressor. 223 The wt gene is strongly expressed in the embryonic kidney where its product appears to be required for the switch from mesodermal to epithelial differentiation that occurs in the developing urogenital system. 224 In addition, it is widely expressed in both male and female genital tract in the embryo and adult. 225 Besides the gross cytogenetic abnormalities occasionally seen, a proportion of sporadic Wilms' tumors show loss-of-heterozygosity at this locus or carry small internal deletions or point mutations in the wt gene, providing support for its role in tumorigenesis. Furthermore, nephroblastoma cells transfected with a normal chromosome 11, are unable to form tumors in mice. 226 The mechanism by which homologous deletion or

252

PHILLIP M. COX

rearrangement of the wt gene allows uncontrolled'proliferation of nephroblasts is unknown, but one might speculate that the normal WTI protein stimulates their differentiation into nephrons, possibly by repressing genes which stimulate growth and which remain active when wt is disrupted. One candidate target for WTl-mediated repression is the insulin-like growth factor II (IGFII) gene, whose product is consistently overexpressed in Wilms' tumors and which is normally repressed by WT1.227 Other Krfippel-like zinc finger proteins have been shown to be dominant oncogenes. The gene glil, on chromosome 12, is amplified in a proportion of human malignant gliomas. 228 Evidence suggests that the GLII protein may be involved in normal development since it is most closely related to the Krtippel zinc finger proteins 229-231 involved in Drosophila segmentation and is produced by embryonal carcinoma cells but not by adult tissues. In addition, disruption of the very similar gli3 gene is responsible for Greig's syndrome, a disorder of craniofacial and limb development. 232 Another ZF gene, evi-l, is activated by integration of ecotropic retrovirus or by the Friend murine leukemia virus in a number of interleukin-3 (IL-3)-dependent myeloid leukemia cell lines. 233-235 It encodes a 120-kDa protein containing 10 Krfippel-like ZF's and an acidic putative activation domain. Bacterially expressed, evi-I specifically recognizes the consensus sequence TGACAAGATAA which is found in the regulatory region of a number of genes. 236 High-level expression is seen in a small number of specific regions in the embryo and exhibits temporal regulation, suggesting that it plays an important developmental role, while widespread low-level expression is found in adult tissues. 237

The Steroid Receptor Family The best characterized of the oncogenes belonging to the steroid receptor class of proteins is v-ErbA, one of the transforming genes of the avian erythroblastosis virus which encodes a truncated thyroid hormone receptor, 238"239'242a relative of the steroid hormone receptors. 24~ As a result of a small C-terminal deletion, the viral ErbA protein shows no affinity for the thyroid hormone T3 in mammalian cells. It does, however, bind correctly to thyroid hormone-responsive DNA elements, functioning as a constitutive repressor of thyroid hormone responsive genes and competitive antagonist of the normal thyroid hormone receptor/ligand complex, 242 a capacity which correlates with transforming ability. 243 This implies that v-ErbA may transform erythroblasts by blocking thyroid hormone-mediated differentiation, placing it in the dominant negative class of oncogenes. This may not be the complete story since when v-ErbA is expressed in yeast cells it is able to respond to thyroid hormone, suggesting that other factors may influence it in mammalian cells. In acute promyelocytic leukemia (APL; FAB M3), tumor cells carry the reciprocal translocation t( 15:17)(q22;q I 1.2-q 12) in approaching 100% of cases. TM The chromosome 17 breakpoint has been characterized in a number of cases and shown to

Transcription and Cancer

2 53

fall in the first intron of the retinoic acid receptor ~-gene (RARA),245 whose product is a member of the steroid hormone receptor family. The translocation separates the first exon, which encodes the transcription activation domain, from the DNA-binding and ligand-binding domains which become part of the derivative chromosome 15. The gene to which they are attached (pml/myl) is a member of a third group of proteins with a cysteine-rich, proposed zinc-binding domain. 246 This structure appears distinct from the Krtippel-like and steroid receptor zinc finger motifs and probably mediates DNA binding since the family comprises known yeast and mammalian transcription factors, Drosophila developmental genes and a recombination-activation protein involved in immunoglobulin gene rearrangement. 247-249 pml is a myeloid specific gene and is presumably freed of its normal regulation by the translocation, causing it to become a dominant oncogene; however, the fusion protein is responsive to retinoic acid and high dose RA therapy will cause remission in patients with APL, possibly by inducing myelocytic differentiation. Other oncogenic members of the same group ofcysteine-rich, putative zinc-binding transcription factors such as PML have been described, including bmi-I and reel-18. The gene bmi-1 is a common site of integration for the Moloney murine leukemia virus in a transgenic mouse line carrying an activated c-myc gene, expressed under the control of an immunoglobulin heavy chain enhancer. The cooperative effect of myc and bmi-1 leads to the development of B-cell lymphomas with a much shorter latency than those formed with c-myc alone. 25~ The closely related mel-18 is a murine neural crest-specific gene expressed at high level in melanoma cells and other tumors, and thus is suggested to play a role in cell proliferation. 252'253 Two T-ALL-associated translocations involving chromosome 11, bands p15 and p13, activate expression of the genes rhom-1 and rhom-2, respectively, both of which encode proteins with cysteine-rich putative zinc-binding LIM domains related to the ZF. 254--256Both genes are expressed in the embryonic mouse nervous system and, in addition, rhom-2 is present in the developing lung, kidney, and spleen. 255'257 Presumably aberrant expression of either of these proteins in T cells as a result of the translocations interferes with normal differentiation or stimulates T cell proliferation. It is clear that the proteins described above are not likely to be the only developmentally regulated transcription factors with a role in transformation, since any such protein which can promote replication of embryonic cells or maintain such cells in an undifferentiated state would have equally serious consequences if expressed inappropriately in adult tissues.

D. Homeobox Genes The homeobox motif is widely distributed in genes with roles in development and cellular differentiation; 86'1~ however, few examples of such genes being oncogenic have been identified.

PHILLIP M. COX

254

In childhood pre-B-acute lymphoblastic leukemia (pre-B-ALL), the tumor cells of 30% of patients carry the translocation t(l:l 9)(q23:pl 3.3). 260 The translocation results in a chimeric gene from which a potentially functional transcription factor is synthesized, 261'262 with the N-terminal part of the novel protein deriving from the E2A gene on chromosome 19 which encodes the Myc-related proteins E 12 and E47. These are bHLH proteins capable of heterodimerizing with a variety of other family members forming enhancer binding factors active on a variety of genes including the kappa immunoglobulin promoter in B cells. 196'263 The part of the protein retained in the chimeric factor is the potent transcriptional activator domain. The C-terminus is encoded by pbx, a gene on chromosome 1, normally inactive in pre-B lymphocytes, which contributes its homeodomain. The result of the pre-BALL translocation is a protein which may be capable of strongly activating a gene or group of genes involved in the control of normal differentiation and not normally expressed at this stage of lymphocyte ontogeny. The homeobox gene hox-2.4, is constitutively expressed in the mouse myeloid leukemia cell line WEHI-3B as the result a rearrangement caused by insertion of a transposable DNA element of the intracistemal A-particle type. 264 The oncogenic potential of this gene may extend beyond haematopoietic cells since when the activated hox-2.4 is introduced into NIH3T3 fibroblasts the resultant cells form fibrosarcomas in nude mice. 265 In approximately 5% of T-ALLs the translocation t(10;14)(q24;ql 1) is present, which juxtaposes the tcr-5 chain gene with another homeobox gene, hoxll (tcl3), expressed in normal liver, but not in the thymus or in T lymphocytes. 266-268 It is presumed that deregulated expression of hoxll interferes with the normal process of T-cell development and thus causes transformation.

E. rei and its Relations ReVT is an avian retrovirus which causes rapidly progressive lymphoid tumors (reticulendotheliosis) in birds. It carries the v-rel oncogene 269 whose product is a protein found in the nucleus of some cells and in the cytoplasm of others. 27~The protooncogene c-rel is one of a number of genes related to NFrd3, a multifunctional transcription factor, and dorsal, a Drosophila gene involved in dorsal/ventral axis determination. 271'272 NFr,.B is a heteromeric complex of two polypeptides of the same family, p50 and p65. p50 homodimers can bind to ~r elements (as the factor KBFI) while p65 does not bind DNA on it own. In cells where it is not constitutively active, NFK:B is sequestered in the cytoplasm through the interaction of p65 with I~r another relative, 133 which in turn may be attached to the cytoskeleton via a series of "ankyrin-like" repeats, p50 is produced from a 105-kDa non-DNA binding precursor (p105) also having "ankyrin-like" repeats. Its activity may be regulated partly through the rate of cleavage of this cytoplasmic molecule to p50, which can then localize to the nucleus, associate with p65, and bind to DNA. 271'272Neverthe-

2 55

Transcription and Cancer

less, sequestration of p50 in the cytoplasm by InB is probably the more important regulatory event. The v-rel oncogene differs from c-rel by 14 base substitutions in the Rel-homology domain responsible for DNA-binding and dimerization. It also lacks approximately 100 C-terminal amino acids. The deletion interferes with cytoplasmic retention, but replacement of the missing amino acids does not affect transforming ability. 27~ The c-rel and v-rel oncogenes are both able to interact with other NF~zB-like factors; however, v-rel is a much weaker activator of transcription. This suggests that it may interfere with the normal equilibrium between active and inactive complexes, possibly by acting as a dominant negative transcriptional repressor, or alternatively, by sequestering other important regulatory proteins. For example, v-re! can abolish the powerful stimulatory effect of phorbol esters on a reporter plasmid controlled by a rd3 site, 274 and it is proposed that v-rel forms nonfunctional heterodimers with other family members. From this point of view, v-rel and v-ErbA appear to function in a similar fashion. Interference with Rel family interactions appears to be the mode of action of a naturally occurring differential splice product of the p65 NFK:B subunit, p65A, which lacks amino acids necessary for heterodimerization with p50 and DNA binding. This protein is highly expressed in proliferating hematopoietic cells and will transform RAT-1 fibroblasts, 275 presumably employing a similar mechanism to v-rel. Studies of human lymphoid neoplasms have revealed abnormalities of two other members of the extended Rel family. The first is lyt-1 0, a gene encoding a protein containing a Rel-homology domain and "ankyrin-like" repeats, which is juxtaposed to an immunoglobulin locus by a t(10:14) translocation in a B-cell lymphoma. 276 In the normal Rel-related proteins the "ankyrin-like" repeats perform a regulatory function, preventing DNA-binding either by cytoplasmic sequestration or by physically obstructing the dimerization domain until they are removed by proteolytic cleavage. The translocation results in a Lyt-10 protein lacking this region and thus possessing constitutive transactivating activity. The second gene is bcl-3, which is overexpressed in B-cell chronic lymphocytic leukemia. The product of this gene has homology with I~:B, containing six "ankyrin-like" repeats, and can inhibit the DNA-binding activity of both NFKzB and KBFI in vitro, 277 although its function in vivo is as yet undetermined. This extended protein family is likely to prove of continuing interest for some time, as its complex interactions are unraveled and the functions of the various family members are determined.

F. myb and ets v-myb Two chicken retroviruses, avian myeloblastosis virus (AMV) and E26, carry the oncogene v-myb, a truncated version of the cellular protooncogene c-myb, 278'279

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which encodes a nuclear protein of Mr 75-80 kDa 279-281 expressed in immature, but not in differentiated hematopoietic cells. 282 Both v-Myb proteins lack the N-terminus and most of the first of three N-terminal repeats responsible for DNA-binding, plus a large part of a C-terminal negative regulatory domain. 283'284 In addition, in E26, v-Myb is expressed as a fusion protein with v-Ets. When chicken bone marrow cells are transformed with v-Myb their differentiation is blocked and they have the phenotype of myeloid precursors, 285 while overexpression of c-Myb prevents the induction of differentiation of cultured erythroleukemia cells. 286Furthermore, transformation ofmyeloid cells with v-Myb leads to dedifferentiation, thus myb expression appears incompatible with a differentiated state. v- and c-Myb bind specifically and with similar affinity to the sequence PyAACG/TG (Py = pyrimidine) and activate transcription from reporter genes linked in cis to this motif.283'284'287The structure of the DNA-binding domain has been determined, and comprises a tryptophan-based HTH-like domain. 288'289Myb activates miml, encoding a promyelocyte-specific secretory protein, 29~and it may also instigate an autocrine growth loop by increasing expression of insulin-like growth factor- 1 and its receptor. TM Other targets of Myb activation are not known. Phosphorylation at a number of serine residues in the N-terminus of c-Myb by casein kinase II has a negative effect on transcriptional activation, by preventing DNA-binding. The region ofc-myb containing these residues is deleted from v-myb and activated c-myb genes, 124 thus transformation may be the result of expression of a Myb protein which inappropriately activates transcription because it cannot be prevented from binding to DNA. Alternatively, the disruption of the C-terminal negative transcriptional domain, which results from some retroviral integrations, can also cause transformation, while retroviral vectors carrying C-terminally truncated Myb will immortalize myelomonocytic precursors, albeit at low frequency. Most probably optimal transforming ability requires both truncations. It is not clear whether loss of part of the first repeat of DNA-binding domain is important for transformation; however, a subtle effect on DNA-binding specificity cannot be excluded. Further elucidation of the mechanism by which C-Myb is rendered oncogenic and the genes responsible for the dedifferentiated state of transformed cells should be most illuminating.

The Rapidly-Growing ets Family The second oncogene present in the genome of avian ieukosis virus, E26, which also bears v-myb, is v-ets and both genes are necessary for it to induce erythroblast o s i s . 292-294 The product of the chicken protooncogene, c-ets-l, from which it is derived, is a member of a family of nuclear phosphoproteins of short half-life. 295-297

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Related proteins all sharing a highly conserved region of amino acids with overall positive charge at the C-terminus (the ETS-domain) 298 are also present in insects and other vertebrates. 299-3~ The c-ets-1 gene binds specifically to a cis-acting element centered on the motif GGAA, found in the long terminal repeat (LTR) of the Moloney murine sarcoma virus 3~ and HTLV 1,3~ in the closely related PEA3motif, in the polyoma virus enhancer, in the promoters of extracellular matrix-degrading enzymes, 3~ and in the T-cell receptor-c~ enhancer. 3~ Ets activates transcription from these sequences and, in the polyoma enhancer, functions c90 peratively with members of the Fos-Jun family which bind to an adjacent AP-1 site. 3~ Juxtaposed AP-1 and PEA3 sites are a feature of a number of oncogene-responsi~,e promoters 3~ and may represent an important target for extranuclear oncogenes such as ras. Phosphorylation of c-ets-1 and -2 by a cellular kinase, possibly the protooncogene product rafol, inhibits DNA-binding. 297'3~ The expression of c-ets-1 is high in quiescent T cells and repressed on T-cell activation, 3~ while it is induced in proliferating endothelial cells. 31~ The v-Ets oncoprotein has limited independent transforming ability, and coexpression with Myb has an additive effect; however, the E26 Gag-Myb-Ets fusion protein, has much greater potency, suggesting that it possesses some additional properties over those of the individual oncoproteins which are its constituent parts.311 The genes for two other ETS-domain proteins, fli-1 and spi-1 are activated by integration of two different retroviruses, 3j2'313 while other family members interact with the serum-response factor (SRF) on the promoter of serum-responsive immediate early genes such as c-fos. 314'315 The Ets family members regulate a number of viral and cellular genes and may have important roles in both differentiation and responses to external growth signals, while the expression of c-ets-1 in proliferating endothelial cells and its regulation of extracellular matrix-degrading enzymes may indicate an important function in production of tumor metastases.

G. The

skiOncogene

The nuclear oncogene, v-ski, is less well-characterized than those described above. This oncogene was isolated from an avian leukosis virus in culture, and the protein it encodes has several features which suggest it may be a transcription factor. 316 The function of the c-ski protooncogene from which it is derived is unknown; however transformation of quail embryo cells with v-ski causes them to undergo myogenic differentiation, suggesting a relationship to the MyoD family of myogenic proteins, 196'317 while ski transgenic mice have excessive muscle development due to fiber hypertrophy. 318

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H. Tumor Suppressor Genes and Transcription With the exception of the WT gene, the majority of the discussion so far has been concerned with dominant oncogenes, i.e. genes which cause cellular transformation as a result of deregulated expression or mutational activation. The opposing class of genes are the recessive oncogenes or tumor suppressor genes, whose products act to inhibit cell proliferation and promote differentiation. Transformation occurs when the function of both genomic copies of such genes is lost, either by homozygous deletion, mutation, or a combination of the two. While an increasing number of potential tumor suppressor gene loci has been identified, few are well characterized; however, the products of three of them are localized in the nucleus. One, WTI, has been described already, since it is a member of the ZF protein family, while evidence is accumulating that the others, pRb, and p53, also function as regulators of transcription.

The Retinoblastoma Gene Product The gene for susceptibility to the embryonic ocular tumor retinoblastoma, rb, was the first tumor suppressor gene to be isolated. 319-321 It is homozygously inactivated by deletion or mutation in a high proportion of retinoblastomas and encodes a nuclear phosphoprotein with DNA-binding activity. 321 rb was subsequently shown to have a wider role in neoplasia, being inactivated in a proportion of other human cancers. 322-325 In addition, the pRb product is sequestered by various DNA virus tumor antigens, including Adenovirus E la, SV40 virus large T, and papillomavirus E7 proteins, 326-328 suggesting that disruption of normal pRb function may be advantageous to the process of viral infection. In normal cells the phosphorylation state of pRb varies with the cell cycle and in nuclear extracts it can be shown to complex with a number of cellular proteins. 329-331 One of these is the transcription factor E2F(DRTF) that transactivates the promoters of several proliferation-related genes. 332-334 During G 1 phase of the cell cycle, E2F forms a transcriptionally inactive DNA-binding complex with underphosphorylated pRb. 332 At the G I/S transition phosphorylation ofpRb occurs and the complex dissociates. E2F then becomes associated with cyclin A via p107, an Rb-related protein, 335 in a still inactive complex, only becoming functional when it is released by phosphorylation of cyclin A on entry into G2/M. In resting cells, E2F DNAbinding activity is not detected owing to binding of the pRb/E2F by a putative additional factor. 332 By complexing with pRb and p107, the DNA-viral oncoproteins liberate E2F in its active form. 336 pRb represses the promoters of genes involved in proliferation, such as c-fos and c-myc which contain potential binding sites for E2F. 337'338In both of these promoters the E2F site is necessary for pRb-mediated repression. This is not the sole way in which pRb influences transcription, for in addition to repressing transcription of the c-myc promoter, the c-Myc protein is among the nuclear factors which complex

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with pRb as detected by affinity chromatography. 338 Furthermore, besides inactivating growth stimulating proteins, pRb activates transcription of the gene for the growth inhibitory factor TGF-]]2. This activation is dependent upon an ATF site in the promoter which preferentially binds the LZ protein ATF-2, while an ATF-2GAL4 fusion protein confers pRb inducibility upon a promoter containing a GAL4-binding site. Moreover, interaction between ATF-2 and pRb has been detected in vitro using a pRb column. 339 Thus it appears that pRb has diverse functions in transcriptional regulation of cell proliferation.

p53 The p53 gene is probably the most frequent target of spontaneous mutation in human tumors. Its product, like pRb, is a nuclear phosphoprotein with DNA-binding activity, 34~which is sequestered by DNA virus transforming proteins, including Adenovirus E IB, SV40 T, and papillomavirus E6. 341-344 Unlike pRb, the p53 protein exists in cells as dimers or tetramers. While the normal p53 protein suppresses proliferation, 345-347 mutant forms of p53 have dominant transforming activity, by virtue of their ability to multimerize with the wild-type protein. 348 Structural features of the p53 protein, including its acidic N-terminal domain and basic C-terminal region, are consistent with it being transcriptionally active. 349 Also, a fusion protein of the whole of p53, or its acidic domain with the DNA-binding domain of GAL4, has transactivating activity. In this assay, mutant p53-GAL4 fusion proteins are inactive. 35~ In addition wildtype p53 represses the transcription of several proliferation-related genes by an unknown mechanism. 353-355 Two distinct, specific DNA-binding elements for p53 have been identified" one was found in a GC-rich region of the polyoma virus (SV40) enhancer, and the other is a direct repeat of a TGCCT motif separated by a short spacer. 356'357 These elements occur in close proximity to one another, as a p53-responsive 5' positive enhancer, in the muscle-specific creatine kinase (MCK) gene. 358 Mutant p53 protein fails to activate transcription from this sequence and inhibits transactivation of the polyoma virus promoter by wild-type p53 when the two are coexpressed, probably by preventing DNA-binding of the latter. 359 These findings strongly suggest that the transforming ability of mutant p53 may be due to its interference with the normal transcriptional functions of the wild-type protein.

IV. C O N C L U S I O N It is clear that transcriptional deregulation as a result of abnormalities in the structure or expression of transcriptionally active proteins plays an important role in the genesis of cancer. This is true both for virally induced tumors in animals and for spontaneous human malignancies. Many aspects of transcription factor function

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may be affected and the abnormal or overexpressed proteins may have either dominant or recessive oncogenicity. With recent evidence indicating that both p53 and pRb act via transcriptional regulation, it is apparent that transcriptional deregulation is a very common and important feature of human tumors. A number of unifying themes emerge regarding the mechanisms by which transcription factors are converted into oncogenes. For example, immediate early genes, such as fos, myc, and evi-l, normally expressed only briefly in response to growth stimuli, can become oncogenic as a result of genetic changes which cause loss of normal regulation and constitutive expression. This may result from gene amplification or from translocation or viral transduction leading to expression under the control of a constitutively active promoter. Similar mechanisms may activate developmentally regulated transcription factors, both those expressed during early development of the tissue in which they are oncogenic and those normally active in different tissues. In T-cell leukemias, activation is typically a result of juxtaposition of such a developmental gene to a T-cell receptor gene locus by translocation, while in B-cell tumors the immunoglobulin loci are involved. Other translocations generate chimeric proteins, usually combining a potent transcriptional activation domain from a constitutively active protein, such as that of the bHLH E2 gene products with a developmentally regulated transcription factor and presumably activating genes normally expressed at an earlier stage of development. Transcription factors encoded by tumor suppressor genes may be inactivated by deletion or point mutation, allowing uncontrolled cell proliferation. In the case of p53, the mutant protein is a dominant negative oncoprotein which directly interferes with the function of its normal counterpart. Retroviral oncogenes often exhibit more subtle changes in structure, with a combination of small internal deletions and point mutations, which makes elucidation of the precise mechanism of oncogenic conversion more diMcult. For some, such as v-ErbA and v-Rel, the oncoprotein acts in a dominant negative fashion, interfering with the function of endogenous proteins which are responsible for differentiation. Others, including v-Jun and the v-Myb protein produced by AMV, have been rendered oncogenic by the deletion of a transcriptional repressor domain, thereby generating a constitutive transcriptional activator. From studies of viral oncogenes, it is clear that mutations affecting only one or a few amino acids can have drastic effects on transcription factor function. Such changes are not detectable by cytogenetic analysis and may well be important in human malignancies. In addition to the widespread occurrence of point mutations in the p53 gene, the presence of internal deletions of the tal-I gene in 25% of T-ALL and of point mutations in the WT gene in a proportion of sporadic Wilms' tumors supports this notion. Although much of the evidence for the involvement of transcription factors, apart from p53 and pRb, in human cancer has come from the leukemias owing to their relative ease of study. It is likely that they will prove equally important in the genesis of solid tumors.

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The identification of target genes regulated by the transcription factors involved in neoplasia should provide an improved understanding of aberrant phenotype of tumor cells. Furthermore, detection of the abnormal transcription factors may aid both in the pathological diagnosis of specific tumors and in the assessment of prognosis. For example, immunohistochemical detection of p53 protein, which is only demonstrable in tissue sections when abnormal, may be helpful in mesothelial proliferations in differentiating reactive hyperplasia from malignant mesothelioma, 36~ while in breast carcinoma detectable p53 expression is associated with early relapse and shorter survival. 361 In contrast, detection of oestrogen and progesterone receptor expression in breast carcinoma cells indicates both a good prognosis and predicts a response to hormonal manipulation therapy. In the future, demonstration of tissue-specific transcription factors may provide a more reliable means of determining the cell lineage of a poorly-differentiated tumor and thus lead to implementation of the most appropriate treatment. This may be particularly relevant to soft tissue tumors, in which the tumor cell type may be difficult to determine without resort to electron microscopy. Demonstration of, for example, muscle-specific factors such as MyoD in tumor cells, which do not show expression of muscle structural proteins, could be helpful in reaching a diagnosis and other factors might indicate neural, adipocytic, or vascular origin. Finally, a detailed understanding of transcription factor structure and function and their role in cancer may enable the development of drugs targeted against specific factors (other than the steroid hormone receptor family, which are already frequent targets of therapy in a range of tumors), thereby interfering with vital steps in the pathways leading to uncoordinated proliferation. In all, much will be learned and much is to be gained from the study of transcription factors in cancer.

ACKNOWLEDGMENT The author would like to thank Dr. Colin Goding of Marie Curie Cancer Research for help and advice in the preparation of this manuscript.

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333. Chittenden, T.; Livingston, D. M.; Kaelin, W. G.Jr. The T/E IA-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell 1991, 65, 1073-1082. 334. Mudryj, M.; Hiebert, S. W.; Nevins, J. R. A role for the adenovirus inducible E2F transcription factor in a proliferation dependent signal transduction pathway. EMBO J. 1990, 9, 2179-2184. 335. Mudryj, M.; Devoto, S. H.; Hiebert, S. W.; Hunter, T.; Pines, J.; Nevins, J. R. Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A. Cell 1991, 65, 1243-1253. 336. Chellappan, S.; Kraus, V. B.; Kroger, B.; et al. Adenovirus EIA, simian virus 40 tumor antigen, and the human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. Proc. Natl. Acad. Sci. USA 1992, 89, 4549-4553. 337. Robbins, P. D.; Horowitz, J. M.; Mulligan, R. C. Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature (London) 1990, 346, 668-671. 338. Rustgi, A. K.; Dyson, N.; Bernards, R. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product. Nature (London) 1991, 352, 541-544. 339. Kim, S.-J.; Wagner, S.; Liu, F.; O'Reilly, M. A.; Robbins, P. D.; Green, M. R. Retinoblastoma gene product activates expression of the human TGF-~52 gene through transcription factor ATF-2. Nature (London) 1992, 358, 331-334. 340. Lane, D. P.; Gannon, J. Cellular proteins involved in SV40 transformation. Cell. Biol. Int. Rep. 1983, 7, 513-514. 341. Sarnow, P.; Ho, Y.-S.; Williams, J.; Levine, A. J. Adenovirus Elb-58kd tumor antigen and SV40 tumor antigen are physically associated with the same 54-kDa cellular protein in transformed cells. Cell 1982, 26, 387-394. 342. Lane, D. P.; Crawford, L. V. T antigen is bound to host protein in SV40-transformed cells. Nature (London) 1979, 278, 261-263. 343. Linzer, D. I. H.; Levine, A. J. Characterization of a 54Kda cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal cells. Cell 1979, 17, 43-52. 344. Werness, B. A.; Levine, A. J.; Howley, P. M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990, 248, 76-79. 345. Baker, S. J.; Markowitz, S.; Fearon, E. R.; Wiiison, J. K. V.; Vogelstein, B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science 1990, 249, 912-915. 346. Diller, L.; Kassel, J.; Nelson, M. A.; et ai. p53 functions as a cell cycle control protein in osteosarcomas. Mol. Cell. Biol. 1990, i0, 5775-5781. 347. Mercer, W. E.; Shields, M. T.; Amin, M.; et al. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53. Proc. Natl. Acad. Sci. USA 1990, 87, 6 i 66-6170. 348. Hinds, P. W.; Finlay, C. A.; Quartain, R. S.; et al. Mutant p53 cDNAs from human colorectal carcinomas can cooperate with Ras in transformation of primary rat cells. Cell Growth Differ. 1990, 1,571-580. 349. Pennica, D.; Goeddel, D. V.; Hayflick, J. S.; Reich, N. C.; Anderson, C. W.; Levine, A. J. The amino acid sequence of murine p53 determined from a cDNA clone. Virology 1984, 134, 477-482. 350. Fields, S.; Jang, S. K. Presence of a potent transcription activating sequence in the p53 protein. Science 1990, 249, 1046-1049. 351. Raycroft, L.; Wu, H.; Lozano, G. Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science 1990, 249, 1049-1051. 352. Raycroft, L.; Schmidt, J. R.; Yoas, K.; Hao, M.; Lozano, G. Analysis of p53 mutants for transcriptional activity. Mol. CeiL Biol. 1991, 11, 6067-6074. 353. Santhanam, U.; Ray, A.; Sehgal, P. B. Repression of the interleukin 6 gene promoter by the p53 and retinoblastoma susceptibility gene product. Proc. Natl. Acad. Sci. USA 1991, 88, 7605-7609. 354. Ginsberg, D.; Mechta, F.; Yaniv, M.; Oren, M. Wild-type p53 can down-modulate the activity of various promoters. Proc. Natl. Acad. Sci. USA 1990, 88, 9979-9983.

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355. Mercer, W. E.; Shields, M. T.; Lin, D.; Appella, E.; Ullrich, S. J. Growth suppression induced by wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression. Proc. Natl. Acad. Sci. USA 1991, 88,-1958-1962. 356. Bargonetti, J.; Friedman, P. N.; Kern, S. E.; Vogelstein, B.; Prives, C. Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell 1991, 65, 1083-1091. 357. Kern, S. E.; Kinzler, K. W.; Bruskin, A.; et al. Identification of p53 as a sequence specific DNA-binding protein. Science 1991, 252, 1708-1711. 358. Zambetti, G. P.; Bargonetti, J.; Walker, K.; Prives, C.; Levine, A. J. Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. Genes Dev. 1992, 6, 1143-1152. 359. Kern, S. E.; Pietenpol, J. A.; Thiagalingam, S.; Seymour, A.; Kinzler, K. W.; Vogelstein, B. Oncogenic forms of p53 inhibit p53-regulated gene expression. Science 1992, 256, 827-830. 360. Kafiri, G.; Thomas, D. M.; Shepherd, N. A.; Krausz, T.; Lane, D. P.; Hall, P. A. p53 expression is common in malignant mesothelioma. Histopathoi. 1992, 21, 331-334. 361. Sawan, A.; Randall, B.; Angus, B.; et al. Retinoblastoma andp53 gene expression relate to relapse and survival in human breast cancer: An immunohistochemical study. J. Pathoi. 1992, 168, 23-28.

LOSS OF CONSTITUTIONAL HETEROZYGOSITY IN H UMAN CANCER" A PRACTICAL APPROACH

Jan Zedenius, G0nther Weber, and Catharina Larsson

I. I!. 111. IV. V.

VI. VII. VIII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer: A G e n e t i c Disease . . . . . . . . . . . . . . . . . . . . . . . . . . Oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor Suppressor Genes . . . . . . . . . . . . . . . . . . . . . . . . . .

280 280 281

. . . . . . . .

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Principles of Loss of Heterozygosity . . . . . . . . . . . . . . . . . . . . . . A. D N A P o l y m o r p h i s m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetic Alterations Detectable by L O H . . . . . . . . . . . . . . . . . .

283 283 285

C. Considerations when Performing L O H Studies . . . . . . . . . . . . . . The R e t i n o b l a s t o m a Paradigm . . . . . . . . . . . . . . . . . . . . The M E N Examples . . . . . . . . . . . . . . . . . . . . . . . . . L O H in T u m o r Progression . . . . . . . . . . . . . . . . . . . . . A. The Colorectal T u m o r Example . . . . . . . . . . . . . . . . . . . . . . B. The G l i o m a E x a m p l e . . . . . . . . . . . . . . . . . . . . . .

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Advances in Genome Biology Volume 3B, pages 279-303. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

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IX. Deletion Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Conclusions: The Researcher and the Tumor . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

295 297 298 299

INTRODUCTION

Human cancer arises from irreversible alterations within the genetic content of a single cell that are inherited by its daughter cells. These mutations may activate the so-called proto-oncogenes, causing their overexpression or hyperfunction, and thereby lead to uncontrolled cell growth. Additionally, it has now become evident that, apart from this activation of oncogenes, loss of genetic information may contribute to cancer development. First observed in rare heritable cancer syndromes and eliminating genes that exert cellular control functions, "genetic losses" have now also been found in frequent and non-inherited cancers, as the initial step of tumorigenesis and contributing to tumor progression. Screening for genetic losses is therefore a generally applied method for the characterization of cancer. Apart from methods related to classical cytogenetics, new molecular tools developed in the past decade have dramatically refined the resolution and facilitated the characterization of the tumor cell genotype. DNA markers have been developed that distinguish between the alleles of homologous chromosomes, enabling the investigator to monitor losses of single alleles, and to determine their parental origin. Thus, detection of"loss of heterozygosity" (LOH) has become a widespread method in cancer research, and in the search for the specific genes involved in the genesis and progression of several tumors. In the following, we will give the background to the usage of the LOH technique, try to describe some major technical and interpretative pitfalls when using it, and to give examples of genetic diseases where LOH has decisively contributed to the description of the mechanisms behind them.

II.

CANCER: A GENETIC DISEASE

The hypothesis that cancer is a malady of genes is based on the following observations:

9 Familial aggregation of specific types of tumors. This has been well documented, and familial clustering has been reported in more than 200 different neoplasms or syndromes. 1'2 9 Constitutional chromosomal aberrations that confer an increased risk of developing specific tumors. For example, patients suffering from the rare eye tumor in children, retinoblastoma (RB), sometimes show a constitutional deletion of chromosomal region 13q 14. 3'4

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9 Chromosomal rearrangements in tumor tissue. This was first suggested by Boveri in 1914. 5 Development of the QFQ-banding technique in 1970 permitted detailed karyotype analysis. 6 Specific chromosomal aberrations could then be regularly associated with certain types of neoplasias, the classical example being the Philadelphia chromosome translocation in chronic myeloid leukemia. 7 9 Carcinogenic agents are mutagenic. Different tumor forms have been associated with environmental factors, such as smoking, sunlight, and dietary components. Since most carcinogenic agents also are known to be mutagenic in experimental systems, it is in agreement with the idea that cancer can arise from somatic mutations. 8 9 Constitutional deficiencies in the systems for repair of DNA damage predispose to tumor development. 9 Patients with xeroderma pigmentosum are deficient in their repair of UV-induced DNA damage. They frequently develop skin cancers in sunlight exposed areas of the body, and their cells demonstrate increases of UV-induced mutations in vitro. 9 Identification of two classes of genes involved in tumor development. The oncogenes need to be somatically activated to contribute to tumor growth, while the tumor suppressor genes exert their phenotypic effect by their functional inactivation.

!11. O N C O G E N E S Initially, o n c o g e n e s were identified as t r a n s f o r m i n g c o m p o n e n t s of retroviruses. ~~ Human nontransforming counterparts were then discovered, the protooncogenes, generally believed to be involved in cell proliferation and differentiation. As a result of specific genetic alterations they may gain the capacity to transform a cell into a neoplastic state. These genes act dominantly on the cellular level since only one of the two gene copies has to be activated to transform a cell. The effects are mediated either through overexpression of the normal, or expression of an aberrant protein, l~ These phenomena may be caused by gene amplification, translocation and/or deletion of chromosomal regions, or point mutations within the actual gene. The oncogenes are classified according to the subcellular localization and biochemical function of their products. Some are, for example, found in the nucleus recognized as transcription factors, while others are found on the outside of the cell surface (i.e., growth factors bound to specific membrane receptors).

IV. T U M O R SUPPRESSOR GENES In 1914, Boveri first suggested the existence of "inhibiting chromosomes," with the function to inhibit tumorigenesis. 5 The idea that certain familial cancer syn-

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MUTATION 1

~'

MUTATION 2

CANC~ER

Figure 1. Model of the recessive mechanism of tumorigenesis. In patients with heritable cancer the first mutation is present in all cells. These are therefore predisposed to tumor development, but appear normal. In order for the tumor to develop, a second mutation which eliminates the normal allele/function (+) in one cell, has to occur. Even if the mutation frequency is low, the number of cells is so large that such a mutation most probably will occur in at least one cell. Hence, predisposed individuals typically develop multiple tumors with comparatively early age of onset. However, in sporadic cases, the two mutations must occur in the same somatic cell. This is less likely, and explains why sporadic tumors usually are single and occur later.

dromes result from additional somatic mutations in the individual who already carries a germline mutation was proposed by de Mars in 1970.12 Shortly thereafter, Knudson's epidemiological analyses of RB led him to propose that development of the condition requires two independent mutations. ~3 This theory was later generalized, 14 and is usually referred to as the "two-mutation model" for tumorigenesis (Figure 1). The first evidence for the existence of tumor suppressor genes came from studies of somatic cell hybrids. In 1969, Harris showed that malignancy could be suppressed by the fusion of malignant and nonmalignant cells. 15 This inspired several research groups to search for tumor suppressor genes. The first and probably the best studied example is the RB gene (rb). The cloning of this gene permitted the

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experiment that provided direct evidence for the existence of a tumor suppressor gene, as the introduction of a cloned wild-type rb gene via a retroviral vector into RB cells resulted in suppression of the malignant phenotype. 16 Several other tumor suppressor genes have since been localized and cloned. Examples are the recently cloned genes predisposing to familial adenomatous polyposis (apc gene), 17 neurofibromatosis type 1,18-2~and neurofibromatosis type 2, 21 which were first mapped by linkage analysis in families segregating the disease. 22-26 The Wilms' tumor gene 27'28 was mapped by identification of constitutional chromosomal deletions. 29 Studies of genetic losses in colon cancers led to the cloning of the dcc gene, and also identified the p53 gene to be involved in tumor progression. 3~ The p53 story has truly been puzzling: it was first considered to be a tumor antigen, then a nuclear oncogene, and presently considered as a tumor suppressor gene. 32-34 In the response of a normal cell to DNA damage, the genome guarding function of the wild-type p53 is induced. 35 This leads to cell cycle arrest in G1, enabling DNA repair or inducing apoptosis (cell death). 36 Disturbances in this function may lead to tumor growth. It is now clarified that the p53 gene can also be involved in tumor initiation. In families with the rare Li-Fraumeni syndrome, associated with an increased risk for different cancers, the disease was found to segregate with a point mutation within the p53 gene. 37'38 In all examples of suppressor genes mentioned here, as well in the mapping of others, such as MEN 1, meningioma, and breast carcinoma, a useful tool has been identification of chromosomal alterations by studying the LOH phenomenon in tumor tissue. 39-44

V. PRINCIPLES OF LOSS OF HETEROZYGOSITY The gateway to the studies of LOH was the chromosomal rearrangements seen in cytogenetic studies (i.e., karyotyping) of tumors. Karyotyping makes it possible to detect several types of aberrations, such as numerical deviations, translocations, inversions, large deletions, homogeneously staining regions, and double minutes (Figure 2). However, the method has several drawbacks. Since the cells must be studied in metaphase, the method demands growing cells. Karyotyping of solid tumors often involves tissue culturing, which may lead to selection of cells with a growth advantage in vitro, or the introduction of secondary aberrations that were not present in the primary tumor. Furthermore, the level of resolution is rather limited. A general molecular genetic approach to study chromosomal rearrangements in tumors was introduced in 1983 by Cavenee and co-workers. 45 Here, by comparing constitutional and tumor genotypes, loss of genetic material in retinoblastomas were identified. This method has since been widely applied to several other tumor forms. Comprehensive summaries of allele losses frequently found in different

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Figure 2. Examplesof chromosome abnormalities, demonstrable by karyotyping.

tumors are available. 46'47 In the following we discuss how to detect allele losses, what significance they have for tumor development, and the genetic events they may reflect.

A. DNA Polymorphisms Sequence variations in the human DNA occur every 300 to 500 base pairs (bp), within genes or between them. 48'49 There are two main groups of DNA polymorphisms: single bp alterations and variations in the number of repeat elements. In approximately 5% of the cases, single bp alterations involve the cleavage site of a restriction endonuclease. These enzymes have bacterial origin, and recognize specific bp combinations, usually four or six bases, where they cleave the DNA. Polymorphic restriction sites result in restriction fragment length polymorphism (RFLP), i.e., different lengths of the restriction fragments. These fragments can be considered as alleles for the RFLP locus, and segregate in a codominant manner in families. In case of heterozygosity, one of the fragments represent the maternally, and the other the paternally derived chromosome. The other main group of DNA polymorphisms are due to variations of the number of tandem repeats: the variable number of tandem repeats (VNTRs) usually reflect variations in repeat motifs of 10 to 20 bases per unit, 5~ while the so called micro-satellite markers reflect sequence variations of shorter repeats, usually consisting of two to four nucleotide motifs, e.g., "CA-repeats". 52'53 The VNTRs

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and short microsatellite repeats are scattered over the entire human genome. Each array of repeats is flanked by unique DNA sequences. These unique (single copy) loci located close to a highly polymorphic repeat, constitute a set of very useful DNA markers. Detection of RFLPs and VNTRs demands Southern blot analysis. 54 In short, the cleaved DNA fragments are size-separated by agarose gel electrophoresis. After denaturation, which makes the DNA single stranded, the fragments are transferred to a DNA binding filter. The filter is then used for hybridization with radio labeled fragments of genomic or complementary DNA probes. The hybridizing fragments are subsequently detected by autoradiography. Detection of micro-satellite repeats requires very small amounts of DNA, since the alleles are amplified by a polymerase chain reaction (PCR), and then separated by polyacrylamide gel electrophoresis. PCR is a cyclic reaction used for specific amplification of DNA fragments between two known sequences. Each cycle includes denaturation, annealing of primers, and elongation of the strands. The primers are known complementary sequences used to direct the DNA polymerase enzyme, and the reaction is repeated in 2 0 - 4 0 cycles. The method is particularly useful for amplification of short DNA fragments (<1 kb), DNA from paraffin embedded tissues, and when only small amounts of cells are available.

B. Genetic Alterations Detectable by LOH LOH can only be detected when the DNA marker (probe) is informative, that is, when the alleles show two separate bands on the autoradiogram. RFLP analysis demands a proper choice of restriction enzyme for the DNA probe in question. 55 In general, micro-satellite markers have the highest information content (sometimes more than 10 distinguishable alleles), followed by VNTR probes. In the initial retinoblastoma studies by Cavenee et al., cloned DNA segments homologous to arbitrary sequences along chromosome 13, and which revealed RFLPs, were used to distinguish the alleles representing the two parental chromosomes. 45 The constitutional and tumor genotypes were compared at several loci, and the results indicated some of the putative second mutations, eliminating the normal function of a tumor suppressor gene (Figure 3). LOH on all informative loci along a chromosome is usually interpreted as loss of one chromosome complement (Figure 3a and b). From the intensity of the remaining band/allele, the number of copies of the remaining chromosome is determined. For example, a doubling of the remaining allele indicates that two copies of the chromosome have co-segregated during mitosis (Figure 3b). When LOH is detected at one or a few loci, in combination with others on the same chromosome showing retained heterozygosity, this indicates a deletion or mitotic recombination (Figure 3c and d). Again, this distinction is made by measuring the intensity of the remaining allele, i.e., by visual inspection and densitometry analy-

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JAN ZEDENIUS, GONTHER WEBER, and CATHARINA LARSSON

Figure 3. Examples of different putative second mutations eliminating the function of a tumor suppressor gene. (a) loss of a whole chromosome (b) imperfect cell division with co-segregation of the two copies of one of the homologs, or loss of one chromosome and duplication of the remaining (c) chromosome deletion (d) mitotic recombination (e) point mutation. Cytogenetic analysis would only detect a and possibly c, while molecular analysis can detect both a, b, c, d, and maybe e (if an intragenic marker is used). sis. Point mutations are usually not detectable by LOH studies, unless they result in alteration of a restriction site (Figure 3e). Some of these genetic alterations are demonstrable also by karyotyping, i.e., loss of a whole chromosome or large deletions. In contrast, chromosome loss coupled with duplication of the remaining chromosome, and also mitotic recombination, result in two apparently intact chromosomes, and can therefore not be detected by karyotyping. On the other hand, alterations such as translocations and inversions are readily detectable by karyotyping, while LOH is not feasible in this respect.

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When using the technique for identification of LOH, one should be aware of additional genetic alterations that might occur. For example, trisomy may be identified on the autoradiogram as a doubled signal intensity for one of the alleles, but without loss of the other. In addition, gene amplification (e.g., of an oncogene) can be revealed as increased signal intenslty for one or both alleles. 56

C. Considerations when Performing LOH Studies The experience in LOH studies so far obtained is mainly based upon Southern blot analysis. Basic requirements for this technique are the availability of fresh frozen tumors and corresponding constitutional DNA (e.g., from the patients leukocytes, fibroblasts, or normal tissue surrounding the tumor). The detection of micro-satellite repeats by CR makes it possible to analyze smaller amounts of DNA, and even DNA from paraffin embedded tissues. Today, more than 800 well-mapped, highly polymorphic CA-repeats evenly distributed over the human genome are available. 57 The disadvantages using the micro-satellite techniques are not yet well-defined. However, the comparatively long experience from LOH studies using Southern blotting gives us the opportunity to point out some pitfalls: 1. Loss of genetic material must be distinguished from gain (Figure 4A, G). A frequent finding is that the alleles in the tumor lane differ in intensity relative to the constitutional alleles (i.e., allelic imbalance). This may indicate a true LOH, and/or doubling or amplification of the other allele. This is best distinguished if the amount of DNA loaded in each gel lane is even. From our own experience, we recommend that tumor DNA concentration judgement is based on both spectrophotometry as well as agarose gel electrophoresis evaluation. Standardization between the lanes can be done by comparison with a marker (a) with ascertained retain of heterozygosity, (b) detecting alleles of similar size, and (c) hybridized to the same filter. 2. Correct exposure time of the autoradiogram is crucial. For instance, a short exposure time in combination with allelic imbalance and less DNA in the tumor lane, may give rise to false LOH. Similarly, overexposure may hide an existing allelic loss by saturation of the X-ray film (Figure 4F). 3. Degraded tumor DNA may mimic LOH. This must be taken into account when there is a considerable size difference between the two alleles, and even more so if one of them is very large (Figure 4D). 4. Partial restriction cleavage must be avoided, as this may also mimic LOH (Figure 4E). Note that restriction cleavage of DNA from solid tissue is less efficient than that in DNA from leukocytes. Furthermore, differences in methylation pattern must be taken into consideration, as the restriction enzymes differ in methylation sensitivity. Cleavage control should therefore be performed with parallel reactions including, for example, lambda DNA, in order to monitor specific phage band patterns on agarose gels.

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Figure 4. Examples of true LOH, and also some false positive and false negative results of LOH. (A) True LOH, detected in a sporadic breast carcinoma by RFLP analysis. (B) True LOH in a familial breast carcinoma, detected by PCR-amplification of a micro-satellite marker. (C) Vector contamination of constitutional DNA from a patient with a thyroid tumor. Where are the alleles? (D) False positive LOH due to degradation and partial cleavage of the tumor DNA. (E) False positive LOH due to partial cleavage of the tumor DNA. (F) False negative LOH due to overexposure of the autoradiogram. (G) Allelic imbalance reflecting trisomy 15 in a mouse plasmacytoma. 5. Plasmid contamination of DNA may also make reading of the results more difficult (Figure 4C). Where are the alleles? Are there constant bands? Are there bands from vector contamination? If the latter is suspected, it is possible to overcome the problem by isolating the insert of the probe before hybridization. 6. Contamination of normal tissue DNA in the tumor may cause a false negative result. Many tumor forms show a high grade oflymphocyte infiltration and/or large amounts of fibrous tissue. Others may show a growth pattern with sprouting of tumor cell formations into surrounding normal tissue. Therefore, it is suggested that a representative section is cut out from the tumor piece to be analyzed, and subjected

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to histo-pathological examination. Tumor tissue containing less than 40-50% tumor cells is therefore not well-suited for studies of LOH. When using microsatellite markers, a higher proportion of tumor cells is required. The amplification obtained by PCR is not linear, which may result in selection for normal tissue DNA. 7. Clonality of the tumor cells must be considered. The alteration you are about to detect must be present in at least 40-50% of the cells studied, thus tumors of a polyclonal origin are less recommendable for LOH studies. For the same reason, subclonal events only present in a fraction of the tumor cells may escape detection by LOH analysis.

VI.

THE RETINOBLASTOMA PARADIGM

The first tumor suppressor gene identified was the rb gene responsible for a rare childhood tumor of the eye, retinoblastoma (RB). Ever since, RB has served as prototype for theories of the genetic mechanisms of cancer, and must be considered as the empirical proof for the two-mutation model proposed by Knudson. 13 The first steps in the cloning of the rb gene was the localization of the disease locus to chromosomal region 13q 14 by cytogenetic methods. A small percentage of RB patients were found to have constitutional deletions overlapping the region, 3"4 and tumor cells sometimes showed similar rearrangements of chromosome 13. 58 Family studies showed an association between the disease and an unbalanced translocation lacking the 13q14 region. 59 However, most RB families were cytogenetically "normal," and in tumors the most frequent cytogenetic finding was isochromosome 6. The close linkage of the disease gene to the genetic locus for esterase D (ESD) on 13q 14, provided the possibility to study LOH on the protein level by distinguishing electrophoretic variants of ESD. 6~ One study described a tumor that contained one chromosome 13 and had no ESD-activity, while the patient's constitutional level of ESD was only 50% of the normal. These findings suggested that the tumor had lost the normal chromosome 13 and provided the first experimental evidence that the rb gene is a recessive cancer gene. 61 Cavenee's original studies of LOH in these tumors introduced a general approach to study the second event in tumorigenesis according to the two-mutation model. The constitutional and tumor genotypes were compared at several chromosome 13 loci, and the results indicated some of the possible types of second mutations suggested by Knudson (see Figure 3). 45'14 LOH for chromosome 13 loci were found to occur in approximately half of all RBs. Allele losses were found in both sporadic and heritable forms of the disease, and were present in primary tumor tissue, cultured tumor cells, and tumors passaged through immunodeficient mice. They were also specific in the sense that other chromosomes tested were not found to be affected. 45'62

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RFLP markers were used to determine the parental origin of the rearranged or lost chromosome. As expected, the lost chromosome was always derived from the unaffected parent, while the one remaining carried the mutated retinoblastoma gene (i.e., originated from the affected parent). 63 The key to the cloning of the rb gene was a probe (H3-8) which was derived from a flow sorted chromosome 13 specific library. 64-67 This marker was found to be homozygously deleted in two RB tumors, and a third tumor showed an interstitial deletion with one of the endpoints in vicinity of H3-8. 68 By locus expansion of H3-8, a conserved sequence was identified. Subsequently, corresponding cDNA clones were isolated and sequenced. 65'66 Gene deletions and absent or abnormal expression of the corresponding mRNA supported the authenticity of the gene. 65-67 Finally, the experiment that provided direct evidence for the role of the rb gene in tumorigenesis could be performed. The introduction of a wild-type rb gene into tumor cells reverted the malignant phenotype. ~6

VII.

THE MEN EXAMPLES

The two-mutational model for tumorigenesis implies that it would be possible to determine where the gene for the heritable form of a certain neoplasia is situated, simply by detecting genetic alterations which might reflect the second mutational event. Such information may be available from karyotypes of cultured tumor cells, or by studies of LOH. LOH was used to localize the multiple endocrine neoplasia type 1 (MEN 1) gene, an autosomal dominant predisposition to develop tumors in the parathyroids, the neuro-endocrine pancreas and duodenum, and the anterior pituitary. 69 Cytogenetic analysis of MEN I patients had not revealed any constitutional chromosomal abnormalities that could aid to the localization of the MEN I gene. However, if MEN l-associated tumors result from unmasking of a recessive mutation according to the two-mutation model, chromosomal rearrangements in such tumors might indicate which chromosome the MEN I gene is situated on. Therefore, constitutional and tumor genotypes were compared at different RFLP loci in two brothers with neuro-endocrine pancreatic tumors who had inherited the disease from their mother. Markers on 17 chromosomes showed retained constitutional genotypes, but both tumors had lost one of the constitutional alleles at all informative loci on chromosome 11. The significance of these findings was further supported when the parental origin of the lost chromosome was determined (Figure 5). 39 In both cases, the lost alleles were always derived from the unaffected father. These findings did fit the hypothesis that the tumors resulted from elimination of the normal allele at the MEN I locus, and that the MEN I gene is a tumor suppressor gene located on chromosome 11. Subsequently, MEN 1 was found to be closely linked to the skeletal muscle glycogen phosphorylase (PYGM) locus at 11 ql 3. 39

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Figure5. Lossofthe wild-type allele in MEN1 associated tumors. The pedigree shows an MEN1 family with three affected members (black symbols). The autoradiogram below each family member shows the alleles for the Taql RFLP at the CALCA locus on chromosome 11. The genotypes with the alleles (1 and 2) for each family member are given below the autoradiogram. The affected mother is homozygous (2,2) for this marker and the father is homozygous for the alternative allele (1,1). Hence, all three children are heterozygous (1,2). Tumor tissue (insulinoma) from both affected sons (case A and B) shows loss of the paternal 1-allele.

Several subsequent studies have reported LOH for chromosome 11 markers in both sporadic and MEN 1-associated parathyroid and pancreatic tumors. 7~ While LOH at l lq13 is seen in the majority of parathyroid tumors from MENI patients, it is only found in one-third of the sporadic cases. 71'72'74 Several explanations for this difference are possible. It might reflect a difference in pathogenesis, or that only a subgroup of sporadic parathyroid tumors are caused by genetic alterations at the MEN 1 locus. Cloning of the MEN 1 gene will hopefully permit identification of minor genetic alterations (e.g., point mutations or small deletions) of the gene. Methods with higher resolution than that obtained by LOH will then be required. Although allele losses in MEN l-associated pancreatic tumors were the key to the localization of the disease locus, similar studies on medullary thyroid carcinomas (MTC) and pheochromocytomas did not provide the information that made it possible to localize the multiple endocrine neoplasia type 2A (MEN2A) locus. Epidemiological comparisons of MTC and pheochromocytomas in MEN2A patients, and in patients with sporadic disease, suggested the necessity of two mutational events for oncogenesis. 75 After plotting the age of onset as a function of the fraction of cases not yet diagnosed, it was proposed that the MEN2A gene is a recessive tumor suppressor gene, similar to what was initially hypothesized by Knudson for the rb gene. 75'13 Given the assumption that LOH involving a specific chromosome would indicate the localization of the disease locus, pheochromocytomas and MTCs were screened with highly polymorphic markers on some chromosomes. 76 In 7 of the 14 tumors, reduced signal intensity for a chromosome l p marker was observed, while for the other markers the constitutional genotype was always retained in the tumors.

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Analysis of the parental origin of the lost allele in two families showed that it was derived from the affected parent in one case. 76 However, according to the two-mutation model one would have expected that the tumor lacked the allele derived from the unaffected parent (illustrated for MEN I in Figure 5). Therefore, this indicates that LOH for chromosome 1 does not reflect unmasking of a recessive mutation at the MEN2A locus. When the putative MEN2A gene was localized to the centromeric part of chromosome 10, 77'78 this chromosome was tested for the occurrence of LOH. Surprisingly, analysis of several MTCs and pheochromocytomas could only detect LOH for chromosome 10 markers in a few cases. 79'8~ Before the identification of the gene responsible for MEN2A, the most likely explanation to this was that it could be difficult to delete the centromeric region of chromosome 10 without losing the whole chromosome. If two copies of another gene on chromosome 10 were essential for survival of these cells, the loss of one MEN2A gene by non-disjunction or a large deletion would be rare events. Therefore, the second event for homozygous inactivation of the MEN2A gene might instead have involved small deletions or point mutations, which were not detectable by LOH. Furthermore, the germline chromosomal abnormality could alone be responsible for the polyclonal thyroid and adrenal hyperplasias. The clonal development of MTCs and pheochromocytomas 81 from some of these cells might then involve mutations at loci other than on chromosome 10. This would still be in agreement with the epidemiological studies which had suggested the involvement of two mutational events. 75 Since the establishment of germline mutations in the ret gene being responsible lk)r MEN2A, s2"s3 it is supposed that this is an aclivating mutation causing a product with oncogenic features. This of course explains the lack of LOH in the region of interest, but does not explain the two mutalional events proposed. It therefore seems logical to expect other events, such as the inactivation (or activation) of another gene situated at a different chromosomal region causing the MEN2A phenotype. 84

VIii.

LOH IN T U M O R PROGRESSION

Genetic alterations in tumors are usually divided into the following three types: 85 1. 2. 3.

primary abnormalities---essential in establishing the neoplasm; secondary abnormalities--important in clonal evolution and progression of the tumor; and noiseErandom events due to genomic instability.

To differentiate between these categories it is important to study a large number of tumors, as well as tumors in different malignancy stages. For the same reason a representative part of the genome must beanalyzed.

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A. The Colorectal Tumor Example The best studied example of multistep carcinogenesis is the colorectal tumor. Two features of colorectal cancer have greatly aided the recent progress in understanding its genetics. First, the majority of colorectal cancers arise from premalignant adenomatous polyps allowing the analysis of somatic genetic changes during tumor progression, s6 Second, there are several well-defined inherited syndromes that predispose to colorectal cancer in an autosomal dominant manner, s7 One of the most widely recognized predispositions imposes a striking phenotype of multiple, adenomatous colon polyps (familial adenomatous polyposis coli). This condition may also occur in combination with benign extraintestinal growths including multiple osteomas, epidermoid cysts, desmoid tumors, thyroid tumors, and retinal hypertrophy: Gardner's syndrome. 88'89 Following the demonstration of a constitutional interstitial deletion of 5q in a mentally retarded patient with adenomatous polyposis coli and a desmoid tumor, 90 the APC locus was assigned to 5q22 by linkage analysis. 22'2s LOH was shown in about 20% of colorectal carcinomas, leading to a hypothesized tumor suppressor gene inactivation model for the disease. 91 Vogelstein et al. performed a general search for LOH in colorectal tumors. 9: At least one polymorphic DNA marker on every non-acrocentric chromosome arm was analyzed in a large panel of tumors (i.e., allelotyping). In this study, the most frequent LOH was surprisingly not on chromosome 5q. Thus, LOH on its own would have been unsatisfactory to localize the apc gene. Actually, allele losses in colorectal tumors were most often seen on chromosomes 17 and 18. By determining the minimal region of overlapping deletions, the p53 and the dec genes were shown to be the targets for the 17p and 18q deletions, respectively. 3~ These alterations are regarded as secondary events, demonstrating that tumor suppressor genes may also be involved in tumor progression. Based on the frequencies by which the most common mutational events were detected in the different malignancy grades, a model for progression of colorectal tumors was proposed. 93 The specificity ot'the mutational events was suggested to be more significant than the order (Figure 6).

B. The Glioma Example LOH studies are often based on previous cytogenetic analyses. For example, identification of genetic alterations in gliomas started with karyotyping of the highly malignant forms. The studies revealed the most common changes to be numerical deviations, particularly gains of chromosome 7 and loss of chromosome 10. Other frequent abnormalities were deletions and translocations of 9p and 19q, and the presence of double minute chromosomes. 91 Molecular genetic studies demonstrated LOH on 9p and 10, and amplification of the epidermal growth factor receptor (EGFR) gene on 7, confirming the cytogenetic results. 95'96 However,

Ch n:H'rx:xsome" Alteration Gene"

5<:1 Mutation/loss APC

12p Mutation K-RAS

18q loss DCC

17p Mutation/loss TP53

DNA Hypomethylation

~r

Other alterations

~

Normal gl~Hypeq~roHf,ll~Early epithelium epithelium a ~

r ~

r

J

Intermediate ~ L a t e adenoma aden enoma

Carcinoma ~ M e t a stasis

Figure 6. The Fearon and Vogelstein model for colorectal tumorigenesis.

Loss of Constitutional Heterozygosity in Human Cancer

295

nonrandom LOH was additionally found on 13 and 17. 95 For 17, the pattern of LOH was interpreted as mainly due to mitotic recombination, resulting in retention of heterozygosity at some loci, and at other loss of one allele and duplication of the remaining. 97 This type of alteration is cytogenetically not detectable. These findings have led to the proposal of a progression sequence similar to that of the colorectal carcinoma. LOH on 17p is regarded as an early event, since it is equally frequent in all malignancy grades. Nullizygosity at the interferon gene cluster on chromosome 9p is, on the other hand, only found in the highly malignant glioblastomas, and is therefore suggested to be a late event. Somewhere between these genetic events, chromosome 10 deletions, heterozygous interferon gene deletions, p53 mutations, and EGFR gene amplification occur. 95'96'98

IX.

DELETION MAPPING

When LOH is detected to a significant extent, an important step has been taken in mapping a putative tumor suppressor gene involved in the genetics of a certain tumor type. To further map the region of interest, the consistent step to follow is identification of the minimal region of overlapping deletions, thereby restricting the gene containing area. This is usually referred to as deletion mapping. The more polymorphic DNA markers used for this purpose, the more obvious it is that the deletion patterns are far more complex than originally suggested (see Figure 3). For instance, a detailed deletion mapping of chromosome 10 in gliomas revealed three distinct regions with high incidence of LOH: one telomeric region on 10p, and both telomeric and centromeric locations on 10q. 99'100 However, the studies did not reveal whether translocations, multiple rearrangements on one, or different deletions on both chromosomes 10 occurred. By analysis of the parental genotypes, these alternatives can be distinguished-an approach that was used in familial breast carcinomas. Allele losses in breast cancer mainly involves chromosomes 16 and 17.42-44 Deletion mapping of chromosome 17 revealed two distinct regions on 17p, and two on 17q. 1~ In several cases, two or more of these regions were deleted in the same tumor, while heterozygosity was maintained for other chromosome 17 loci. Determination of the parental origin of the lost alleles showed that deletions affecting these regions were independent events. The LOH shown in Figure 7 can be explained by mitotic recombination, a translocation, multiple deletions on one chromosome 17, or single deletions on both chromosomes. After parental genotyping, only the latter possibility remained. In MEN l-associated tumors, LOH studies revealed three regions with allele losses: additional to the MEN 1 locus on 11q 13 itself, a region telomeric on 11q, and one on 11p.102.74In context to these observations, this particular pattern may reflect that certain regions of chromosome 11 must be maintained and are required for cell survival, or give growth advantage. One of the retained regions actually overlapped the l lq13 amplicon, frequently detected in several other tumor types. ~~176

Figure 7. LOH on chromosome 17 in a case of familial breast carcinoma. RFLP analysis showed LOH (empty circle) at two regions flanking the centromere, and retained heterozygosity (black circle) at the distal part of 17p (grey circles: not informative). Parental genotyping showed that the deletion on 17p eliminated the paternal allele, and that the 17q deletion involved the maternal allele. 296

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297

X. CONCLUSIONS: THE RESEARCHER A N D THE T U M O R To summarize, let us illustrate the advantages and disadvantages of the "loss of heterozygosity" (LOH) technique presented in this chapter by discussing a fictitious situation: You, a researcher in cancer genetics, have in your hands a collection of tumors previously investigated poorly. How do you proceed? A common strategy does not exist. The nature of the disease, the information available, and the quality of the material will determine which methods can be applied. Considerations of this kind before starting an experiment may save both time and money. Does the cancer exist in a heritable form (i.e., are any families available for the studies)? As an example, colon and breast carcinomas do exist in forms with Mendelian inheritance. The "sporadic" forms that appear comparatively often in the population may also comprise hereditary components: the statistical risk of an individual to acquire these cancers increases drastically with every relative affected. In fact, this may be relevant for most cancers. 87'1~176 Linkage is sometimes hidden by heterogeneity of the disease; focusing on families with a homogenous clinical appearance may unmask this phenomenon. For example, linkage in breast cancer to chromosome 17q was established by selecting families with frequent premenopausal tumor onset, l~ Neoplasias with strict inheritance, thus probably of monogenic origin, are thought to be rare. However, they may be more common than generally believed since they can be hidden by variations in their clinical appearance. MEN I serves as a good example: from 130 patients in a large Tasmanian family, only 3 were diagnosed as MEN 1 before kinship was recognized. 1~ The skill of the investigator "asking the patients the right questions" would be crucial in such situations. A tight cooperation between the molecular researcher and the clinician is therefore highly beneficial. Provided no additional information by related methods (see below) is available, today's method of choice for monogenic inheritable cancer is linkage analysis. On the basis of microsatellite markers, linkage is more informative and less time consuming than classical Southern analysis. In addition, linkage analysis is not restricted to genes with certain properties, while LOH is only applicable to tumor suppressor genes. If family history reveals that your material of tumors is non-hereditary, or if the families in question are too small to provide informativity, other approaches than linkage analysis are required. Are the samples suitable for LOH analysis? The method demands tumors of monoclonal origin, of a certain tissue homogeneity, and a minimum quality of undegraded DNA. It also requires availability of constitutional DNA from every patient. The least ambiguous results will be obtained with fresh-frozen tumors and classical RFLP or VNTR markers. Paraffin-embedded tissue DNA, irrespective of its age, is still suitable for microsatellite marker analysis. The method of preparation

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should be known since fixating agents cause DNA breakdown to a different extent, and thus restrict the choice of markers. 1~ Most important, again, is complete access to the clinical data. A "breast tumor" or a "thyroid tumor" is not sufficient. Age and sex of the patient, treatment, survival time, and site and stage of the tumor are essential for interpretation of the LOH results. Again, it is worth emphasizing the cooperation with clinicians and histopathologists. Do other, faster methods give information about the locus of interest? A number of methods exist that may complement and direct LOH studies. One good rectaphase saves a hundred blots: In spite of its limitations, classical cytogenetics gives an immediate view over gross rearrangements within the tumor genome. In addition, new techniques are presently refining cytogenetic analysis. Using fluorescently labeled markers for in situ hybridization (FISH), in context with confocal laser scanning microscopes, a resolution of less than 100 kilobases can be obtained.110,111 Using chromosome specific libraries, and cosmids and yeast artificial chromosomes as markers, even minor chromosome rearrangements can be detected. 122 Single chromosomes can be amplified by degenerated oligonucleotide primer PCR. ll3 Rearranged chromosomes fluorescently PCR-amplified can be hybridized against normal metaphases, thus revealing their derivation. 1~4 Comparative genomic hybridization (CGH) will probably revolutionize molecular genetics, especially in cancer research. With CGH, regions of both gains and losses (deletions, duplications, amplicons) can be detected. ~15'116 In short, tumor DNA and normal reference DNA, differentially labeled with fluorochromes, are hybridized simultaneously to normal metaphase chromosomes. The alterations are detected as changes in the ratio of the intensities of the fluorochromes. The method is very promising, though still in its infancy. In spite of all newly developed techniques, LOH can not yet be replaced. The results are fairly easy to obtain and interpret, and certain genetic alterations can only be detected this way. In fact, the fascinating phenomenon of instability in the replication of dinucleotide repeats was unexpectedly discovered by LOH studies of colon carcinomas. i17.118,119 LOH still holds its position in human cancer research.

ACKNOWLEDGMENTS

This work was supported by the Swedish Cancer Society, the Swedish Medical Research Council, the Magnus Bergwall Foundation, the Marcus BorgstrOm Foundation, the Lars Hierta Foundation, and the Karolinska Institute.

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77. Mathew, C. G. P.; Chin, K. S.; Easton, D. E et al. A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nat,re 1989, 328, 527-528. 78. Simpson, N. E.; Kidd, K. K.; Goodfellow, P. J.; et al. Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage. Nature 1987, 328, 528-530. 79. Landsvater, R. M." Mathew, C. G. P.; Smith, B. A 9et al. Development of multiple endocrine neoplasia type 2A does not involve substantial deletions of chromosome 10. Genomics 1989, 4, 246-250. 80. Nelkin, B. D.' Nakamura, Y." White, R. W." et ai. Low incidence of loss of chromosome 10 in sporadic and hereditary human medullary thyroid carcinoma. Cancer Res. 1989, 49, 4114--4119. 81. Baylin, S. B.; Gann, D. S 9Hsu, S. H. Clonal origin of inherited medullary thyroid carcinoma and pheochromocytoma. Science 1976, 193, 321-323. 82. Mulligan, L. M.; Kwok, J. B. J." Healey, C. S.; et al. Germline mutations of the RET protooncogene in multiple endocrine neoplasia type 2A. Nature 1993, 363, 458--460. 83. Donis-Keller, H 9Dou, S.; Chi, D.; et al. Mutations in the RET proto-oncogene are associated with MEN2A and FMTC. Hum. Mol. Genet. 1993, 2, 851-856. 84. Mulligan, L. M.; Gardner, E." Smith, B. A 9Mathew, C. G. P." Ponder, B. A. J. Genetic events in tumour initiation and progression in multiple endocrine neoplasia type 2. Genes Chrom. Cancer 1993, 6, 166-177. 85. Heim, S.; Mitelman, E Cancer Cytogenetics. Alan R. Liss, New York, 1987. 86. Tierney, R. P." Ballantyne, G. H.; Modlin, I. M. The adenoma to carcinoma sequence. Surg. Gyn. Obstr. 1990, 171, 81-94. 87. Bishop, D. T." Thomas, H. J. W. The genetics ofcolorectal cancer. CancerS, ra,. 1990, 9, 585-604. 88. Gardner, E. J. A genetic and clinical study of intestinal polyposis, a predisposing factor for carcinoma of the colon and rectum. Am. J. Hum. Genet. 1951, 3, 167-176. 89. Camiel, M. C.' Mule, J. E.; Alexander, L. L." Benninghoff, D. k Association of thyroid carcinoma with Gardner's syndrome in siblings. N. Engi. J. Med. 1968, 278, 1056-1058. 90. Herrera, L.; Kakati, S.; Gibas, L.; Pietrzak, E." Sandberg, A. Gardner syndrome in a man with an interstitial deletion of 5q. Am. J. Med. Genet. 1986, 25, 473-476. 91. Solomon, E.; Voss, R." Hall, V.; et al. Chromosome 5 allele loss in human colorectal carcinomas. Nature 1987, 328, 616-619. 92. Vogeistein, B 9Fearon, E. R.; Kern, S. E." et al. Allclotype of colorectal carcinoma. Science 1989, 244, 207-210. 93. Fearon, E. R.; Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell 1990, 61, 759-767. 94. Bigner, S. H.; Mark, J.; Burger, P. C.; et al. Specific chromosomal abnormalities in malignant human gliomas. Cancer Res. 1988, 48, 405-411. 95. James, C. D.; Carlbom, E." Dumanski, J. P." et al. Clonal genomic alterations in glioma malignancy stages. Cancer Res. 1988, 48, 5546-5551. 96. James, C. D.; He, H.; Carlbom, E.; Nordenskjtld, M.; Cavenee, W. K.; Collins, V. P. Chromosome 9 deletion mapping reveals Interferon ct and Interferon 13-1 gene deletions in human glial tumors. Cancer Res. 1991, 51, 1684-1688. 97. James, C. D.; Carlbom, E.; Nordenskjtld, M 9Collins, V. P.; Cavenee, V. K. Mitotic recombination of chromosome 17 in astrocytomas. Proc. Natl. Acad. Sci. USA 1989, 86, 2858-2862. 98. Sidransky, D.; Mikkelsen, T,' Schwechheimer, K.; Rosenblum, M. L." Cavenee, W." Vogelstein, B. Clonal expansion of p53 mutant cells is associated with brain tumor progression. Nature 1992, 355, 846-847. 99. Rasheed, A. B. K.; Fuller, G. N.; Friedman, A. H." Bigner, D. D.; Bigner, S. H. Loss of heterozygosity for 10q loci in human gliomas. Genes Chrom. Cancer 1992, 5, 75-82. 100. Karlbom, A. E." James, C. D." Bo~thius, J." et ai. Loss of heterozygosity in malignant gliomas involves at least three distinct regions on chromosome 10. Hum. Genet. 1993, 92, 169-174.

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101. Lindblom, A.; Skoog, L.; lkdahl Andersen, T.; Rotstein, S.; Nordenskj61d, M.; Larsson, C. Four separate regions on chromosome 17 show loss of heterozygosity in familial breast carcinomas. Hum. Genet. 1993, 91, 6-12. 102. Larsson, C.; Weber, G.; Janson, M. Sublocalization of the multiple endocrine neoplasia type 1 gene. Henry Ford Hosp. Med. J. 1992, 40, 159-161. 103. Lammie, G. A.; Peters, G. Chromosome l lq13 abnormalities in human cancer. Cancer Cells 1991, 3, 413-420. 104. Gaudray, P.; Szepetowski, P.; Escot, C.; Birnbaum, D.; Theillet, C. DNA amplification at 1lq13 in human cancer: from complexity to perplexity. Murat. Res. 1992, 276, 317-328. 105. Lynch, H. T.; Watson, P.; Lynch, J. F. Epidemiology and risk factors. Clin. Obst. Gyn. 1989, 32, 750-760. 106. Houlston, R. S.; McCarter, E.; Parbhoo, S.; Scurr, J. H.; Slack, J. Family history and risk of breast cancer. J. Med. Genet. 1992, 29, 154-157. 107. Hall, J. M.; Lee, M. K.; Newman, B.; et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1990, 250, 1684-1689. 108. Shepherd, J. J. The natural history of multiple endocrine neoplasia type 1--highly uncommon or highly unrecognized? Arch. Surg. 1991, 126, 935-952. 109. Greer, C. E.; Peterson, S. L.; Kiviat, N. B.; Manos, M. M. PCR amplification from paraffin embedded tissues. Amen. J. Clin. Path. 1991, 95, 117-124. !10. Pinkel, D.; Straume, T.; Gray, J. W. Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci. USA 1986, 83, 2934-2938. 11 I. Wiegant, J.; Kalle, W.; Mullenders, L.; et al. High-resolution in sittt hybridization using DNA halo preparations. Htlm. Mol. Genet. 1992, 1,587-591. ! 12. Cremer, T.; Lichter, P.; Berden, J.; et al. Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Htlm. Genet. 1988, 80, 235-246. 113. Telenius, H.; Pelmear, A. H.; Tunnacliffe, A.; et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chrom. Cancer. 1992, 4, 257-263. 114. Blennow, E.; Telenius, H.; Larsson, C.; et al. Complete characterization of a large marker chromosome by reverse and forward chromosome painting. Httm. Genet. 1992, 90, 37 !-374. 115. Kallioniemi, A.; Kallioniemi, O-P.; Sudar, D.; et ai. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992, 258, 818-821. 116. du Manoir, S.; Speicher, M. R.; Joos, S.; et al. Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Htun. Genet. 1993, 90, 590-610. 117. Thibodeau, S. N.; Bren, G.; Schaid, D. Microsatellite instability in cancer of the proximal colon. Science 1993, 260, 816-819. 118. Aaltonen, L. A.; Peltomfiki, P.; Leach, E S.; et ai. Clues to the pathogenesis of familial colorectal cancer. Science 1993, 260, 812-816. 119. Lindblom, A.; Tannergfird, P.; Werelius, B.; Nordenskj61d, M. Genetic mapping of a second locus predisposing to hereditary non-polyposis colon cancer. Nat,re Genetics 1993, 5, 279-282.

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THE ROLE OF THE BCR/ABL ONCOGENE IN H U M A N LEUKEMIA

Peter A. Benn

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Philadelphia Chromosome Positive Leukemias . . . . . . . . . . . . . . IIl. Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The A B L Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. The B C R Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. B C R / A B L Fusions in C M L . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. B C R / A B L Fusions in Acute Leukemias . . . . . . . . . . . . . . . . . . . . . VIII. The Role of B C R / A B L in Blast Crisis C M L . . . . . . . . . . . . . . . . . . . IX. Experimental Studies with B C R / A B L . . . . . . . . . . . . . . . . . . . . . . X. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

305 306 309 310 311 312 320 321 324 325 320

I. INTRODUCTION C a n c e r is the c o n s e q u e n c e o f an a c c u m u l a t i o n o f m u l t i p l e critical g e n e t i c a l t e r a t i o n s that c o n f e r a g r o w t h a d v a n t a g e . T h e c o m p l e x i t y o f the p r o c e s s is a p p a r e n t f r o m both the d i v e r s i t y o f g e n e s that are i n v o l v e d in the p r o c e s s ( o n c o g e n e s and t u m o r

Advances in Genome Biology Volume 3B, pages 305-335. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

305

306

PETER A. BENN Table 1. Common Abbreviations*

Abbreviation ABL ABL c-abl BCR BCR

Alternative Forms c-abl, c-ABL abl protein, P145, P145 ABL bcr, bcr gene, phi, PHL bcr protein, P160 and PI80 or PI90, P 160 BCR and P 180 BCR or P 190 BCR

M-bcr

bcr, bcr, bcr-1

m-bcr

m-bcr-l, bcr-2

K-28 L-6 Ph

phi, Ph', 22q-

Brief Definition Human ABL gene Human ABL protein Normal abl gene (other than human or viral) Human BCR gene Human BCR protein

Major breakpoint cluster region, 5.8 kb including exons 12-15 of BCR Minor breakpoint cluster region, 3' end of intron 1 of BCR m-RNA with a junction of BCR exon 14 to ABL exon 2 m-RNA with a j unction of BCR exon 13 to ABL exon 2 The Philadelphia chromosome

Note." *The abbreviations used in this review for the BCR, ABL, and fusion BCR/ABL genes. Some of the most common alternative forms used in other publications are also listed.

suppressor genes with diverse functions), the mechanisms of genetic alteration (amplification, overexpression, deregulation, deletion, point mutation, and translocation), and from the subtleties of the alterations arising within individual genes that can elicit a proliferative advantage. In this review, one example of an oncogene activation by translocation is considered in detail. The translocation fuses two human genes, BCR and ABL, and this juxtaposition appears to be a critical change in some leukemias. The gene fusion was the first recognized example of a specific genetic change in a human cancer. Following the cytogenetic identification of the translocation, the genetic rearrangement was characterized precisely by molecular techniques. This review discusses the role of the BCR/ABL fusion in the pathogenesis of human leukemia, emphasizing both the common features of the genetic alteration in different patients and the diversity that can exist within this single alteration in cancer. Because abbreviations used by different investigators vary and have led to some confusion, a summary is provided in Table 1 of the terms used in this chapter together with same of the alternate forms commonly encountered in the literature.

!1. T H E P H I L A D E L P H I A C H R O M O S O M E POSITIVE LEUKEMIAS Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease of a pluripotent stem cell. !-3 The early stage of the disease is relatively benign ("chronic phase") characterized by an overproduction of granulocytes. Myeloid cells in the

The BCR/ABL Oncogene

307

peripheral blood show all stages differentiation. Progressive splenomegaly and occasionally hepatomegaly occur as increasing myeloid cells are produced. The rate of cell division of myeloid cells is not increased but the life-span of the cells appears to be extended. 4 The median age of onset of the disease is approximately 50 years with similar numbers of male and female patients. 5 The duration of the chronic phase is highly variable with a median of approximately 3 years. 6 In approximately 50% of patients with CML, the disease progresses from the chronic phase to an "accelerated phase." This is characterized by splenomegaly and leukocytosis and cells may become resistant to chemotherapy. Basophils and eosinophils may increase and thrombocytosis and myelofibrosis may develop. 8'9 In most patients the disease progresses to an acute terminal phase, referred to as "blast crisis." Blast crisis may follow an accelerated phase or the patient may progress directly from chronic phase. Generally, blast crisis is characterized by greater than 30% blast cells in peripheral blood, bone marrow, or both. 8'1~Extramedullary blastic transformation may occur in which a localized tumor mass (myeloblastoma) is present at a lymph node or elsewhere. 11 The blast cells are myeloid in approximately 70% of cases and lymphoid in approximately 30% of cases. 12Rare cases of biphenotypic or mixed lymphoblastic/myeloblastic cell types have also been observed. 13-15 Median survival after the onset of blast crisis is only approximately 3 to 6 months. 6 In many respects, blast crisis CML is similar to some acute leukemias. Greater than 90% of cases of CML contain a Philadelphia chromosome (Ph--see Section III). 16 However, the chromosome is also found in 2-6% of childhood acute lymphoblastic leukemias (ALL) and 19-30% of adult ALL. 17-18 These leukemias are often phenotypically B or pre-B cell, 13 although T-cell acute leukemias may also have a Ph chromosome. 19 Ph-positive ALL may have a worse response and survival than Ph-negative ALL. z~ Morphologically, these leukemias are usually classified as L 1 or L2 and are terminal deoxynucleotidyl transferase (TdT) positive. 2~ At least some of the patients noted with Ph-positive ALL might be considered as patients with lymphoid transformation of CML with a short or unrecognized chronic phase, z2 However, based on clinical response and relapse criteria, as well as some ancillary cytomorphologic criteria, some patients with Ph-positive ALL appear to be quite distinct from blast crisis CML patients and are much more typical of other ALL patients. 23 Rare cases of AML are also Ph positive. 24 These leukemias have been typed morphologically as M 1, M2, and M4. 25 The induction of all types of leukemia appears to involve both genetic and environmental factors. 5'z6 The association between radiation exposure and leukemia incidence is well established, and it is likely that other agents which cause chromosome breakage are leukemogenic. It is, therefore, perhaps not surprising that specific chromosome rearrangements are implicated in the development of leukemias.

Figure 1. (a) Karyotype prepared from the bone marrow cells of a patient with CML. The Philadelphia (Ph) chromosome and derivative chromosome 9 (9q+) are identified by arrows. (b) Diagram illustrating the reciprocal translocation t(9;22)(q34;q11) that gives rise to the Ph chromosome and 9q+ chromosome. 308

The BCR/ABL Oncogene

309

III.

CYTOGENETICS

In 1960, Nowell and Hungerford noted that a specific marker chromosome was present CML. 27 The marker chromosome was named the Philadelphia chromosome (Ph) after the city of its discovery. The Ph chromosome was noted to be present in approximately 90% of cases of CML. 16Following the introduction of chromosome banding, it was observed that the Ph chromosome was usually the result of an apparently balanced reciprocal translocation between a chromosome 9 and a chromosome 22. 28 The translocation involves band q34.1 of chromosome 9 and band q11.21 on chromosome 22 (Figure 1). 29 Approximately 4% of CML patients show "complex" exchanges involving three or more chromosomes. 3~ In such cases, chromosome 9 and 22 are still involved and the Ph chromosome appears to be identical to that seen in simple t(9;22) translocations. In a further 3-4% of patients, chromosome 9 does not appear to be obviously involved although the Ph chromosome again appears to be identical to that seen with simple t(9;22) translocations. 32 This latter group is sometimes referred to as "simple variant" translocations. 32 "Masking" of Ph chromosome by additional chromosomal exchanges also sometimes occurs. 33 Although some cases diagnosed as Ph chromosome negative CML are in fact other hematologic disorders, it is clear that Ph-negative CML does exist. 34 In at least some of these cases, BCR and ABL are juxtaposed with the exchange occurring below the level of resolution used by light microscopy. 35-37 Additional cytogenetic changes are frequently seen in CML, particularly during blast crisis. 16 These additional changes are nonrandom in nature (Table 2) with considerable diversity in the karyotype of blast crisis CML from patient to patient.

Table 2. Additional Chromosome Abnormalities in CML* Chronic Phase

Blast Crisis

+Ph

3

32

+8

3

29

i(17q)

0

13

+19

1

14

+21

3

8

-Y

3

2

Other

5

25

Any

11

67

Note." *Percentage of cases of CML with additional cytogenetic changes in chronic phase and blast crisis. Based on data in Ref. 197.

PETER A. BENN

310

Clonal evolution probably also involves additional genetic changes such as p53 38'39 and ras gene mutation 4~ not detectable by cytogenetic analysis. The Ph chromosome in Ph-positive ALL is indistinguishable cytogenetically from that in CML. 17-18 Like CML, complex and variant translocations have been observed in Ph-positive ALL. 42 However, Ph-positive ALL patients may more frequently show some normal metaphases in their bone marrow cells at diagnosis. 43 Another difference between Ph-positive ALL and CML is that nonrandom secondary cytogenetic changes (extra Ph, +8, i(17q), and +19) frequently seen in blast crisis CML are often not present in ALL. As will be discussed in detail later, a further distinction between Ph-positive CML and some Ph-positive ALL can be seen at the molecular level in terms of breakpoint locations. Before discussing BCR/ABL fusions, the normal structure and function of these two genes is reviewed.

IV. THE ABL GENE Before considering the structure and function of the human ABL gene, a brief review of the transforming v-abl gene of Abelson murine leukemia virus (AMuLV) is necessary. 44 A number of comprehensive reviews of A-MuLV have been published in recent years 45-48 and only a brief discussion of the virus will be included here. AMuLV is a retrovirus that is capable of rapidly inducing lymphomas in mice. 44 The virus is also capable of transforming mouse fibroblasts and lymphoid cells in vitro. 49-51 The transforming property of A-MuLV is associated with the v-aM viral gene that encodes a tyrosine kinase. 52'53 Tyrosine kinases catalyze the transfer of the terminal phosphate of adenosine triphosphate to the hydroxyl group of tyrosine. A number of other oncogenes are tyrosine protein kinases as are several growth factor receptors. 54 A-MuLV is thought to have originally arisen as a result of a recombination event between the less oncogenic Moloney murine leukemia virus (M-MuLV) and the mouse c-ab! gene. 44'55'56 As a result of the recombination, the v-abl gene contains a 5' sequence coding for a gag viral structural protein moiety and a 3' sequence coding for part of c-abl. The gag viral protein contains a myristylation signal that probably directs the v-abl protein to the membrane. 57 Control of A-MuLV v-abl expression is mediated by promoter and enhancer signals originating from the virus. 48 A second viral form of v-abl is found in the feline Hardy-Zuckerman (HZ-2) virus. 58 HZ-2 was originally isolated from a fibrosarcoma in a Siamese cat. In HZ-2 the v-ab! is the result of a recombination between the viral gag gene and the feline c-abl. Precise recombination sites differ in the two viruses with involvement of a second feline virus gene, pol at the 3' recombination site in HZ-2. 59 The human gene homolog to the mouse c-abl gene is referred to as ABL. In contrast to v-abl, the normal cellular mouse c-abl and human ABL proteins have

Figure 2. Diagram of the BCR gene (chromosome 22) and ABL gene (chromosome 9) showing the location of the major breakpoint cluster region (M-bcr), minor breakpoint cluster region (m-bcr),and the position of the ABL breakpoints.

312

PETER A. BENN

only weak tyrosine kinase activities. 6~ The humanABL gene has been extensively characterized. The ABL gene is at least 230 kilobases (kb) in size 62 with its 5' and towards the centromere of chromosome 9, at band q34 (Figure 2). 63 There are 12 exons with two alternate forms of the 5' most exon. 64 Exons 2-12 are sometimes referred to as the "common" exons since they are present in all mRNA' S transcribed from ABL. The alternative forms of exon 1 are 1A (located approximately 19 kb 5' ofexon 2) and IB (located at least 200 kb 5' ofexon 1A). The substantial distance between these two alternate exons is unusual and the mechanisms for transcription over such distances are unclear. 62 The two types of mRNA's produced by alternate splicing are identifiable by their size difference (6.0 kb for exon IA usage and 7.0 kb for exon I B) (Table 3). 64 Based on studies on mice, alternate forms of mRNA can be found in a wide variety of tissues, but particularly in testis, thymus, and other lymphoid tissues. There is some evidence for variation in the relative amount of each type of transcript in different tissues. 65-67 The proteins produced by human ABL have a molecular mass of approximately 145 kDa and are referred to as p145. 68 The tyrosine kinase function of the protein is defined by the so-called SHI domain (src homologous domain 1),54 and it is this region of the protein that shows the strongest homology between species.64 The majority of this region is essential for the transforming capability of v-abl. 69 The SH2 domain includes amino acid sequences found in GTPase-activating proteins (GAPs) and other proteins that may bc involved in signaling pathways. 48 The SH3 domain contains sequences shared with other oncogenes, although the presence of the entire SH3 domain does not appear to be essential for the transformation properties of the protein. 48 In fact, the SH3 domain is a negative regulator of the kinase activity of the abl protein. Deletion of the SH3 domain in mouse c-abl results in activation of the transformation ability of the protein. 7~The amino acid terminus of the protein is the end where substitutions of gag are found in v-abl and where BCR is juxtaposed in human leukemias (sec Section VI). The carboxyl terminus does not appear to be required for kinase activity although the region does show a relativcly highly conserved sequence. 48

Table 3. Transcripts and Translation Products* Gene

mRNA

Protein

BCR

4.5 kb, 6.0 kb

PI60, PI80 or PI90

6.0 kb, 7.0 kb

P145

ABL BCR/ABL (CML)

8.5 kb (K-28), 8.5 kb (L-6)

P210

BCR/ABL (ALL, AML)

8.5 kb (K-28), 8.5 kb (L-6)

P210

7.0 kb

P 190

Note: *Summary of the transcription (mRNA) and translation (protein) products for the various BCR, ABL, and BCR/ABL genes. Sizes of the mRNA and proteins are approximate and different authors cite somewhat variable numbers in their designations of these products.

The BCR/ABL Oncogene

313

Despite the large amount of information available relating to the human ABL gene, its normal function in cells is unknown. Among the other well-analyzed tyrosine kinases, ABL is most similar to the oncogene FES. 54 Another gene, ARG (ABL-related gene) has been described that shows substantial homology to ABL. 71 ARG is located on chromosome 1, band q24 or q25. This observation is of interest since this region has been associated with specific chromosome rearrangements in cancer. 72 This includes a specific translocation t(1;6)(q23-q25; p21-25) noted in occasional cases of myeloproliferative disorder. 73'74 While the precise details of the functions of ARG other putative tyrosine kinases are unclear, the insights gained from the study of ABL activation may be extremely helpful in unravelling parallel pathways to neoplasia.

V. THE BCR GENE The BCR gene is located on chromosome 22 with the 5' end of the gene closest to the centromere (see Figure 2). 75 The gene was named following the observation that the breakpoints on chromosome 22 in Ph-positive CML were clustered within a limited region, the breakpoint cluster region, or M-bcr. 76 The BCR gene is also frequently referred to as phi. The normal BCR gene is approximately 130 kb in size with approximately 21 e x o n s . 75'77'78 Like ABL, there is a large separation between exons 1 and 2. 79 In the case of BCR this distance is approximately 68 kb. The gene appears to be evolutionally conserved with substantial homology between mouse, chick, and human sequences.8~ Two RNA transcripts are made (4.5 and 6.0 kb in size) 81 with two BCR protein products of molecular mass approximately 160 and 180 kDa, or 190 kDa (see Table 3). 82`83 BCR transcripts are detectable in human fibroblasts, B and T lymphoid, myeloid, and erythroid cell lineages, v5'77'8~The protein does not have tyrosine kinase activity (in contrast to ABL). In immunoprecipitates of BCR, a serine kinase activity has been detected 83 but this may represent a contaminant since there is no homology between BCR and any known serine kinase gene. Sequence analysis of the BCR gene indicates that there is homology between BCR and GTPase-activating proteins (GAPs), and BCR appears to be a member of this interesting group of polypeptides. 84 GAPs accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins leading to down regulation of the active GTPbound form. 85 The ras oncogenes regulate many processes of eukaryote cells including cell growth, cytoskeletal organization, transportation, and secretion. GAPs may act as tumor suppressor genes limiting the level of Ras oncoprotein activities. 8 It has been shown that the Ras-related protein Rac has increased GTPase-activity in the presence of BCR. 84 It is the carboxyl terminal domain of BCR that appears to have this GAP characteristic. Although the function of Rac is unknown, its homology with other Ras oncoproteins imply an important role in

PETER A. BENN

314

cellular function. The rac messenger RNA is present in a wide variety of cell types and there is some evidence the levels may be higher in myeloid cell lineages. 87 Thus BCR appears to a regulatory factor for Rac, a protein that can be speculated as playing an important role in cell growth or differentiation of eukaryote cells. Another closely related GAP, n-chimerin exists which appears to be related to BCR. 84 However n-chimerin has not been implicated in fusion with ABL. BCR shows homology with three other loci (BCR2, BCR3, and BCR4) which are all closely linked with BCR on chromosome 22. 88 It is not known what is the function of these additional related genes or even whether they are expressed.

VI.

BCR/ABLFUSIONS

IN CML

Following the localization of ABL chromosome 9, 89 it was established that in CML a substantial part of ABL is translocated to the Ph chromosome. 63'9~ Breakage on chromosome 22 was clustered within a "breakpoint cluster region ''76 and, as a result of the genetic rearrangement, a novel mRNA species was produced, 81'92-95 with an associated fusion protein. 83'96-97 These studies unequivocally established the important mechanism of gene fusion in the pathway to neoplasia. The breakpoint cluster region (M-bcr) of chromosome 22 was originally defined as a 5.8-kb segment that included four exons of the BCR gene. 76 This region contains nearly all the chromosome 22 breakpoints in patients with CML. The four exons are often referred to as M-bcr exons l to 4 and correspond to exons 12 to 15 of the BCR gene, based on the characterization of the gene by Heisterkamp et al. 79 Within the M-bcr, the vast majority of breakpoints occur between exon 13 and exon 15. 98 Localization of breakpoints can be achieved by Southern blot analysis with combinations of multiple restriction enzyme digests of leukemic cell DNA and using multiple M-bcr probes (Figure 3). 99-100 Intbrmation on breakpoints may also be deduced by a polymerase chain reaction (PCR) technique amplifying cDNA produced from BCR/ABL transcripts, l~176 although interpretation is subject to modification as a result of alternate splicing of mRNA. 1~ In the PCR technique, primers to a BCR exon sequence and an ABL exon sequence are used to selectively amplify eDNA sequences where these two exons are juxtaposed (Figure 4). Both techniques indicate that breakpoints are clustered on either side of exon 14. Rare cases have been assigned breakpoints that are 5' of exon 13, l~176 but at least some of these may be due to technical problems associated to the assignment of breakpoints. 1~ Breakpoints 3' of exon 15 also seem to be rare. i~176 Mills et al. ll3 have pointed out that sequence data indicate that an out-of-frame mRNA with an early stop codon would result from a splice between BCR exon 15 and ABL exon 2. This is in contrast to the in-frame mRNAs produced as a result of splicing between BCR exon 13 or 14 and ABL exon 2. Thus, in the absence of additional mutation, the sequence and the requirement for a biologically active BCR/ABL protein product appear to define permissible breakpoints.

(a)

Figure 3. (a) Results of a Southern blot analysis for M-bcr rearrangement using a 1.2 kb Hind III/Bgl II probe on DNA digested with the restriction enzyme Bgl II. Lane 1 shows lambda DNA digested with Hind Iii. Lane 2 and 9 show molecular weight

markers. Lane 3 is a control specimen; Lanes 4 and 5 are blood and bone marrow from one patient; Lanes 6 and 7 are blood and bone marrow from a second patient and Lane 8 is blood from a third patient. Each patient shows its own characteristic rearrangement autoradiograph band (in addition to the normal 5.0 kb unrearranged M-bcr band). The rearrangement band is indicative of a change in the distance between restriction enzyme sites as a result of translocation. (b) Same as (a) but with DNA digested with the restriction enzyme Hind III. (c) Restriction enzyme map of the M-bcr with the location of the restriction enzyme sites marked. From the results in (a) and (b) the location of breakpoint can be deduced. For example, patient 1 shows rearrangement with Bgl II digestion but not with Hind II!. Thus, the breakpoint is likely to be between the two Bgl II sites shown but also 5' of the second Hind III site.

(continued)

315

316

PETER A. BENN

(b)

(c)

1" 9 Kb

1 "2 Kb

3"

5' B

Bg H

=

Bam

HI

= Bgl

II

=

Hind

III

Figure 3.

(continued)

In contrast to the tightly clustered range of breakpoints seen on c h r o m o s o m e 22 in CML, the breakpoints on chromosome 9 are to be tbund in a substantial region spanning 200 kb or more. 62 It was originally thought that breakpoints were always 5' of A B L exon 2, the first c o m m o n exon, and that the breakpoints could be 5' of exon I A or exon lB. Breakpoints appear to be mostly between exon I B and exon

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Figure 4. Diagrammatic illustration of the PCR technique for the detection of bcr rearrangement. In this approach RNA is extracted from cells and incubated in the presence of an oligonucleotide primer (primer 1) and the enzyme reverse transcriptase. This produces a strand of cDNA. Synthesis from this cDNA can be achieved using a second primer (primer 2) and DNA polymerase. The number of copies of the two strands of cDNA can be increased exponentially (ex) by repeated dissociation of the double stranded DNA and repeated rounds of synthesis.

IA and may be clustered in three regions: 30 + 5 kb, 100 + 13 kb, and 135 + 8 kb downstream from exon,lB, ll4 Recently, a case of CML has been described with a chromosome of breakpoint between A B L exon 2 and A B L exon 3. It5 Similar observations have been made for patients with ALL. 196 It is of interest to note that these downstream breakpoints result in the removal of 17 amino acids encoded by exon 2 in the BCR/ABL fusion protein. The 17 amino acids correspond to part of the SH3 domain of ABL, and since this domain is thought to have a negative regulatory effect on the kinase (SHI)domain, 78 the change may well have biological significance. The frequency of these 3' breakpoints remains to be established. Although the translocation t(9;22)(q34;q22) appears to be balanced by cytogenetic analysis, molecular analyses indicate that there are often substantial deletions o f sequences at the breakpoi nts. l~6 Using Southern blot analyses with M-bcr probes, Popenoe et al. 116 noted that deletions can be detected in 10-20% of CML patients. Sequence analysis also indicates that deletions are present 75 and these probably

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occur for both chromosome 9 and chromosome 22. Additional sequences of undetermined origin may also be present at the breakpoints in the derivative chromosomes. 75 While there is a requirement for in-frame mRNA produced by the biologically significant BCR/ABL fusion chromosome (the Ph chromosome), no such constraint exists for the reciprocal rearrangement chromosome (the 9q+ chromosome). In fact, no transcripts are consistently detected from the ABL/BCR fusion on 9q+. No substantial homology between BCR and ABL sequences have been reported despite the fact that both the carboxyl terminal of BCR and the SH3 domain of ABL have sequences associated with GAPs. 48'84 At least among newly diagnosed CML, breakpoints in the exon 13 to exon 15 segment of BCR appear to be random, although the number of cases studied is small. ! 17 An Alu repeat sequence is present between BCR exon 14 and e x o n 15, 75'118 and Alu sequences are also found within ABL. 119-121 However, based on the analysis of the sequences at the t(9;22) breakpoints in patients with CML, there is no evidence for sequence-specific recombination involving Alu or other j22 common repeat sequences. 75 Unlike some other tumor-associated specific chromosome rearrangements, there do not appear to be any known recombinase-specific sequences present in the vicinity of breakpoints to account for t(9;22) exchanges. An additional consideration in evaluating the consequences of different breakpoints on chromosome 9 and 22 is the alternate splicing of mRNA transcribed from the BCR/ABL fusion gene. 1~ Alternate splicing can sometimes occur around BCR exon 14 in patients with a 3' breakpoint. That is, if a patient has a BCR/ABL rearrangement in which the chromosome 22 breakpoint is downstream of exon 14, the cells may contain BCR/ABL mRNAs with or without BCR exon 14 encoded sequences, or may have both species of mRNA. The two types of mRNA that are usually produced are sometimes referred to as the K28 type with BCR exon 14 sequence linked to ABL exon 2 sequence and L-6 when there is a BCR exon 13 to ABL exon 2 junction. Early studies using an RNase protection assay indicated that alternate splicing was common, based on the presence of both species of mRNA in a high proportion of patients. 123 Data from PCR amplification of cDNA (see Fig. 4) has confirmed that alternate splicing takes place although the frequency of this may be less than previously thought (see Section VIII). 101-105'124 The extent to which other alternate splicing or incomplete splicing may occur is not well established. Romero et al. 125 studied the transcripts produced by the cell line KBM-5 (established from a CML patient with a myeloid blast crisis) and observed that two alternative BCR exon 1 sequences were sometimes utilized. The transcripts were also detected in two other blast crisis CML patient samples, but not in 12 other CML patients of various clinical stages or in six acute leukemias. Whether or not variation in breakpoint or splicing has a significant effect on the properties of the BCR/ABL fusion protein is also unknown. The presence or absence of BCR exon 14 corresponds to 25 amino acids in the BCR/ABL fusion protein and the absence of ABL exon 2 sequence deletes 58 amino acids from

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Figure 5. Some of the types of mRNA observed in the cells of patients with BCR/ABL gene fusions, el . . . . denotes BCR exons and a2 . . . . denotes ABL exons. The first two types of mRNA shown are the most common and are found in nearly all cases of CML and approximately 50% of Ph chromosome positive acute leukemias. The third type of mRNA is seen in rare cases of CML. 195 The fourth mRNA lacks ABL common exon 2 and has been observed in CML and ALL. 115'196 The fifth and sixth mRNAs appear to be largely confined to acute leukemias.

BCR/ABL. 75'77'78'126 The novel transcripts described by Romero et al. 125 with alternate BCR exon 1 sequences result in even larger changes to BCR/ABL protein structure. Two patients with CML have been described with breakage 5' of BCR exon 19 resulting in an in-frame fusion mRNA 540 kb longer than usual and a resulting protein with an additional 180 amino acids (Figure 5). 195 Unfortunately, sensitive quantitative assays for BCR/ABL protein amount and activity are not yet available. While the BCR/ABL protein can be isolated by immunoprecipitation, protease activity or inhibition of the kinase activity have made quantitation problematical. 97"j~ The proteins isolated by immunoprecipitation have a molecular mass of approximately 210,000 kDa, but minor variations are not easily detectable. 83'96'97These proteins are referred to as P210. Van Denderen et al. 129have described the production of a polyclonal antiserum that will specifically recognize the junction site of a BCR exon 13/ABL exon 2 fusion protein. The antiserum did not react with BCR exon 14/ABL exon 2 fusion protein. The production of antibody specific to particular types of fusions may be extremely useful in increasing an understanding of the effects of the various types of BCR/ABL proteins, as well as being potentially useful diagnostic reagents. 130

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Variation in breakpoints and transcripts does not pose a substantial problem for the routine diagnosis of CML by Southern blot analysis 1~176 or PCR. l~176 Some myeloproliferative syndromes are clinically difficult to distinguish from CML and detection of M-bcr rearrangement can confirm a diagnosis of CML. 132 A small proportion of cases of CML show an apparently normal karyotype, yet do have M-bcr rearrangement with rearrangement involving chromosomal segments too small to be identified cytogenetically. 35'36 Other M-bcr rearrangement positive cases show complex translocations without obvious involvement of chromosome 22 or with exchanges that mask the Ph chromosome. 36 Since Ph chromosome negative CML has previously been regarded as a subgroup of patients with poorer prognosis, additional molecular characterization in this group of patients is required to reevaluate this issue. 133 Despite the reports that (atypical) M-bcr rearrangement negative CML exists, 132'134 Dr. J. Rowley has proposed that the sine qua non for CML is the juxtaposition of BCR and ABL. 135 In interpreting diagnostic tests that utilize Southern blot analyses with bcr probes, rare polymorphisms need to be considered since these can lead to false positive results. 136-139 Quantitation of autoradiograph band intensities can be useful for monitoring disease 14~and residual disease following bone marrow transplantation can be detected using the PCR technique. TM Occasional cases of CML may give a false negative result in PCR analysis due to degradation of mRNA, expression of unusual transcripts, or for other unknown reasons. 1~ In situ hybridization techniques that take advantage of the juxtaposition of BCR and ABL may also be useful in the future for diagnosis of CML. 142

VII.

BCR/ABLFUSIONS

IN ACUTE LEUKEMIAS

Approximately 50% of patients with Ph-positive ALL have chromosome 22 breakpoints within the M-bcr. 143'144 The remaining 50% of patients have breakpoints within the first intron of the BCR gene. 145,146 Surprisingly, breakage is nonrandomly distributed in this region and breakpoints are clustered to a segment referred to as m-bcr. 145'148This is an unexpected finding since breakage anywhere throughout the intron might be expected to result in the same BCR/ABL mRNA. Furthermore, no specificity is apparent in the location of the breakpoints within the introns in CML (see above). Heisterkamp et al. 146 observed that six out of six breakpoints in ALL were at the 3' end of the first intron, a region consisting of about 35 kb. Denny et al. 147 noted that for eight patients studied with BCR intron 1 breakpoints, all were within a 20-kb 3' segment of intron 1, while Chen et al. 148 noted that six of seven patients had breakpoints within a 10.8-kb segment that they termed bcr-2. Combining these results, it would appear that there is a distinct clustering of breakpoints, although there may not be an exclusive precisely-defined region of BCR intron 1 that is involved. Chromosome 9 breakpoints appear to be similar to that seen in

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CML, with most cases showing involvement of the region 5' of exon 2 and rare cases showing breakage between exon 2 and exon 3 of ABL. 196 The reason for preferential location of breakpoints within BCR intron 1 has been the subject of some speculation. Denny et al. 147 have pointed out that BCR intron 1 contains a 1-kb deletion polymorphism with approximately one-third of chromosomes 22 lacking the 1-kb sequence that includes Alu repeats. They proposed that this sequence may in some way result in increased susceptibility toward rearrangement. Although breakage appears to be near or within Alu sequences in the few cases where breakpoints have been sequenced, the exchange does not necessarily involve recombination between homologous sequences. 119-221 Papandopoulos et al. ~21 proposed that Alu or other common sequences may represent potential binding sites for proteins involved in DNA cleavage or proteins that alter chromatin structure in a way that favors cleavage. Alternatively, for reasons unknown at this time, the retention of a large BCR intron 1 sequence may be essential for BCR/ABL functional protein and that the clustering of breakpoints at the 3' end of BCR intron 1 could reflect the need to maintain specific intron sequences. In view of the widespread distribution of Alu sequences in the human genome, the location of these within the BCR and ABL genes could be coincidental. Breakage within BCR intron 1 and joining to ABL results in a mRNA of 7.0 kb. 145'150-153 The M-bcr and m-bcr breakpoints are consistently found in Ph-positive acute leukemia but are not usually present in Ph-negative acute leukemia. ~46 Ph-negative acute leukemia is not, in general, therefore attributable to a "masking" of the genetic exchange or to rearrangement of minute sized segments of chromosomes as characterizes many cases of Ph-negative CML. Thus, the acute leukemias appear to reflect a heterogenous group of disorders that do not all have a BCR/ABL gene fusion as a component of their etiology. It is not known whether there is any distinct clinical differences between acute leukemias with BCR/ABL rearrangements involving M-bcr and those involving m-bcr. Based on a small number of patients, Secker-Walker et al. 154 could not identify any clear distinction between the two types of Ph-positive acute lymphoblastic leukemia patients. They noted that M-bcr rearrangement was sometimes confined to the lymphoblastic component of marrow or blood and suggested that heterogeneity in the translocation target cell in terms of its differentiation potential could be important in determining disease outcome. Because of the rarity of Ph-positive acute myeloid leukemia, relatively few cases have been studied. 98 Subclassification of additional acute leukemias on the basis of their BCR breakpoints is required and such studies could well help better delineate disease subtypes.

VIIi.

THE ROLE OF

BCR/ABL IN BLAST CRISIS CML

Since each case of CML has slightly different breakpoints on chromosomes 9 and 22, the size of the genomic BCR/ABL junction fragments differ for each fusion (see

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Fig. 3). Serial studies on patients indicate that, in general, no alteration in breakpoints occurs as patients progress from chronic phase through blast crisis, l~ This appears to be the case even when cytogenetic studies indicate that a second Ph chromosome has arisen during clonal evolution. 11~ The second Ph chromosome would therefore represent a duplication of the initial Ph chromosome rather than the product of additional gene rearrangement. For patients sequentially studied during disease progression, a number of exceptional or unusual situations have been described. Bartram et al. 156 reported a case of CML in which blast crisis was characterized by two Ph chromosomes and the presence of an additional M-bcr rearrangement that was associated with a novel 10.3-kb mRNA. There are at least two reports of cases of CML in which there appeared to be a loss of BCR/ABL fusion DNA. 157'158Cytogenetic evidence for loss 159 of the Ph chromosome has been presented but such cases seem to be rare. Retention of the normal BCR allele is also the usual situation although one case has been described in which a progressive loss of germline BCR DNA was observed as the patient progressed to blast crisis. 16~ There is controversy regarding reports that some BCR/ABL fusions may be associated with shorter chronic phase durations than other fusions. A number of studies have suggested that the median chronic phase in the population of patients with a breakpoint at the 5' end of M-bcr is longer than the median chronic phase durations for patients with 3' M-bcr breakpoints. ~~0,~l~,~~3,~~7,1~8Other studies have failed to confirm this trend 1~ and it is clear that there are many exceptional patients who may have 5' breakpoints and short chronic-phase durations as well as patients with 3' breakpoints and unusually long chronic phases. 167"168Heterogeneity in the populations and ascertainment biases within the patient studied clearly exist; there is a need for careful prospective studies to resolve this question. 169'17~Amore rapid disease course in the population of patients with 3' M-bcr breakpoints relative to those with 5' M-bcr breakpoints could account for the relatively large number of 3' breakpoints seen in patients with blast crisis CML. 11~ It has been suggested that alternate splicing of BCR/ABL may occur more frequently during blast crisis compared to during the chronic phase. 172 Lee et al. 172 observed the simultaneous presence of the two mRNA species corresponding to the presence or absence of BCR exon 14 sequences (K-28 and L-6 junctions) in 3 of 9 blast crisis patients compared to only 2 of 21 chronic phase patients. Morgan et al. 173 observed a less striking difference in the frequency of dual expression of transcripts: 3 of 9 chronic phase patients compared to 5 of 11 blast crisis patients. Since M-bcr breakpoints were not given for the patients, it is not clear what proportion of patients had the potential for dual expression and whether the observed differences are in fact confirming that 3' breakpoints tend to be relatively more common in blast crisis CML patients. Interestingly, Lee et al. 174 have presented preliminary data that many patients with expression of an mRNA that includes BCR exon 14 (K-28 junctions) have higher platelet and white cell counts

The BCR/ABL Oncogene

3 23

and do not respond as well to interferon therapy compared to patients with mRNA lacking BCR exon 14 (L-6 type). In general, progression to blast crisis is not associated with a switch to production of the P 190 protein that is associated with acute leukemias. Hooberman et al. 1~ described a case of ALL that expressed both the mRNA associated with M-bcr rearrangement positive CML and the mRNA characteristic of M-bcr rearrangement negative, Ph-positive ALL. They speculated that the acquisition of the ALL-type of transcript might be one mechanism whereby acute lymphoid transformation can occur. In a series of 37 accelerated and blast crisis CML patients, dual expression of mRNA encoding for P210 and PI90 was observed only in 3 cases. 175 In two of these three cases samples studied at the time of initial chronic phase diagnosis, only mRNA corresponding to P210 was expressed. Expression of the mRNA corresponding to P 190 in blast crisis CML does not appear to necessarily correspond to the acquisition of a second Ph chromosome. 175 The level of expression of the various fusion genes may also change during disease progression. One of the most common additional cytogenetic abnormalities that characterizes blast crisis is the acquisition of a second Ph chromosome 16 and resulting increased levels of BCR/ABL fusion protein could possibly contribute to the more aggressive leukemic cell phenotype. In the cell line K562, originally derived from a patient with blast crisis CML, 176 the BCR/ABL rearrangement is amplified four- to eightfold with corresponding enhanced expression of mRNA. 177'178 Furthermore, the level of expression of the BCR/ABL mRNA appears to be higher for some other cell lines established from acute phase CML patients. 81'92 Collins and Groudine 155 have described a patient who presented in lymphoid blast crisis with amplification of the rearranged BCR/ABL gene in both blast cells and granulocytes and with overexpression of BCR/ABL transcripts in blast cells relative to granulocytes. Weinstein et al. 36 described a patient with Ph-negative CML who had a BCR/ABL rearrangement that appeared to be duplicated (relative to the copy number for the unrearranged BCR gene) and who rapidly progressed to blast crisis. In a series of 10 patients with blast crisis CML, Andrews and Collins 177 observed significantly elevated levels of transcription of BCR/ABL in 4 patients. No elevation in the transcription of BCR/ABL was observed in a further 7 chronic phase patients. These observations suggest, but do not prove, that the levels of expression of the BCR/ABL fusion gene could sometimes be important in determining disease progression. In summary, blast crisis CML appears to be associated with some degree of alteration in BCR/ABL gene expression which can arise through duplication of the Ph chromosome, changes in splicing of mRNA transcripts, and deregulation of gene expression. Whether or not these are critical in determining a more aggressive cell proliferation or whether other cytogenetic changes, oncogene activations or other changes are of greater importance remains to be determined.

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IX. EXPERIMENTAL STUDIES WITH

BCR/ABL

Cloned cDNA derived from BCR/ABL fusions can be used in experimental studies to assess the effects of the introduction of the fusion gene in various cell types. Plasmid constructs are made where BCR/ABL cDNA is joined to appropriate replication and promoter sequences together with genes encoding for antibiotic resistance to assist in selection of transfected cells. When NIH 3T3 fibroblasts are transfected with a construct that encodes P210, expression of P210 can be demonstrated, but the cells show no morphological signs of transformation. 18~ However, when a gag determinant is fused to BCR/ABL, transformation of NIH 3T3 cells is achieved with cells showing altered morphology similar to that seen when cells are infected with A-MuLV. 18~These results indicate that myristylation-dependent membrane localization is probably necessary for BCR/ABL transformation of fibroblasts. In contrast to the experiments with NIH 3T3 cells, a gag determinant does not to appear to be necessary for the transformation of bone marrow cells. Transformation characteristics are seen when fresh bone marrow cells are infected and maintained under the conditions used for long-term culture of B-lymphoid cell lines. TM Variability is observed in the ability to grow in soft agar and in the induction of lymphomas after inoculation of the transformed cells into syngeneic mice. Transformation is also achieved with long-term B-cell cultures and long-term myeloid cell cultures although the type of transfomaed cell appears to be an early B cell for either type of culture. 182 A murine bone marrow derived cell line thought to be of early B-cell lineage referred to as Ba/F3 can also be transformed by BCR/ABL retroviral constructs. 183 This cell line normally has a requirement for interleukin-3 for growth but after transformation becomes independent of the growth factor. This observation suggests that the P210 protein provides an alternative to interleukin-3 for growth stimulation. The transformed Ba/F3 cells induced tumors in nude mice after a short latency. Differences appear to exist between the P210 and PI90 proteins in terms of their potency to transform cells. ~84 Using retrovirus vectors with structures that differed only in the size of the BCR gene component, McLaughlin et al. ~84 compared the outgrowth of cells transformed by the two types of construct and showed that the growth stimulating effect of P 190 was significantly greater than that for P210. P 190 appeared to be more effective than P210 in the induction of tumors following inoculation into mice, but, as in the case of P210, the presence ofP 190 in transfected cells did not invariably result in tumors. The stronger stimulating effects of P I90 compared to P210 would be consistent with the more aggressive nature of the leukemias associated with P I90 (ALL and AML) compared to most of the leukemias associated with P210 (CML) at presentation. The fact that tumors are not invariably present in mice carrying the transformed cells would seem to indicate a need for additional oncogene activations for tumor formation. ~84

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In the in vitro studies described above, the cell phenotype of transformed bone marrow cells was lymphoid in nature with the induction of lymphomas when cells were reintroduced to mice. In transgenic mice containing BCR/v-abl constructs, T-cell lymphomas and some pre-B-cell lymphomas were found. 185 An alternative model system has been developed which does not limit the tumor type to lymphomas. 186-189 When mouse bone marrow is treated with 5-fluorouracil, transfected, and then returned to irradiated recipients, a diverse set of transformed hematologic cell types is seen. Elefanty et al. 186noted macrophage, erythroid, mast cell, pre-B lymphoid, T-lymphoid, and mixed lineage tumors in their experiments. They noted that the spectrum of tumors observed differed substantially in different stains of mice. Using BALB/c mice, Daley et al. 187 have noted the induction of three disease types: (1) a myeloproliferative syndrome with a mean latency of approximately 9 weeks, (2) an acute lymphoblastic leukemia arising after a mean of 14 weeks, and (3) macrophage derived tumors after an average of 16.5 weeks. Similar results were reported by Kelliher et al. 188 The myeloproliferative disorder strongly resembles chronic phase CML with the animals showing enlarged spleen, increased granulocytes in peripheral blood, and hypercellular bone marrow containing myeloid cells at all stages of differentiation. If the CML-like disorder is transplanted to irradiated syngeneic recipients, a small proportion of animals continue to demonstrate the chronic CML-like disorder or may show an acute leukemia of lymphoid or myeloid typemi.e., the disorder appears to evolve to resemble blast crisis CML. 189 The development of the animal model for CML will clearly be helpful not only in further understanding the role of BCR/ABL fusions but also in providing a model for testing various therapeutic strategies. Selective inhibition of BCR/ABL fusion gene under in vitro conditions provides additional evidence that expression of BCR/ABL is directly associated with the leukemic cell phenotype. Szczylik et al. 19~prepared oligodeoxynucleotides complimentary to mRNA (specifically, the sequences corresponding to the BCR/ABL junctions) and tested the effect of these antisense oligomers on the outgrowth of in vitro colonies from CML blast crisis patients. Fewer colonies with reduced numbers of cells were seen in cultures where a matched antisense oligomer was present compared to control cultures. Residual colonies in the treated cultures lacked detectable BCR/ABL mRNA. Studies of this type clearly have important therapeutic implications; gene-targeted anti-leukemic therapy appears to be possible.

X. OVERVIEW Studies on colon carcinomas and other tumor types has led to a model for cancer in which there is an accumulation of critical mutations with gradual progression to full expression of the cancer cell phenotype. 191 Often, the order in which the mutations arise is unimportant. 192 The BCR/ABL fusion that characterizes many

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human leukemias differs in that this alteration is usually present at diagnosis with subsequent additional mutations occurring during evolution. A few cases of CML preleukemia and acute leukemia have been described that appear to be Ph-negative 193at diagnosis and subsequently acquired a Ph chromosome. The rarity of these cases attests to the role o f BCR/ABL as a very early event in the disease pathogenesis in the vast majority of cases. Whether or not BCR/ABL fusion is truly the seminal change cannot be easily proven. Fialkow et al. 194 noted in a Ph-positive CML patient, a high proportion of Ph-negative B-lymphoid clones showing a particular glucose-6-phosphate dehydrogenase (G6PD) isozyme, identical in type to that seen in the Ph-positive clone. From this observation they suggested that a primary alteration causing proliferation of a pluripotent stem cell proceeds the acquisition of a Ph chromosome. However, distortion in G6PD isozyme types within any particular tissue may not necessarily indicate a neoplastic change that enhanced the proliferative capacity of a stem cell clone. Currently, there remains little data to support the concept of specific alterations preceding the BCR/ABL gene fusion. It is also apparent that retention of the BCR/ABL fusion gene is the usual situation. A few patients have been described in which there may be a loss of the Ph chromosome. 157-159Given the fact that a BCR/ABL fusion is not essential for acute leukemia, and that there must be alternative pathways to develop the leukemic cell phenotype, it is not unexpected that during clonal evolution additional changes could occur within a Ph-positive population that provides an equal or greater opportunity for uncontrolled cell proliferation. Within such an evolving cell clone, the selective advantage in retaining the Ph chromosome may no longer exist. The fact that a single oncogene activation usually characterizes the earliest stages of a disease and that the change usually persists within the clone may eventually prove to be advantageous. While traditional therapeutic strategies have concentrated on abnormal cell eradication, new approaches such as the use of antisense oligomers raise the exciting possibility of selectively inhibiting the production or activity of common aberrant oncoproteins. 190 Clearly, advances in therapeutics can only be made with a thorough understanding of the details of the abnormalities in the cells. The advancements made in the understanding the role of the BCR/ABL oncogene have provided a paradigm for cancer research.

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156. Bartram, C. R.; De Klein, A.; Hagemeijer, A.; Carbonell, E; Kleihauer, G.; Grosveld, G. Additional c-abi/bcr rearrangements in a CML patient exhibiting two Ph I chromosome during blast crisis. Leukemia Res. 1986, 10, 221-225. 157. Bartram, C. R.; Janssen, J. W,; Becher, R. Persistance of CML despite deletion of rearranged bcr/c-abl sequences. Hematol. Bluttransfus 1987, 31, 145-148. 158. Laneuville, P.; Sullivan, A. K. Clonal succession and deletion of bcr/abl sequences in chronic myelogenous leukemia with recurrent lymphoid blast crisis. Leukemia 1991, 5, 752-756. 159. Hagemeijer, A. E.; Smit, M. E.; Lowenberg, B.; Abels, J. Chronic myeloid leukemia with permanent disappearance of the Ph I chromosome and development of new c lonal subpopulations. Blood 1979, 53, 1-14. 160. Reeve, A. E.; Morris, C. M.; Fitzgerald, P. H. Acquired homozygosity of the rearranged bcr allele during the acute leukemic phase of a patient with Ph-negative chronic myeloid leukemia. Blood 1988, 72, 24-28. 161. Ogawa, H.; Sugiyama, H.; Soma, T.; Massaocka, T.; Kishimoto, S. No correlation between locations of bcr breakpoints and clinical states in Phi-positive CML patients. Leukemia 1989, 3, 492-496. 162. Przepiorka, D. Breakpoint zone of bcr in chronic myelogenous leukemia does not correlate with disease phase or prognosis. Cancer Genet. Cytogenet. 1988, 36, 117-122. 163. Jaubert, J.; Martiat, P.; Dowding, C.; lfrah, N.; Goldman, J. M. The position of the M-bcr breakpoint does not predict the duration of the chronic phase or survival in chronic myeloid leukaemia. Br. J. Hematol. 1990, 74, 30--35. 164. Morris, S. W.; Daniel, L.; Ahmed, C. M. I.; Elian, A.; Labowitz, P. Relationship of the bcr breakpoint to chronic phase duration, survival and blast crisis lineage in chronic myelogenous leukemia patients presenting in early chronic phase. Blood 1990, 75, 2035-2041. 165. Tein, H. F.; Wang, C. H.; Chen, Y. C.; et al. Chromosome and bcr rearrangement in chronic myelogenous leukaemia and their correlation with clinical states and prognosis of the disease. Br. J. Hematol. 1990, 75, 469-475. 166. Tefferi, A.; Bren, G. D.; Wagner, K. V.; Schaid, D. J.; Ash, R. C.; Thibodeau, S. N. The location of chromosomal breakpoint site and prognosis in chronic granulocytic leukemia. Leukemia 1990, 4, 839-842. 167. Dreazen, O.; Berman, M.; Gale, R. P. Molecular abnormalities of bcr and c-abl in chronic myelogenous leukemia associated with a long chronic phase. Blood 1988, 71,797-799. 168. Nowell, P. C.; Jackson, L.; Weiss, A.; Kurzrock, R. Historical communication: Philadelphia-positive chronic myelogenous leukemia followed for 27 years. Cancer Genet. Cytogenet. 1988, 34, 57-61. 169. Birnie, G. D.; Mills, K. I.; Benn, P. Does the site of the breakpoint in chromosome 22 influence the duration of the chronic phase in chronic myeloid leukemia? Leukemia 1989, 3, 545-547. 170. Mills, K. 1.; Benn, P.; Birnie, G. D. Does the breakpoint within the major breakpoint cluster region (M-bcr) influence the duration of the chronic phase in chronic myeloid leukemia? An analytical comparison of current literature. Blood 1991, 78, 1155-1161. 171. Schaefer-Rego, K.; Dudek, H.; Popenoe, D.; et ai. CML patients in blast crisis have breakpoints localized to a specific region of the BCR. Blood 1987, 70, 448-455. 172. Lee, M. S.; LeMaistre, A.; Kantarjian, H. M.; et al. Detection of two alternative bcr/abl mRNA junctions and minimal residual disease in Philadelphia chromosome positive chronic myelogenous leukemia by polymerase chain reaction. Blood 1989, 73, 2165-2170. 173. Morgan, G. T.; Hernandez, A.; Chan, L. C.; Hughes, T.; Martiat, P.; Wiedemann, L. M. The role of alternative splicing patterns of BCR/ABL transcripts in the generation of the blast crisis of chronic myeloid leukemia. Br. J. Haematol. 1990, 76, 33-38. 174. Lee, M.; Kantarjian, H.; Deisseroth, A.; Freireich, E.; Trujillo, J.; Stass, S. Clinical investigation of BCR/ABL splicing patterns by polymerase chain reaction (PCR) in Philadelphia chromosome (Ph l) positive chronic myelogenous leukemia (CML). Blood 1990, 76, 294a.

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175. Dhingra, K.; Talpez, M.; Kantarjian, H.; et al. Appearance of acute leukemia-associated P190 BCR-ABLin chronic myelogenous leukemia may correlate with disease progression. Leukemia 1991, 5, 191-195. 176. Lozzio, C. B.; Lozzio, B. B. Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 1975, 45, 321-334. 177. Collins, S. J.; Groudine, M. T. Rearrangement and amplification of c-abl sequences in the human chronic myelogenous leukemia cell line K-562. Proc. Natl. Acad. Sci. USA 1983, 80, 4813-4817. 178. Leibowitz, D.; Cubbon, R.; Bank, A. Increased expression of a novel c-abl-related RNA in K562 cells. Blood 1985, 65, 526-529. 179. Andrews, D. F.; Collins, S. J. Heterogeneity in expression of the bcr-abl fusion transcript in CML blast crisis. Leukemia 1987, 1, 718-724. 180. Daley, G. Q.; McLaughlin, J.; Witte, O. N.; Baltimore, D. The CML-specific P210 bcr/abl protein, unlike v-abl does not transform NIH/3T3 fibroblasts. Science 1987, 237, 532-535. 181. McLaughlin, J.; Chianese, E.; Witte, O. N. In vitro transformation of immature hematopoietic cells by the P210 bcr/abl oncogene product of the Philadelphia chromosome. Proc. Natl. Acad. Sci. USA 1987, 84, 6558-6562. 182. Young, J. C.; Witte, O. N. Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures. Mol. Cell, Biol. 1988, 8, 4079-4087. 183. Daley, G. Q.; Baltimore, D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210 bcr/abl protein. Proc. Natl. Acad. Sci. USA 1988, 85, 9312-9316. 184. McLaughlin, J.; Chianese, E.; Witte, O. N. Alternative forms of the BCR-ABL oncogene have quantitatively different potencies for stimulation of immature lymphoid cells. Mol. Ceil, Biol. 1989, 9, 1866-1874. 185. Hariharan, I.K.; Harris, A. W.; Crawford, M.; et ai. A bcr-v-abi oncogene induces lymphomas in transgenic mice. Mol. Cell, Biol. 1989, 9, 2798-2805. 186. Elefanty, A. G.; Hariharan, I. K.; Cory, S. bcr-abl the hallmark of chronic myeloid leukemia in man, induces multiple haematopoietic neoplasms in mice. EMBO J. 1990, 9, 1069-1078. 187. Daley, G. Q.; Van Etten, R. A.; Baltimore, D. Induction of chronic myelogenous leukemia in mice by the P210 bcr/abl gene of the Philadelphia chromosome. Science 1990, 247, 824-830. 188. Kelliher, M. A.; McLaughlin, J.; Witte, O. N.; Rosenberg, N. Induction of a chronic myelogenous leukemia-like syndrome in mice with v-abl and BCR/ABL. Proc. Natl. Acad. Sci. USA 1990, 87, 6649-6653. 189. Daley, G. Q.; Van Etten, R. A.; Baltimore, D. Blast crisis in a murine model of chronic myelogenous leukemia. Proc. Natl. Acad. Sci. USA 1991, 88, 11335-11338. 190. Szczylik, C.; Skorski, T.; Nicolaides, N. C.; et al. Selective inhibition of leukemia cell proliferation by BCR-ABL antisense oligodeoxynucleotides. Science 1991, 253, 562-565. 191. Vogelstein, B.; Fearon, E. R.; Hamilton, S. R.; et al. Genetic alterations during colorectal-tumor development. N. Engl. J. Med. 1988, 319, 527-532. 192. Vogelstein, A. Deadly inheritance (editorial). Nature 1990, 348, 681-682. 193. Sandberg, A.A. The Chromosomes in Human Cancer and Leukemia, 2nd ed. Elsevier, New York 1990, p. 479. 194. Fialkow, P. J.; Martin, P. J.; Najfield, V.; Penfold, G. K.; Jacobson, K. J.; Hansen, J.A. Evidence for a multistep pathogenesis of chronic myelogenous leukemia. Blood 1981, 58, 158-163. 195. Sagio, G.; Guerrasio, A.; Rosso, C.; et al. New type of bcr/abl junction in Philadelphia chromosome-positive chronic myelogenous leukemia. Blood 1990, 76, 1819-1824. 196. Soekarman, D.; van Denderen, J.; Hoefsloot, L.; et al. A novel variant of bcr-abi fusion product in Philadelphia chromosome positive acute lymphoblastic leukemia. Leukemia 1990, 4, 397-403. 197. Ishihara, T.; Sasaki, M.; Oshimura, M.; et al. A summary of cytogenetic studies in 534 cases of chronic myeloid leukemia in Japan. Cancer Genet. Cytogenet. 1983, 9, 81-93.

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A D V E N T U R E S IN M Y c - O L O G Y f

Paul G. Rothberg and Daniel P. Heruth

1. II.

II1.

Introduction

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The Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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O v e r e x p r e s s i o n o f c - m y c is C a r c i n o g e n i c : You C a n H a v e T o o M u c h o f a G o o d T h i n g . . . . . . . . . . . . . . . . .

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The myc Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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What Does myc Do?

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Global myc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Breast

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Cervix, Ovary, and Uterus

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Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Prostate

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Testes

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Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Bladder

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Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Advances in Genome Biology Volume 3B, pages 337-414. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8

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K. Stomach and Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . L. Colon and Rectum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Putting Things in Perspective: Does myc Cause Cancer Everywhere? . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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tThis chapter is a review of selected topics concerning the myc oncogene. It does not deal in any way with fungi.

INTRODUCTION Why another review of the myc gene? This gene has been well reviewed in the past. General reviews have been published 1-4 and reviews of specific areas have been published in abundance. 5-16 On the other hand, the field moves so fast that there is a need for frequent updates. The literature on the myc gene is huge and fascinating. It is beyond the capabilities of the authors to cover the field in its entirety. In this review we will focus primarily on the involvement of this gene in abnormal growth. Thus, this paper will be a specific review of the literature on the involvement of the c-myc gene in disease. Related issues will be discussed, such as tissue specificity of the regulation of c-myc expression, and the clinical implications of alterations in the structure and expression of the c-myc gene. Whenever appropriate we will refer to other reviews for detailed background arguments and references to points which have been well covered in the recent literature. However, we will neglect some very interesting areas like the still very active research on the mechanisms of control of myc gene expression. We will also concentrate on the c-myc gene, neglecting the other members of the myc gene family. In Section II of this review, we will discuss the foundations of the study of the myc oncogene which were derived mostly from studies on fibroblasts and hematopoietic cells. In Section III, we will focus on other tissues which are less well understood, but extremely important in order to appreciate the importance of the c-myc gene in neoplasia. il.

C L A S S I C MYC

A. The Basics The human c-myc gene consists of three exons and covers about 5200 nucleotides. It is located on the q24 band of chromosome 8.17-19 The entire gene and surrounding region have been sequenced. 2~ There are two main promoters for transcription located 160 base pairs (bp) apart, and several other promoters which are used less frequently: one upstream of the gene and a cluster in the first intron. 21'23-25 Two main proteins are encoded by the c-myc gene: one starts at an A U G codon in exon 2, and the other uses the more unusual C U G codon for initiation in exon 1.26 The protein products have estimated molecular masses between 62 and

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68 kDa in human cells and are phosphorylated. 27-29 There is evidence for an additional protein product encoded by the first exon. 3~ The main protein products of the c-myc gene are located in the nucleus of the cell and are loosely associated with chromatin, except during mitosis. 33-36 During mitosis, c-myc protein is more highly phosphorylated and it relocates throughout the cytoplasm. 34'37 Both c-myc RNA and protein are very unstable with estimated half-lives of 15-30 minutes.28,29,38-41

B. Ancient

myc-Ology

Many of the now familiar concepts concerning the molecular biology of cancer were derived from the study of viral oncology. This includes a major contribution to the list of the dominantly acting transforming genes, called oncogenes. Although the study of the viral oncogenes today is not the dominant theme in the molecular biology of cancer, an understanding of the contributions from viral research is a necessary element in understanding any of the oncogenes. In this section we will briefly review the lessons from viral oncology with respect to the c-myc gene. The myc gene was discovered in the avian acute transforming retroviruses CMII, MC29, MH2, and OK I0. 42 The viruses in this group cause a broad spectrum of malignancies in vivo, including sarcomas, carcinomas, and myelocytomas, and also possess the ability to transform fibroblasts, epithelial cells, and bone marrow cells in culture. 43 As for all of the other retroviral oncogenes, the viral myc gene (v-myc) was derived from the host cell genome by a recombination event between a replication competent retrovirus and a preexisting host cell gene, in this case the cellular myc gene (c-myc). As a result of the recombination, the virus lost some of the functions required for its own propagation; thus, most of the acute transforming retroviruses replicate as two-virus systems with the transforming component containing the host cell derived oncogene, and a replication competent helper virus that supplies in trans the functions needed for viral propagation. As an example, the MC29 virus contains approximately 1600 bases of myc-specific sequences in a 5700-base genome. 44'45 The viral pol (reverse transcriptase) gene and parts of the gag and env genes were deleted. The myc gene is expressed as a fusion protein with the remaining portion of the gag gene. 46 The CMII, MH2, and OK10 viruses have different structures from MC29 and from each other; thus, they were born in separate myc transduction events. 47-52 A detailed description of the discovery, structure, and expressed oncoproteins of the v-myc family of avian retroviruses is contained in the review of Erisman and Astrin. 2 How frequently do retroviruses arise with a myc oncogene in nature? As you will see in the remainder of this review, this event takes place frequently in laboratories. Infection with feline leukemia virus, a replication competent retrovirus that does not carry a host cell derived oncogene, has been found to result in the production of a recombinant provirus containing the cat c-myc gene in about 10% of the

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induced T-cell lymphomas. 53-57 Thus, the finding of several independent retroviruses with a myc oncogene has been seen in two species, chickens, and cats. There is ample evidence that the v-myc sequences are responsible for the ability of MC29 to cause tumors. Three mutants of MC29 have been isolated that were capable of transforming fibroblasts in vitro, but were severely deficient in transforming macrophages, as compared to wild-type virus. 58 The mutants also had a severely lower pathogenic potential in vivo. 59 Analysis of the RNA from these mutants showed overlapping deletions between 200 and 600 bases in length. 6~The protein products of the mutant viruses were smaller than the wild-type 110-kDa gag-myc fusion protein, with molecular masses of 100, 95, and 90 kDa. 61 Studies of both RNA and protein demonstrated that the deletions occurred in myc-specific sequences and did not involve the gag domain. 6~ Another group showed that a deletion in the 3' portion of the v-myc gene caused a reduction in the ability of MC29 to transform fibroblasts. 62 In addition, the v-myc sequences in MH2 have been shown to be responsible for its ability to transform fibroblasts. 63 From these studies we can conclude that v-myc sequences are an important determinant in transformation and that separate functional domains of the v-myc gene may be involved in transformation of fibroblasts and macrophages. We know that v-myc can cause cancer in chickens and that this gene was obtained from the host genome. This leads to two questions: (1) what is different about the v-myc gene that makes it a potent carcinogenic agent?, and (2) does the host cell c-myc gene cause cancer? We will answer the first question here and attempt to answer the second question in the remainder of this review. A comparison of the nucleotide sequences of 4 avian v-mycs and chicken c-myc revealed several differences: 44'45"48'49'52'64-67 I. 2. 3.

The v-myc genes lack the introns and the noncoding first exon of c-myc. Some of the v-myc genes were expressed as g a g - m y c fusion proteins. Point mutations that change amino acids in the encoded protein have occurred in the v-myc genes.

These structural changes are also accompanied by a change in regulation, because the viral myc genes are under control of the viral regulatory elements and removed, in large part, from the host cell systems that normally regulate the expression of the c-myc gene. A number of experimental approaches have revealed that aberrant regulation of expression of the myc gene is a critical event in its activation to a carcinogenic agent. However, in comparing the biological potency of v-myc and c-myc there is some evidence for structural changes in v-myc that may be considered activating. Several biological assays can distinguish the transformation potency of chicken c-myc from the potency of the v-myc gene in MC29 and MH2 when expressed similarly. 68'69 A comparison of the activity of MC29 v-myc and chicken c-myc in their ability to transform myelomonocytic cells from mice revealed that point mutations in MC29 v-myc contributed to its transforming potential.V~ A

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mutation at position 61 of the chicken c-myc gene, a position that was found mutated in MC29, MH2, and OK10, activated its ability to transform fibroblasts. 71 Is v-myc sufficient for transformation? We will return to this theme of biological potency, or spectrum of carcinogenic activity, of the myc gene repeatedly throughout this review as one of its central themes. The v-myc gene, as described earlier, is responsible for the ability of the MC29 virus to cause several malignancies in vivo and to transform cells in culture. However, the MC29 virus was a much less potent carcinogenic agent in Japanese quail than the MH2 virus. 72 This may be accounted for by the fact that MH2 has a second host cell derived oncogene, called mil o r mht. 47'49 The mouse homolog of the mil gene (raf) was transduced by a murine retrovirus (murine sarcoma virus 3611) in which this gene is an oncogene in its own right. This story was further complicated by evidence that the mil gene does not contribute significantly to the biological activity of MH2. 63'72 The ability of the v-myc gene to transform fibroblasts has been questioned. Although primary chick embryo and quail embryo fibroblasts that have been infected with MC29, or other members of the myc group of avian defective retroviruses, appeared transformed by several criteria, they were not tumorigenic in syngeneic animals or nude mice, respectively. 73'74 The v-myc gene derived from OK I0, in a murine retroviral construct, was able to transform established and primary mouse fibroblasts by the criteria of anchorage independent growth, but the colonies were not as large as ras or src transformed cells, and tumorigenicity was not evaluated. 75 It seems that there are limitations to the biological potency of the myc oncogene.

C. Overexpression of c-myc is Carcinogenic: You Can Have Too Much of a Good Thing Mice that lack both copies of the c-myc gene do not progress past day 10.5 of gestation. 76 Thus, myc null mutants are genetic lethals, and the myc gene is essential. Therefore, we are not going too far afield in calling the myc gene a good thing. However a tremendous body of experimental evidence has been amassed which supports the theory that overexpression of the myc gene contributes to malignancy. In this section, we will review some of the evidence for the importance of the control of expression of a nonmutated myc gene in hematopoietic cells and fibroblasts. In other sections we will review the evidence for other cell types. The avian leukosis virus (ALV), which is a replication competent retrovirus that does not carry an oncogene, can cause bursal lymphoma in susceptible strains of chickens only after a long incubation. In most of the induced lymphomas an ALV regulatory element such as the promoter or an enhancer was found in the immediate vicinity of the c-myc gene and caused a big increase in its expression. 77'78 The chick syncytial virus, which is unrelated to ALV, caused bursal lymphomas with apparently similar integration events. 79 Similar events have been shown to occur in mouse T-cell lymphomas induced by replication competent murine retroviruses. 8~ The

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PAUL G. ROTHBERG and DANIEL P. HERUTH

viral integration events were shown to cause an increase in expression of the targeted gene. These viral systems serve to provide evidence that the host cell c-myc gene can become a carcinogenic agent when its normal regulation is disrupted, without having to be transduced by the virus. More detailed information on this type of oncogenesis can be found in several reviews? '83'84 If a virus can integrate near the c-myc gene and activate its expression can a genetic rearrangement within a cell generate an activated myc gene without involving a virus? The answer to this question is yes, and was provided by the study of Burkitt's lymphoma and mouse plasmacytoma. In these diseases a chromosome translocation is commonly found which has been shown on a molecular level to result in the juxtaposition of the c-myc gene with an immunoglobulin gene. 18'85-88 The immunoglobulin genes are normally expressed in cells of the B-cell lineage, and the c-myc gene comes under the control of this active area of chromatin as the result of the translocation. The translocations themselves are probably the result of errors in the DNA rearrangements which generate functional immunoglobulin genes. The details of the rearrangements and the consequences for control of c-myc expression is an extensive story that has been well reviewed. 4'15 To evaluate the effects of abnormally regulated c-myc expression on lymphoid cells, several groups have made transgenic animals with a c-myc gene linked to an immunoglobulin heavy-chain enhancer. 89-91 These transgenic mice and rabbits got lymphoid malignancies which were mono- or oligoclonal. 89-91 The fact that the tumors started from at most a few of the susceptible lymphocytes carrying the transgene indicated that other events must occur to get a malignancy besides deregulation of myc expression. In transgenic mice an early event was found to be a large expansion of actively growing pre-B lymphocytes. 92 The transgenic work confirms the implication from the findings in Burkitt lymphoma and mouse plasmacytoma that abnormal regulation of the c-myc gene is a carcinogenic event in lymphocytes. Myeloid cells are also susceptible to transformation by c-myc. A recombinant retrovirus containing the c-myc gene was able to cause clonal monocyte-macrophage tumors in infected m i c e . 93'94 Mouse bone marrow cells infected in vitro gave rise to partially transformed cells that required CSF-I for growth. 94 The situation in fibroblasts is equally interesting. Increasing c-myc expression in established rodent fibroblasts by inserting an exogenous c-myc gene driven by a strong promoter caused the acquisition of tumorigenicity without causing gross morphological alterations. 95- 97 In the experiments described in the last section which showed a lack of tumorigenicity in cells containing v-myc, primary cells were used as opposed to the established cell lines used here. The host cell is a critical variable in this type of experiment. Zerlin et al. 98 found that early and late passages of the same rat fibroblast cell line differed in the degree of transformation in response to forced expression of an exogenous c-myc gene. The cells which had been in culture for longer periods were more susceptible to transformation by c - m y c . 98

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In primary rodent fibroblasts, the c-myc gene activated by way of strong expression, together with a Ha-ras gene activated by mutation, were necessary to get morphological transformation, while each gene alone was not sufficient. 99 Using this cotransformation assay, the level of expression of the c-myc gene was shown to be the most critical variable for demonstrating its potential carcinogenic activity.lOO,lol Another event which can result in enhanced expression of a gene is an increase in the number of genes, which is referred to as gene amplification. In the classic case, selection of cells for resistance to methotrexate often results in increased expression of dihydrofolate reductase, the enzyme which is inhibited by methotrexate, due to an amplification of the gene encoding this enzyme. 102 Similarly elevated expression of the c-myc gene can sometimes be attributed to an accompanying amplification of the gene. The first discovered example of c-myc gene amplification was in the human myeloid leukemia cell line HL60.1~176 Throughout this chapter we will review the incidence of c-myc gene amplification in each malignancy covered. For most tumor types, amplification of the c-myc gene is an occasional or a rare finding. For the purposes of this section, however, myc gene amplification provides evidence for the importance of quantitative alterations in the expression of the myc gene. Among tumors with a high level of expression of the c-myc gene some have amplification or rearrangement of the gene, but many have no detectable cis alteration (many references throughout); the elevated expression in these tumors is likely caused by a trans mechanism. The usual control mechanisms which regulate expression of the c-myc gene in the immortal, but nontumorigenic, mouse fibroblast cell line A31 were lost in its tumorigenic descendants without any detectable alteration in the encoding gene. 1~ The expression of the c-myc gene in these cells was stuck at the high end of the normal transient increase seen in quiescent cells stimulated to proliferate. 1~ This type of finding leads to use of the term deregulated~applied when an abnormally high level of expression of the c-myc gene is found in a tumor. The term, deregulated, is not quite correct since the gene is still under some type of control, but this control is not producing the same effect on expression of the gene as the regulatory mechanisms operative in the corresponding nonmalignant tissue. An interesting contribution to this area is the finding that the autoregulatory suppression of c-myc expression exerted by the c-Myc protein product is absent in many malignant cell lines. 1~176 It will be interesting when the trans deregulation of c-myc expression is understood to the extent of tracing the causality back to a carcinogenic mutational event. As the (patient) reader will note in the second part of this chapter, the c-myc gene is expressed at an elevated level in some fraction of cases in many types of neoplasia. In some cases the reason for overexpression is obvious, but in many cases the reasons are yet unknown.

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D. The myc Family The c-myc gene, like most genes, has a family of related sequences. The N-myc gene was discovered because of its amplification in some neuroblastomas, and the L-myc gene was discovered because of its amplification in some small cell lung cancers. 1~176 Both genes have been shown to have biological activity similar to the c-myc gene. lll-ll3 Other members of the family are the B-myc and S-myc genes.114,115 The B-myc gene does not have a DNA binding and dimerization motif as in c-, L-, and N-myc, but it does have a transcriptional activation domain, ll6 The B-myc gene inhibited transformation by c-myc in the ras cotransformation assay. I16 The S-myc gene suppressed tumorigenicity in the rat neural tumor cell line RT4A C . 114 However, this cell line has a neu gene activated by point mutation. 117 In view of the finding that overexpression of the c-myc gene suppressed the expression of the neu gene, and transformation induced by an activated neu gene, in NIH/3T3 cells, lIB the suppression of tumorigenicity by S-myc in the RT4-AC cells should not be interpreted as suggesting a generalized tumor suppressor function for the S-myc gene. The P-myc and R-myc genes are less well studied, but an initial report revealed transforming activity for the R-myc gene. il9

E. What Does c-myc Do? The c-myc gene, when expressed at a high level and removed from its normal control mechanisms, is carcinogenic. However, it must have some other function in control of cellular growth and differentiation besides causing cancer. In this section we will review the work which demonstrates a role for c-myc in cell growth and differentiation, a role in cell death, our present understanding of the biochemical function of its protein product, and its impact on response to cancer therapy.

Proliferation and Differentiation What does myc do to a cell to make it transformed? Here we will look at the changes in proliferative behavior of cells in response to deregulated c-myc expression. Primary cells have a limited proliferative potential in culture. Elevated expression of a transfected c-myc gene has been associated with immortalization of primary fibroblasts, l~176176 In fact, the spontaneous immortalization of three different strains of rodent fibroblasts into immortal cell lines was accompanied by an increase in the expression of c-myc RNA. 121 However, the association of deregulated c-myc expression with tumorigenicity in established rodent fibroblast cell lines, which are already immortal, reveals that the immortalizing function of the c-myc gene is not the whole story. The decision of a resting fibroblast to proliferate involves two steps: the acquisition of competence, which can be initiated by platelet derived growth factor (PDGF); and the decision to enter S phase and synthesize DNA, which can be

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induced by several agents called progression factors. The finding that the competence inducing growth factor PDGF also caused an increase in expression of the c-myc gene suggested that myc may mediate some or all of the effects of PDGF on cellular proliferation. 122 This has been tested in several ways: 1.

Expression of a transfected c-myc gene or microinjection of Myc protein caused a reduction in the need for PDGF to get proliferation of rodent fi broblasts. 123,124

2.

Expression of an exogenous c-myc gene caused an increase in DNA synthesis without an increase in cellular proliferation in cells grown in a defined medium without additional progression factors. 125 The response of several rodent fibroblast cell lines to stimulation by a progression type growth factor was enhanced by elevated expression of an exogenous myc gene. 126,127 Forced elevated expression of the c-myc gene could mediate some of the aspects of competence, but did not completely replace PDGF.123'I27

3.

4.

In a number of systems the transition of a resting cell to active proliferation is associated with an increase in c-myc expression that is usually transient. The association of PDGF with an increase in expression in fibroblasts was described above. Also in fibroblasts, the mitogens bombesin, transforming growth factor c~, and epidermal growth factor caused an increase in expression of the c-myc gene. 128-131 In B-lineage lymphocytes c-myc expression was induced by the mitogens lipopolysaccharide, interleukin-7, anti-immunoglobulin antibodies, or an appropriate antigen. 122'132-134 In T lymphocytes, c-myc expression was induced by concanavalin A and monoclonal antibody OKT3 which reacts with the antigen receptor. 122'135 In erythropoietin sensitive cells of the erythroid lineage, erythropoietin caused an increase in expression of the gene. 136 In all cases examined, the response of the c-myc gene occurred before the cells actually began to make DNA. Other examples of this type of experiment will be described in later sections of this review. These experiments tell us that c-myc expression is associated with the decision to proliferate in many cell types. The induction of differentiation in vitro is associated with a reduction in expression of the c-myc gene in many cell types including myeloid leukemia, erythroleukemia, myoblasts, teratocarcinoma, thyroid carcinoma, and colon carcinoma, 105'137-149 although there are a few exceptions, such as mouse keratinocytes, chicken lens epithelium, the human teratocarcinoma cell line Tera-2, and chronic lymphocytic leukemia cells. 15~ The association of differentiation with lower c-myc expression in the majority of cases suggests that myc may have something to do with maintaining the dedifferentiated state. Direct evidence for this has been found in mouse preadipocyte, erythroleukemia, and myoblast cultures in which forced elevated expression of an exogenous c-myc gene blocked differentiation. 154--16~

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Another line of experimentation which reveals the biological activity of the c-myc gene involves reducing the level of myc expression with antisense nucleic acids. The antisense nucleic acid has a sequence complementary to the c-myc mRNA and presumably binds to it and prevents translation, or possibly promotes degradation of the RNA by a double-strand specific ribonuclease. The antisense nucleic acid may be DNA or a variant which facilitates entry into the cell and resistance to nucleases. The addition of an antisense nucleic acid to a cell with a consequent reduction in expression of Myc protein has been seen to cause a reduction in proliferation of T lymphocytes, F9 teratocarcinoma cells, mouse erythroleukemia cells, human myeloid leukemia cell line HL60, human keratinocytes, a breast cancer cell line, COLO320 colon carcinoma cells, muscle cells, and ras transformed NIH/3T3 mouse fibroblasts. 161-171 In many cases, the growth arrest was accompanied by signs of differentiation. 162-165 The bottom line of the experiments described or listed in this section is that the c-myc gene is an essential element in promoting cellular proliferation in many cell lineages. Control ofmyc expression is tantamount in these cells to control of growth. Deregulated expression of this oncogene also inhibits differentiation, either as a direct effect or as a consequence of promoting proliferation.

Apoptosis The biological activity of the c-myc gene is usually connected with cellular proliferation. However, a body of evidence is accumulating which suggests a role for this gene in programmed cell death, a process called apoptosis. The sequence of events seen in this process involves a condensation of the cell nucleus and degradation of nuclear DNA to nucleosome sized fragments. 172 When either a mouse myeloid cell line or a rat fibroblast cell line were growth arrested and strong exogenous c-myc expression was induced, the cells underwent apoptosis. 173'174The process was dependent on both growth arrest and expression of a functional c-myc gene. 173'174 In order for the myc gene to cause neoplastic transformation of a cell type that is susceptible to myc induced apoptosis, the process of apoptosis must be defeated. One mechanism for stopping apoptosis that was found to be effective in both fibroblasts and lymphocytes is the expression of the bcl-2 oncogene. 175,176 Another way to overcome apoptosis is to proliferate continuously, due either to a constant extracellular proliferative stimulus or another mutation in a growth controlling gene. The involvement of the c-myc gene in apoptosis may be a type of anti-cancer safety valve in which a cell that has lost control of its c-myc gene, but is not yet fully transformed, dies before another mutation(s) completes the process. On a more speculative note, this finding may provide one of the reasons why toxic agents, which cause a compensatory increase in cell division, are carcinogenic. 177 These agents may allow cells with deregulated myc expression to avoid apoptosis and expand in number. This would provide more time and more targets for further mutational events that complete the carcinogenic process.

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Inhibition of c-myc expression using an antisense oligonucleotide blocked apoptosis in a T-cell hybridoma when the apoptosis was induced by activation of the CD3 T-cell receptor, but not when apoptosis was induced by dexamethasone. 178 Thus, c-myc expression is necessary for some pathways of apoptosis but not others. The involvement of the c-myc gene in inducing apoptosis may explain a number of interesting experimental results. Expression of very high levels of Myc protein in Chinese hamster ovary fibroblasts appeared to be toxic. 179'j80The rat fibroblast cell line 208F, in which a highly expressed human c-myc gene had been introduced, produced rapidly growing tumors in immune-suppressed mice. 181 These tumors had a high mitotic rate, but also a high rate of apoptosis, compared with the parental cell line and tumors induced by expression of a Ha-ras gene. 181 Transformation of chicken embryonic bursal lymphocytes to a preneoplastic state with v-myc made the cells more susceptible to apoptosis induced by gamma radiation, or disruption of cell to cell contacts, than normal bursal lymphocytes. 182 Invasive lymphomas derived from v-myc transformed preneoplastic cells were resistant to the induction of apoptosis. 182 Is the c-myc gene involved in apoptosis in epithelial cells? This question has not been answered yet; however, several experiments suggest that the answer may be yes. In transgenic mice in which c-myc expression was directed to the acinar cells of the pancreas an early event was increased apoptosis in the pancreas and smaller than normal organs in some mice. 183 In rat ventral prostate induced to atrophy by withdrawal of androgen, c-myc expression increased as the secretory epithelial cells underwent apoptosis. 184'185 Similarly, the expression of c-myc RNA increased in nude mouse xenografts of the estrogen-dependent breast cancer cell line MCF-7 during the apoptosis that preceded the regression of the tumor in response to withdrawal of estrogen. 186 In HeLa cells exposed to y-interferon, expression of the c-myc gene was increased at 24-40 hours, which was prior to cell death by 72 hours. 187 A most recent example is the finding that there was an increase in c-myc RNA in nasopharyngeal carcinoma cells preceding apoptosis induced by vitamin K3.188

Biochemical Aspects The evidence for the protein product of the c-myc gene (Myc) being involved in DNA synthesis has been well reviewed. 1 The most recent work implicates Myc as a sequence specific DNA binding protein which controls the expression of other genes, possibly including itself. However, this evidence does not preclude the possibility that Myc is involved in DNA synthesis. The protein product of the c-myc gene is located in the nucleus. 33'35'36The amino acid sequence of Myc contains the basic region helix-loop-helix and leucine zipper motifs which have been associated with sequence-specific DNA binding proteins. 8 The Myc protein has been shown to bind to the sequence 5'-CACGTG, 189-191 and is capable of activating transcription. 192-194 It binds as a heterodimer with a protein, called Max, which is required

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for transactivation of gene transcription in vivo. 194-196 Max can form homodimers with itself that bind to the same sites as the Max/Myc heterodimer. 197'198 However, the Max protein does not stimulate transcription. The Max/Max homodimer has the opposite effects as the Max/Myc heterodimer, perhaps by binding and not activating transcription in competition with the Max/Myc heterodimers which bind and enhance transcription. 193'194'198-2~ If Myc is a transcriptional activator, which genes are activated? (1) ~-prothymosin, a nuclear protein associated with cell proliferation, (2)ornithine decarboxylase, (3) the p53 tumor suppressor gene, (4) the 70-kDa human heat-shock protein gene, (5) plasminogen activator inhibitor 1, (6) cyclin A, and (7) cyclin E were all activated by Myc. 2~176 For ornithine decarboxylase and p53, the activation by Myc required the CACGTG binding site, which in both cases was located in the first intron. 2~176 Thus, the effect of Myc on the p53 and ornithine decarboxylase genes is likely to be a direct effect. Myc expression was also associated with decreased expression of some genes. Expression of the neu, cyclin D l, metallothionein I, HLA class I, LFA-I, two variants of histone H 1, and several collagen genes have been shown to be decreased by Myc. 118'2~176 In the case of the collagen genes, a likely mechanism involves an alteration in the CCAAT transcription factor/nuclear factor I 213 It is not yet clear if all the genes whose expression is inhibited by Myc involve an indirect mechanism as is likely for the collagen genes. 213 The Myc protein product has been implicated in control of the stability of intranuclear RNAs. 214'215 In particular, the plasminogen activator inhibitor 1 RNA in rat fibroblasts 2~ and ribosomal RNAs in Xenopus oocytes 215 were stabilized. Does Myc induce the transcription of a protein(s) that controls the stability of these RNAs, or is there a direct effect of a Myc protein on RNA stability ? Consistent with the latter is the intranuclear colocalization of Myc protein in the small nuclear ribonucleoprotein particles of rodent fibroblasts 36 and the nucleoli of Xenopus oocytes. 216 Myc is unlikely to be the only factor regulating the genes listed above. Therefore, the finding that Myc is involved in regulation of a given gene in one experimental system may not be applicable in another cell type or state of differentiation in which the milieu of other factors regulating gene expression will be different. Myc protein also regulates the expression of its own encoding gene. 217-221 Myc proteins have been shown to cause a decrease in transcription of the c-myc gene. 217'221 In some tumors with elevated c-myc expression the autoregulatory mechanism seems to be inoperative. 1~176 Some workers have found that Myc protein causes an increase in expression of the c-myc gene under certain conditions.2222 23 Which of the putative Myc functions are responsible for the ability of the c-myc gene to transform cells? Regions in Myc that are essential for cellular transformation have been identified by directed mutagenesis and testing of the mutants using the ras cotransformation assay on primary rat fibroblasts, and/or transformation of

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established rat fibroblasts, while other regions have been identified as being dispensable for these biological activities. 224'225 The domains of Myc involved in heterodimer formation, sequence-specific DNA binding, transcriptional activation, and autoregulation were shown to be essential regions for transformation. 192'199'2~ A correlation has also been demonstrated between the regions of the c-myc gene needed for inhibition of adipocyte differentiation and the regions needed for transformation of an immortalized rat fibroblast cell line. 227

Cancer

Therapy

Does the level of expression of the c-myc gene have an impact on the sensitivity of a cell to cancer therapy? In other sections of this chapter we will address the impact of alterations in the structure and expression of the c-myc gene on prognosis in various types of cancer. Here we will review the evidence, based on direct experiments, for a role of the c-myc gene in altering the way a cell responds to chemotherapy and radiation. Primary rat embryo cells, transfected with a mutationally activated Ha-ras gene, became slightly resistant to X-radiation compared to the parental cells, an effect which was enhanced by cotransfection of a v-myc gene, while the v-myc gene alone did not alter sensitivity to radiation. 228 Increased c-myc expression was also associated with resistance to y-radiation in human fibroblasts derived from the skin of individuals with the Li-Fraumeni syndrome. 229 It is interesting that variant small cell lung cancer cell lines, which frequently have elevated c-myc expression, also are relatively resistant to X-rays. 23~ Another group inserted a c-myc construct, which was inducible by zinc ions due to a metallothionein promoter, into rat embryo cells and found that inducing increased c-myc expression increased the yield of cells having methotrexate resistance and amplification of the dihydrofolate reductase gene when the cells were cultured under selective conditions. 232 This result may be related to the finding that transfection with a construct that causes expression of a v-myc or c-myc gene in rat fibroblasts caused an increase in sister chromatid exchange and aneuploidy. 233 Unequal sister chromatid exchange is one of the events that can cause gene amplification. Increased myc expression in NIH/3T3 cells, due to transfection of a construct with the c-myc gene under control of a retroviral promoter, was associated with an increase in resistance to several chemotherapeutic agents including cisplatin and adriamycin. 234 This result was not due to increased expression of the multiple drug resistance gene or the glutathione S-transferase-n: gene. TM Similar results were obtained using a murine erythroleukemia cell line in which increased expression of an exogenous c-myc gene caused increased resistance to cisplatin, while decreased c-myc expression caused by transfection of a c-myc antisense construct resulted in increased sensitivity to cisplatin. 235 The one discordant study in this group was the finding that two drug resistant variants of Chinese hamster lung cells had lost an amplification and overexpression of the c-myc gene which was present

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in the parental line. 236 Insertion of a construct that caused increased c-myc expression in one of these drug resistant variant lines resulted in an increase in sensitivity to several drugs such as vincristine and actinomycin D. 236

III.

GLOBALMYC

The majority of human cancer strikes epithelial cells. In this section, we will look at several different tissues, mostly epithelial, to evaluate the frequency of occurrence of derangements in the structure and expression of the myc gene, the clinical correlates of such alterations, the biological consequences of intentionally deregulating c-myc gene expression, and the tissue-specific aspects of control of c-myc expression. Not every aspect will be covered with every tissue, mostly because not every aspect has been studied. One of the lessons learned by the authors is that despite the enormity of the literature on this gene there is still much that is unknown and in dispute.

A. Lung Clinical Associations The c-myc oncogene has been frequently found amplified in cell lines derived from small cell lung cancer (SCLC), particularly in a variant type that grows faster, has an increased ability to form colonies in semisolid medium, and lacks some of the differentiated neuroendocrine features found in classical SCLC, like expression of L-dopa decarboxylase. 230'237-243 In fact the association of c-myc amplification with the variant form of SCLC was one of the first such consistent associations of a myc gene alteration with a human carcinoma. 23~Amplification of the c-myc gene in a cell line established at relapse was a statistically significant negative prognostic indicator for the patient from whom the cell line was derived. 240 In surgical specimens of SCLC the incidence of c-myc amplification has been found to be much lower; only 4 out of 122. 241-245 This discrepancy is probably due to the increased ease of establishing cell lines from tumors with amplification. 241 Amplification of the c-myc gene was found to be more common in tumors from patients who had been treated prior to biopsy. 241 Overexpression of the c-myc gene in SCLC has been demonstrated on both the RNA and protein level. 230'238'243'246'247 Elevated expression of the gene has been demonstrated in the absence of gene amplification. 243'248 In one cell line this was shown to be due to increased initiation of transcription compared to SCLC lines that do not express the c-myc gene, and increased transcriptional elongation compared to a SCLC line that had both amplification and elevated expression of the gene. 248 In a series of 18 cell lines derived from small cell lung cancers there was a statistically significant correlation of increased expression of c-myc RNA

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3 51

with increased proliferative index, defined as the fraction of cells in the S, G2, and M phases of the cell cycle. 249 The c-myc-related genes, N-myc and L-myc, have also been found amplified in human SCLC. 110,250In fact, the L-myc gene was discovered because of its amplification in SCLC cell lines and its sequence similarity to the c-myc gene. 11~The association of c-myc amplification with the variant form of SCLC was not seen with N-myc and L-myc. 11~176 In nonsmall cell lung cancer the c-myc gene has been found to be overexpressed in some fraction and occasionally amplified or rearranged. Using only primary tumors, the c-myc gene has been found amplified in 4 out of 43 adenocarcinomas, 4 out of 28 squamous cell carcinomas, and in 2 out of 13 large cell carcinomas of the lung. 242'251'252 Immunohistochemical analysis of the expression of c-Myc protein in lung cancer showed an association between overexpression and poorly differentiated squamous cell carcinoma: 14 of 20 poorly differentiated tumors had elevated Myc protein compared with 6 out of 19 well and moderately differentiated tumors. 253 Using in situ hybridization, the association of strong c-myc expression with poorly differentiated squamous cell carcinoma was confirmed. TM Other subtypes of lung cancer also had detectable c-Myc overexpression, including 13 of 22 adenocarcinomas.253 In giant cell carcinoma of the lung, two examples of c-myc rearrangement have been found" one in a cell line and one in a primary tumor. 255'256In the primary tumor, a repeated DNA element of the L 1 (Kpn) class was inserted upstream of exon 1.257 In addition, one giant cell lung cancer cell line, C-Lu99, had an elevated level of c-myc RNA without apparent amplification or rearrangement of the encoding gene. 255 Myc overexpression has been detected in nonmalignant tissue from lung cancer patients. 25s This may reflect a preneoplastic alteration in the lung tissue from which the tumor arose or, alternatively, growth factors produced by the tumor may cause increased myc expression in nearby nonmalignant tissue.

Bioassay The significance of elevated expression and amplification of the c-myc gene in human lung cancer is supported by direct experiments. Transfection of the classic SCLC cell line H209 with a human c-myc gene, in order to increase its expression, resulted in clones with some similarities to the SCLC variantsY 9 In particular, the transfected clones grew faster in culture and formed colonies in soft agar at an increased frequency compared to the parental cells, had a morphology like the variant, and a histology in nude mouse xenografts intermediate between the classic and variant lines. 259 Thus, at least some of the differences in phenotype between the classic and the variant SCLC cell lines can be attributed to differences in the expression of the c-myc gene.

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Human bronchial epithelial cells immortalized by the SV40 large T antigen were made tumorigenic in nude mice by transfection of the c-myc and rafoncogenes. 26~ Both oncogenes were required for tumorigenicity. The resultant tumors were described as "multidifferentiated," with markers of squamous, glandular, and neuroendocrine differentiation, similar to some small cell lung cancers seen in vivo. 261

Regulation In two experimental systems derived from epithelial cells of the lung the usual association of decreased c-myc expression with decreased proliferation was not observed. Type 2 epithelial cells (which line the alveoli) freshly isolated from neonatal rats grow in tissue culture in the presence of serum. 262 When serum was withdrawn, the cells stopped proliferating, but the expression of c-myc RNA remained constant. The same cell type isolated from adult rats did not proliferate in culture, but still had the same level of c-myc RNA as the proliferating neonatal cells. 262 This corresponds to the in vivo situation in humans in which the type 2 cells had the strongest expression of c-myc RNA in the alveolar region, without detectable expression of the Ki-67 antigen which is a marker of proliferating cells. 254 In another system, the SCLC variant cell line NCI-N417 had a constant level of c-myc RNA when proliferating or when growth was inhibited by human recombi-

nant leukocyte or-interferon, or the ornithine decarboxylase inhibitor difluoromethylornithine. 263 These agents have been shown to lower myc expression in other systems (see section on colon and rectum and Refs. 187, 264, and 265). Thus, epithelial cells of the lung may differ from other cell types in their regulation of c-myc expression. One possible explanation is that the c-myc gene is being regulated at a translational level 262 although in a series of cell lines and xenografts derived from 23 small cell lung cancers, the expression of c-myc RNA was consistent with the level of c-Myc protein. 249 This area of investigation certainly deserves more attention. B. Breast

Clinical Associations Studies of the c-myc gene in human breast cancer have revealed amplification of the gene in several cell lines including SKBR-3, SW613-S, HBL100, VHBI, and B S M Z . 266-271 This reflects the fact that the c-myc gene is amplified in many primary human breast carcinomas, 2v2-293 but there is a great deal of disparity in the frequency, ranging from 1% in a survey of 99 tumors 2s9 to 41% in a survey of 48 tumors. 281 A very large survey of 1052 human breast tumors showed 17.1% had amplification of the c-myc gene. 282 Studies of c-myc expression in human breast

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cancer have shown that the encoded RNA and protein is frequently expressed at a higher level in malignant than nonmalignant mammary tissue. 277'278'290'294-299 In tumors with amplification of the c-myc gene it was almost always expressed at an elevated level, but it was also overexpressed in breast tumors with no amplification or rearrangement. 277'278'29~ The reason for elevated expression in breast cancers with a c-myc gene of apparently normal structure and quantity is unknown. On a contrary note, one study found that cell lines made from norrfial human mammary epithelium expressed the c-myc gene at a higher level than a panel of breast cancer cell lines. 3~176 Whether this reflects a need for elevated myc expression in order to grow in culture or a normal consequence of growth stimulation of mammary epithelial cells is not known. Rearrangement of the c-myc gene has been found in a small number of breast tumors. 279'281'283'3~ The three rearrangements that have been characterized all involved the 3' portion of the gene, 279'283'301 but the significance of this unusual geography of myc rearrangement is unknown. In one interesting rearrangement a Line-I repetitive element was found inserted into the second intron of the c-myc gene from an aggressive breast carcinoma, but was absent from nearby nonmalignant tissue. 283 This is not the only example of the mobilization of repetitive sequences that find the c-myc gene as a target. In canine transmissible venereal tumor a Line-1 element was found to be inserted 5' of the coding region. 3~ The search for prognostic indicators in breast cancer is particularly intense. A number of studies have attempted to link alterations in the structure and expression of many genes to clinical parameters. Amplification and/or overexpression of the c-myc gene has been correlated with increased age, 279 lack of progesterone receptors, 272'291 inflammatory carcinoma, 288 high S-phase fraction, 284 high proliferative activity as measured by expression of the nuclear antigen Ki-SI, 3~ well differentiated tumors, 298 larger tumors, 285'299 and positive lymph nodes. 284'285'290'295'304 Most importantly, c-myc amplification has been found to be a negative prognostic indicator by a number of investigators. 272'273'277'284'285'3~176 Myc has also been found to be expressed at an elevated level in some specimens of fibrocystic disease of the breast, particularly in subtypes associated with proliferation and considered to be premalignant. 297'3~ A correlation has been found between elevated expression of the c-myc gene in benign breast discase and a first-degree family history of breast cancer. 3~

Bioassay The clinical studies provide evidence for an association between c-myc activation and neoplasia of the mammary epithelium. A number of studies have been done which provide direct evidence for the biological consequences of altering the control of expression of the c-myc gene in this tissue. In one study the human breast cancer cell line SW613-S, which has an amplification of the c-myc gene, was subcloned into several sublines that differed with respect to the degree of myc

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amplification and expression. 3~176 The sublines with 30- to 60-fold amplification were tumorigenic in nude mice, while the sublines with 2- to 3-fold amplification were not tumorigenic. When the nontumorigenic cells were transfected with a c-myc gene under the influence of the powerful SV40 early region enhancer, they gained the property of tumorigenicity. 3~176 In a similar experiment a c-myc coding region under the control of a retroviral enhancer/promoter altered the properties of an immortalized nontumorigenic mouse mammary epithelial cell line so that it acquired the ability to grow in soft agar and produce tumors in nude mice. 31~ Another nontumorigenic mouse mammary epithelial cell line did not gain the ability to produce tumors when it was transfected with a v-myc gene under control of a similar retroviral enhancer/promoter, although it did gain other properties such as a limited ability to grow in soft agar and altered responsiveness to lactogenic hormones. 311 As seen below, an activated c-myc gene alone cannot produce a mammary carcinoma. Thus, the apparent discrepancy between the two latter transfection experiments probably lies in the genomes of the host cells used. This type of discrepancy is reminiscent of the findings from studies in fibroblasts in which the apparent biological activity of the c-myc gene was strongly dependent on the host cell. Among the most convincing work comes from transgenic mouse experiments. Two groups have produced mice with c-myc transgenes that are expressed in mammary epithelium: In one set of experiments the c-myc gene was linked to the transcriptional control machinery of mouse mammary tumor virus (MMTV) which is inducible by glucocorticoids, 312 and in the other the c-myc gene was linked to the promoter and upstream region of the whey acidic protein (WAP) gene which is normally expressed in lactating mammary gland. 313 Both transgenics produced an excess of adenocarcinoma of the mammary epithelium, but only after one or more pregnancies. 3~2-3~4 In the transgenic mice that expressed the WAP-myc construct, 80% got palpable tumors after two lactation periods. 313 The tumors expressed the transgene as well as the endogenous WAP gene and the ]]-casein gene, which is another lactation specific gene. This indicates that an event(s) occurred which caused deregulation of the expression of the lactation-specific genes, together with the c-myc transgene, all of which were under control of similar regulatory elements. In the MMTV-myc transgenic mice, only about 10% of the mice developed mammary cancer, again only after one or more pregnancies, indicating a need for hormonal stimulation of the transgene in order to get neoplasia. In both transgenic systems the tumors grew out from nonmalignant mammary epithelium which expressed the transgene; thus, abnormal c-myc expression by itself was not sufficient to cause neoplasia. When the myc transgenic mice were crossed with mice containing a Ha-ras gene activated by point mutation (MMTV/v-Ha-ras or WAP/human mutant Ha-ras), double transgenics were generated that developed adenocarcinoma of the mammary epithelium with a very high frequency. Close to 90% of the MMTV double transgenics developed breast cancer, including the males. 315 In the WAP double

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transgenics all the females developed multiple mammary tumors after several pregnancies, and normal mammary epithelium failed to develop, so these double transgenics were unable to nurse. 316 In both cases nonmalignant mammary epithelium that expressed both the ras and myc transgenes were found, indicating that additional steps were still required to get a fully malignant adenocarcinoma of the breast.315.316 In the transgenic mouse experiments all of the cells in the mammary epithelium carried the transgene. In nature, single cells that acquire mutations are surrounded by normal cells which may have a suppressive effect on growth of the premalignant cell. 69'317'318 In order to model this situation in breast carcinogenesis, mammary epithelial cells were manipulated in short-term primary culture, then implanted into the cleared mammary fat pads of immature mice. 319 In this system normal cells grow into a normal gland. When the cells were exposed to a recombinant defective retrovirus containing a v-myc gene, only some of the cells got infected and contained the activated myc gene. Transplants of these cells produced mammary glands with areas of hyperplasia, but no neoplasia, unlike the transgenic mouse models in which an activated c-myc gene can produce an increase in mammary neoplasia. 319 Similar experiments with v-Ha-ras produced no tumors, but infection with both v-Ha-ras and v-myc produced mammary tumors in the majority of animals. 32~Thus, the transplantation experiments support the transgenic mouse and DNA transfection experiments in providing a role for the myc gene in mammary carcinogenesis. Infection of newborn mice with a recombinant murine retrovirus containing the MC29 v-myc gene resulted in a variety of tumors including adenocarcinoma of the mammary gland. 321 These tumors were clonal and did not develop immediately after infection, which indicates that other events are probably needed to get a malignancy out of a cell which expresses a high level of v-myc. 321 These experiments support the conclusion that an activated myc gene is a carcinogenic agent in mammary epithelial cells, but it is not sufficient to produce a tumor alone.

Regulation The level of c-myc RNA in cultured mammary carcinoma cell lines which are dependent on estrogen for growth, like MCF-7 and T47D, was low when the cells were in a quiescent state induced by deprivation of estrogen, but was transiently stimulated by estrogen. 268'322-324 The effect was shown to be due to increased transcription of the c-myc gene. 323 Exposure to an antisense oligonucleotide that inhibits expression of the c-myc gene, inhibited the proliferative response of MCF-7 cells to estrogen. 168 This indicates that the estrogen stimulation of c-myc gene expression is a necessary step in estrogen-dependent proliferation. Breast cell lines that are not estrogen-dependent (e.g., MDA-MB-231, BT-20, and HBL-100) have a constitutively high level of c-myc expression. 268'323 This may mean that deregulation of c-myc expression is a step in going from an estrogen-dependent to an

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estrogen-independent tumor. However, when an activated c-myc gene was transfected into MCF-7 cells, estrogen was still required for tumorigenicity in nude mice. 325 Thus, deregulation of myc expression was not sufficient to create a fully estrogen-independent phenotype. The effects of estrogen on growth of mammary epithelial cells involves more than just stimulation of expression of the c-myc gene. Tamoxifen is an agent which blocks the growth effects of estrogen. In MCF-7 and T47D cells tamoxifen caused a decrease in the level of c-myc RNA. 268'322'326 The anti-estrogen ICI 164,384, as well as tamoxifen, blocked the effect of estradiol on increasing the expression of c-myc RNA in MCF-7 cells. 327 A similar result was obtained from an in vivo experiment in which breast cancer biopsies from women pretreated with tamoxifen had significantly less c-myc RNA, measured by in situ hybridization, than a similar population with breast cancer that was untreated before biopsy. 328 In vivo, progesterone is believed to promote the growth of mammary epithelium, even though it inhibits the growth of most breast cancer cell lines including T47D. The synthetic progestins medroxyprogesterone acetate (MPA) and ORG 2058 caused a rapid transient increase in expression of c-myc RNA in T47D cells. 326'329 However, the initial effect of MPA was to cause a slight increase in the number of cycling cells, so this does not contradict the usual finding of an increase in c-myc expression associated with the transit of cells from a quiescent to a cycling state. Intcrestingly, there is an apparent contradiction to this association when the agents interferon-y or epidermal growth factor caused an increase in the level of myc RNA in the MDA-468 breast carcinoma cell line even though they decreased the growth of these cells. 330"331

C. Cervix, Ovary, and Uterus

Clinical Associations, Cervix The c-myc gene has been found to be frequently amplified in carcinoma of the uterine cervix. 10'332Amplification was found more frequently in advanced stage III and IV tumors (49%) than in early stage I and II t u m o r s (6%). 10'333 In a survey done in China, no amplification of the c-myc gene was seen in the 17 patients studied. 334 It is not clear if this was due to geographic differences in the pathology of the disease or to the collection of earlier stage tumors. Although almost all tumors with amplification of the c-myc gene had elevated expression of its RNA, only about 25% of tumors with elevated expression had amplification; 333 thus the reason for elevated expression of the c-myc gene in these tumors is not yet explained. The gene was similarly overexpressed in a number of cervical carcinoma cell lines, also without amplification or rearrangement. 334 An examination of archival biopsies of cervical cancers using flow cytometry of nuclei showed a higher level of Myc protein in nonmalignant cervix than in most tumors. 335 The apparent contradiction to the studies showing high levels of c-myc

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RNA and frequent gene amplification is unresolved. However, an immunohistochemical approach using the same monoclonal antibody revealed elevated but variable expression of Myc protein in 11 invasive tumors compared to no staining in six specimens of normal cervical epithelium. 336 In advanced tumors there was no prognostic significance to Myc protein expression detected by immunohistochemistry. 337 However, in early invasive cervical carcinoma, an elevated level of c-myc RNA was shown to be a strongly negative prognostic indicator. 333'338'339 In a study of 93 patients, multivariate analysis revealed that greater than 5-fold elevated expression of the c-myc gene gave a 3.7-fold greater risk of relapse. 333,339

Clinical Associations, Uterus Amplification of the c-myc gene has been found in endometrial cancer: 10 out of 16 tumors in one survey, 34~ and 1 out of 3 in another. 341 Amplification was associated with advanced stage, lack of differentiation, and serous papillary adenocarcinoma histology. 34~

Clinical Associations, Ovary The c-myc gene was also found amplified in about one-quarter of the ovarian carcinomas that have been reported. 342-346 Amplification had no prognostic value in one survey of 17 tumors, 342 while in another survey of 16 adenocarcinomas c-myc amplification was associated with poor differentiation, high number of mitoses, and a high degree of nuclear atypia. 343 myc RNA was shown to be overexpressed in more than one-third of human ovarian t u m o r s , 347-349 with stage III serous adenocarcinomas having an 84% incidence of overexpression. 348 In a study of radiationinduced ovarian carcinomas in mice, 25% (7/28) had overexpression of c-myc, but no amplification or rearrangement was seen. 35~Overexpression of c-myc RNA was associated with progression of disease in a survey of 28 stage III and IV human ovarian carcinomas. TM Myc protein was expressed in the tumor cells, not stromal elements. 348 A study of c-Myc protein by flow cytometry of nuclei from archival specimens of serous papillary ovarian cancer revealed that about two-thirds had a c-Myc protein signal twofold or more above the median signal from normal ovary. 352 In mucinous tumors of the ovary, malignancy was associated with the presence of c-Myc protein in the cytoplasm and the nucleus, instead of the more usual finding of an exclusively nuclear location. 353-

Regulation The mammalian uterus and the avian oviduct are estrogen-responsive tissues, which respond with cellular proliferation. Estrogen caused an increase in expression of c-myc RNA in the prepubertal rat uterus 5'354-356 and the avian oviduct.~57

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Table 1. Differentiation Agents which Cause a Reduction in c-myc Expression in Ovarian and Cervical Cancer Cell Lines Cell Line HOC-7, ovarian cancer

HeLa, cervical carcinoma HeLa, cervical carcinoma

Agent

Reference

Dimethylsulfoxide Dimethylformamide Retinoic acid Transforming growth factor-I] 1 Interferon-], Tumor necrosis factor Dimethylsulfoxide

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363 362

Progesterone, which inhibits estrogen-induced proliferation in the uterus, caused a transient reduction in the level of c-myc RNA in estrogen-stimulated chicken oviduct. 358 Vanadate caused a transient increase in expression of c-myc RNA in CaOv ovarian carcinoma cells. 359 Vanadate is a tyrosine phosphatase inhibitor that is mitogenic in many cells. This experiment implicates tyrosine phosphorylation in the control of c-myc expression in an epithelial cell. Dexamethasone, a glucocorticoid, causes inhibition of growth of the avian oviduct and also caused a transient drop in the expression of c-myc RNA. 36~The ovarian cancer cell line HOC-7, and the cervical carcinoma cell line HeLa, responded to several agents (see Table 1) with a concomitant reduction in both growth and level of c-myc gene expression. 361-363 Thus, the tissues of the female reproductive tract are consistent with the general trend of c-myc expression increasing in response to growth stimulation and decreasing in response to growth inhibition. Tumor necrosis factor (TNF) and interferon-y (IFN-y) both reduced the expression of the c-myc gene in HeLa cells by reducing transcription. 363 Interestingly, the effects of these two agents followed different pathways: The effect of IFN-y was inhibited by the protein synthesis inhibitor cycloheximide, which indicates the need for the synthesis of a new protein to lower the expression of c-myc RNA, while the effect of TNF was not inhibited by cycloheximide, indicating a more direct pathway. Addition of saturating amounts of each agent together resulted in an additive effect; thus, the rate limiting step for each agent is different. 363

D. Brain Clinical Associations

Medulloblastoma is a rare type of brain cancer that is predominately pediatric. The c-myc gene was found to be expressed at an elevated level in the majority (4/7) of medulloblastomas that have been reported. 364'365Amplification of the c-myc gene has been found frequently in cell lines and xenografts derived from medulloblas-

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tomas, but much less frequently (less than 10%) in primary tumors. 364'366-371 The difference between primary tumors and cell lines in amplification of the c-myc gene is likely accounted for by the outgrowth in culture of an undetectably small fraction of cells which did have amplification in the original tumor. 367 Medulloblastomas are capable of metastasis, and it will be interesting to see if some metastatic medulloblastomas are different from the original tumor with respect to myc amplification. Rearrangement of the c-myc gene was reported in one medulloblastoma, 367 and mutation of the first exon was reported in another. 365 Amplification, rearrangement, and elevated expression of the c-myc gene was found in a cell line derived from a glioblastoma multiforme. 372 However, the c-myc gene was not found amplified in patient specimens of 81 other glial tumors. 364'366'373 Expression of the c-Myc protein was shown to be elevated in glial tumors and glial tumor cell lines over that in normal brain and nonmalignant glial cell lines, with an increased incidence of overexpression in higher grade tumors. 374'375 A malignant meningioma cell line has been established with amplification of the c-myc gene. 376 In a study of 19 primary tumors, the expression of c-myc RNA was 5-fold or more above nonmalignant brain tissue in 12, but none of the tumors in this series had amplification or rearrangement of the encoding gene. 377 There was a 10-fold decrease in the level of c-myc RNA between newborn and 3-week-old whole mouse brain. 378 This decrease was shown to be due to a complex set of regulatory events involving decreased transcriptional elongation and initiation as well as posttranscriptional events. 378 The contribution of different tissues to the results in whole brain may contribute to the apparent complexity. In situ analysis of c-myc expression during development of the human and mouse brain revealed increased expression of c-myc RNA in both actively proliferating regions, and differentiating postmitotic z o n e s . 379'380 For example, on the 10th day after birth of the C57BL/6 mouse, c-myc was found in the actively proliferating external granular layer of the cerebellum, as well as in the Purkinje cells which were differentiating and no longer proliferating at this stage. 38~

Bioassay Several experimental systems have been established which demonstrate the biological activity of a strongly expressed exogenous c- or v-myc gene in cells of the central nervous system. MC29, the prototypical avian retrovirus with a myc oncogene, has been shown to induce transformation in neuroretina cells from 7-day-old chicken embryos and in neural crest cells derived from 2-day-old quail embryos. 38t'382 In both cases differentiation was not inhibited: The chick neuroretina cells produced both neural and glial cell types, 381 while the neural crest cells expressed catecholaminergic traits. 382 The latter result was also obtained after infection with a recombinant retrovirus expressing a c-myc oncogene. 382 Recombinant retroviruses with the v-myc and v-rafoncogenes, or a c-myc oncogene alone, have been shown to immortalize primary cultures of murine microglial cells and

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neuroepithelial cells from the mesencephalon of mouse embryos, respectively. 383'384 The immortalized microglial cells retained a number of differentiated functions including phagocytosis, 383 and one of the immortalized neuroepithelial cell lines was capable of differentiating to glial or neural cells. 384 In a transgenic mouse experiment, two transgenic lines were crossed, one with a human T-cell leukemia virus (HTLV) tax gene driven by an immunoglobulin promoter, and the other line with a c-myc gene driven by a HTLV promoter, which is trans-activated by the tax gene product. 385 Mice with both transgenes were produced which had a 76% incidence of brain tumors by 90 days. The histology of the tumors most resembled the human primitive neuroectodermal brain tumors (PNET), a tumor type which includes the medulloblastoma. It is possible in this transgenic experiment that the tax gene had a role both in activating the myc transgene and also in activating some other cellular gene(s) in order to get such a high rate of tumorigenesis. 385 In a cell transplant type of approach, fetal rat brains were removed, made into a single cell suspension, and infected with recombinant retroviruses, then reinjected into the cranium of an adult syngeneic rat. 386 One of 13 transplants that had been infected with a v-myc retrovirus developed a PNET from the transplanted cells. This is the same type of tumor described earlier in the transgenic experiment. 385 When the transplanted cells had been infected with a recombinant retrovirus that expressed both v-myc and v-Ha-ras, all the rats developed, within 2-4 weeks, multiple highly malignant tumors some o1" which eventually expressed glial fibrillary acidic protein, a marker of gliai cells. Infection with ras alone gave tumors in only half the recipient rats with a much longer latency. 386 Thus, several lines ot" experimentation reveal a potent biological activity for the myc gene in tumorigenesis ot" both neural and gliai cells in the central nervous system. E. Prostate

Clinical Associations Expression of c-myc RNA was elevated 5-fold over normal prostate in 4 out of the 7 human prostatic carcinomas that were analyzed in one study, 387 and 2-fold or more over the level of myc RNA in benign prostatic hypertrophy in 10 out of 12 prostatic carcinomas in another. 388 myc RNA levels were also elevated in benign prostatic hypertrophy, but not as much as in the carcinomas. 387 Using in situ hybridization, c-myc RNA was localized to the prostatic epithelial cells in benign prostatic hypertrophy. 389 Growth in nude mice of the PC-3 prostatic carcinoma cell line resulted in tumors with a 10- to 12-t'old amplification of the c-myc gene; however, amplification of the c-myc gene was not detected in the parental cell line. 39~ Presumably an undetectably small fraction of cells with amplification of the c-myc gene in the original cell line had a selective advantage for growth in nude mice. 39~ There was no correlation between expression of c-Myc protein, detected

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by immunohistochemistry, and prognosis, in a survey of 45 early (stage A1) adenocarcinomas of the prostate. 391

Bioassay The biological activity of the c-myc oncogene in the epithelial cells of the prostate has been demonstrated in an organ reconstitution experiment in which fetal cells from the urogenital sinus of a C57BL/6 mouse were infected with retroviral vectors containing v-Ha-ras, v-myc, or both, and then reimplanted into the renal capsule of syngeneic hosts. 392The ras oncogene induced dysplasia; the myc oncogene induced hyperplasia; and the cells infected with the combination of both ras and myc developed adenocarcinomas. The tumors were clonal, and not all cells that expressed the oncogenes formed tumors. Thus, although the combination of ras and myc were necessary for tumorigenesis, they were not sufficient. 392 When this experiment was done using cells from B ALB/c mice, instead of C57BL/6, the result of infection with ras + myc was focal epithelial hyperplasias and very rarely carcinomas. 393 This result emphasizes the importance of the host cell when evaluating biological activity. Four cell lines made from the hyperplasias induced by v-Ha-ras in the C57BL/6 mice all had mutations in the p53 gene, while nine cell lines made from the adenocarcinomas induced by v-Ha-ras + v - m y c all lacked mutations in the p53 gene. 394 The activated myc gene seems to have made p53 mutations unnecessary in this system.

Regulation The prostate depends on androgen for growth and maintenance of the adult organ. Castration results in atrophy of the ventral prostate accompanied by the death of numerous epithelial cells. Castration of the rat resulted in a paradoxical increase in expression of c-myc RNA, which was due to the regressing epithelial cells. 184'185 Administration of androgen revised the increase in c-myc RNA. 184 This system seems to be an in vivo manifestation of the c-myc function involved in apoptosis. This increase in myc expression is probably directly involved in the programmed death of the prostatic epithelial cells when deprived of androgen. After castration, readministration of androgen causes a regrowth of the regressed prostate. In rats that had been castrated 7 days previously androgen caused a transient increase in the expression of c-myc RNA. 395 Insight into the regulation of c-myc gene expression by androgen was gained from a model system in which mibolerone, a synthetic androgen which inhibits growth of the prostate carcinoma cell line LNCaP, caused a decrease in expression of c-myc RNA due to a decrease in its synthesis. 396 Thus, myc appears to be involved in both proliferation and apoptosis of prostatic epithelial cells. It is hard to reconcile the latter experiments showing the usual association of increased c-myc expression and proliferation, and the earlier described experiments

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showing a role for expression of the myc gene in prostate carcinogenesis, with the experiments showing increased c-myc expression in cells undergoing apoptosis. Other factors are involved in the decision to proliferate or die in cells that have increased expression of the c-myc gene. This point was illustrated by experiments using two transplantable adenocarcinomas of the prostate that were produced in the organ reconstitution experiments described above. 392An increase in myc expression in response to androgen deprivation was seen in both carcinomas, one of which was androgen sensitive and another that was androgen insensitive for growth in syngeneic mice. 397'398However, the androgen sensitive cells also induced a gene associated with apoptosis of the prostate, while the androgen insensitive tumor cells did not induce this same gene in the castrated host. 398 It seems that expression of the c-myc gene is necessary, but not sufficient, for both apoptosis and proliferation in the prostate. F. Testes

Clinical Association The protein product of the c-myc gene was expressed at an elevated level in testicular cancer compared to normal testes. 399 Seminomas and teratomas with intermediate differentiation and yolk sac elements had the highest levels of c-Myc protein. 399'4~176 Interestingly, increased expression of the c-myc gene was correlated with a better prognosis in testicular cancer! 4~176

Bioassay A transgenic mouse line, MMTV-myc, which expressed the c-myc transgene in a variety of tissues, including testes, developed Sertoli cell testicular neoplasms in four of 35 mice studied. 314

Regulation In mouse testes the association between rapid growth and elevated expression of the c-myc gene did not hold for spermatogonia which actively proliferate and have very low levels ofc-myc RNA. 4~ Other cell types in the testes, such as Leydig cells and Sertoli cells, expressed abundant c-myc RNA when actively proliferating. 4~ Leydig cells from the mouse and the pig in vitro, and the rat in vivo, responded to human chorionic gonadotropin, which promotes proliferation of this cell type with a transient increase in expression of the c-myc gene. 4~176 The freshly isolated pig Leydig cells also responded to the progression factors epidermal growth factor and basic fibroblast growth factor with an increase in the level of c-myc RNA. 4~ The rate-limiting step for the stimulation of c-myc gene expression by chorionic gonadotropin differs from that for epidermal growth factor and basic fibroblast growth factor. 403

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G. Kidney Clinical Associations The c-myc oncogene has been found overexpressed with a high frequency in human renal cell carcinomas, 4~176 and the cell lines derived from this malignancy. 4~ An association was found between elevated c-Myc protein, detected by immunohistochemistry and nuclear pleomorphism. 4~ It was also expressed at an elevated level in kidney tumors induced by estrogen in Syrian hamsters. 4~ The c-myc gene was not found amplified or rearranged in any of the six human renal cell carcinomas with an elevated level of c-myc RNA that were analyzed.a~ A family with inherited renal cell carcinoma that cosegregated with a chromosomal translocation [t(3;8)(p 14.2;q24.1)], which relocated the c-myc gene to chromosome 3, was found to have its chromosome 8 breakpoint at least one-half million bp distant from the gene. 41~ Thus, it is doubtful that the translocation directly caused an alteration in the regulation of the c-myc gene; however, very long-range effects cannot be completely dismissed. The analysis of the structure and expression of the myc gene in renal malignancies is still very incomplete. However, polycystic kidney disease holds a fascinating story on the potential impact of deregulation of myc expression in a nonmalignant proliferative disease. The polycystic kidney diseases are a collection of disorders which are characterized by the growth of a multitude of cysts, lined with epithelial cells, that cause enlargement and eventual failure of the kidney. In humans the most common type is an adult onset autosomal dominant inherited condition. However, there is a rare autosomal recessive form of the disease with a very early onset and a mouse model. In autosomal recessive polycystic kidney disease of the C57BL/6J mouse, the c-myc oncogene has been shown to be highly overexpressed; 3-week-old affected whole kidneys had 25- to 30-fold overexpression compared with normal kidney of the same age. 411'412 The c-myc gene was shown to be expressed in proliferating cells in the normal developing mouse kidney, but by 3 weeks of age its expression was greatly decreased. 4~3'424 However, in the polycystic kidneys c-myc RNA was increasingly expressed in the cells lining the cysts, and in the 3week-old animals even the apparently uninvolved proximal tubules expressed more c-myc RNA than in normal kidney. 414 The expression of c-myc RNA increases with proliferation in adult mouse kidney (see below), but 3-week-old polycystic mouse kidneys were not in a state of very active proliferation. 411'412 It seems that the c-myc gene may play a role in the pathogenesis of this disease. Perhaps the gene for autosomal recessive polycystic kidney disease is a negative regulator of c-myc gene expression, that is specific for kidney epithelial cells.

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Bioassay One important question is: Can deregulated c-myc expression, by itself, cause polycystic kidney disease? This question was answered by accident in a transgenic mouse experiment in which the c - m y c gene was linked to the promoter from the 13-chain of hemoglobin, and an SV40 enhancer, with the intention ofoverexpressing the myc gene in cells of the erythroid lineage. 415 The gene was instead expressed in epithelial cells of the kidney, probably because of the creation of unexpected signals when the globin, SV40, and c-myc sequences were linked. All 18 transgenic lines developed a disease that was very similar to adult polycystic kidney disease, and died of renal failure within 6 weeks to 3 months of birth. 415 Thus, deregulated c-myc expression is both necessary and sufficient to initiate polycystic kidney disease.

Regulation Unilateral nephrectomy induces a compensatory hypertrophy in the remaining kidney. This was accompanied by a small increase in the expression of c - m y c RNA in both mice and rabbits that was not much different from the small increase in c-myc expression induced by a sham operation. 412'416'417 This contrasts with the situation in liver in which partial hepatectomy causes a big increase in m y c expression (see Section III.I). However, partial hepatectomy causes increased proliferation, not just hypertrophy. Folic acid is a toxic agent which causes the death of kidney cells and regenerative cell proliferation. This was shown to be accompanied by an increase in the expression of c-myc RNA, up 25-fold at 12-18 hours in mice, and up 6-fold at 4 hours in rabbits. 412'417 The increase in the abundance of c-myc RNA in tblic acid damaged mouse kidney was due to posttranscriptional events. 418 Growth hormone also induced an increase in the level of c - m y c RNA in rat kidney. 419

H. Bladder

Bioassay The potential biological activity of the c-myc gene on bladder epithelial cells has been demonstrated in a reconstituted organ experiment. 42~ When mouse bladder urothelium was removed, infected with a recombinant retrovirus containing a v - m y c gene, and placed into the renal capsule of a syngeneic mouse, the cells produced a hyperplasia relative to control cells infected with the vector virus. When a similar experiment was done using a v-src oncogene, hyperplasia and dysplasia were produced. However, when a vector containing both myc and src oncogenes were used focal tumors were produced. 4"~ The expression of the introduced genes could not be measured in the nonmalignant tissue to determine if myc and src together were sufficient to get a tumor, or if further events were needed.

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Clinical Association Although we know that activation of the c-myc gene is potentially a neoplastic event in bladder, we do not yet have a complete view of how often it is involved, nor do we know a great deal about how it is regulated in bladder urothelium. The c-myc gene was not amplified in four stage-II bladder carcinomas. 286 The 3' half of the c-myc gene was found to have a lower level of DNA methylation in tumors, an alteration which was more prominent in tumors of increasing stage. 421 Lower levels of DNA methylation are associated with increased expression, but expression was not measured in the same series of tumors. 421Ahigh level ofc-myc RNA was found in hyperplastic and transitional cell carcinoma cell lines generated from carcinogen treated rats. 422 The level of c-myc RNA was also elevated in several human bladder cell lines: T24, Hu609, HCV29T, and Hu609T. 423'424 Interestingly, the urothelial cell line HCV29 has much less c-myc RNA than its tumorigenic derivative HCV29T. 423 The enhanced expression of the c-myc gene in the Hu609 and T24 cell lines was shown to be due to a trans-effect, and not to amplification, rearrangement, or mutation. 424'425

I. Liver

Clinical Associations Several groups have reported higher levels of c-myc RNA in most human liver carcinomas than in nonmalignant human liver. 426--431 One group found the opposite result of less c-myc RNA in the carcinomas. 432 The level of c-myc RNA in normal liver, but not in carcinomas, has been found to increase during surgery. 433 Thus, the circumstances under which the control liver specimen was collected will have a big impact on interpretation of the results in this type of experiment. The expression of c-Myc protein, measured by Western blot or immunohistochemistry, was also found elevated in hepatocellular carcinomas. 429"431'434'435 Elevated expression of the cmyc gene was associated with several preneoplastic states like chronic active hepatitis B infection, 435 and cirrhosis. 428'430'431'434'435 The level of c-myc RNA was found to be very high in the tumorigenic human hepatoma cell line Hep G2, in which myc expression was constitutive and not dependent on the growth state of the cells. 436'437 However, these cells did not have amplification or rearrangement of the encoding gene, leaving open the issue of what caused the apparent deregulation. 437 Hypomethylation is often associated with gene expression. Two groups have found evidence for a relative hypomethylation of the c-myc gene in hepatocellular carcinoma compared to nonmalignant liver. 438'439 A potential methylation site in exon 3 was found methylated in normal liver and hypomethylated in 88% of the tumors. 439 Neither group determined whether increased expression of the c-myc gene accompanied the hypomethylation. The methylation experiments would have

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revealed gene amplification, but it was not seen in any of the data shown, nor was such a finding pointed out in the articles. 438'439 In view of the frequent finding of amplification of the myc gene in rodent liver cancer (see below), this point needs further study in humans. It seems there may be more known about the structure and expression of the c-myc gene in liver cancer in rats than in humans. Morris hepatomas are rat liver tumors induced by feeding the hepatocarcinogen N-(fluoren-2-yl)phthalamic acid, and maintained by intramuscular transplantation in syngeneic rats. 44~Seven Morris hepatomas were studied and found to have an elevated level c-myc RNA compared to normal rat liver. 44~ One of these tumors was found to have a 5- to 10-fold amplification of the c-myc gene. 44~ Carcinogenesis with N-methyl-N-nitrosourea resulted in tumors, some of which had elevated c-myc expression, and amplification or rearrangement of the gene. 442 The rearrangements were mapped to the 5' untranslated part of the first exon or just upstream. 442 Seven rat tumors induced with the hepatocarcinogen aflatoxin B~ all had a variable, but elevated, expression of c-myc RNA, with one tumor having a 2- to 4-fold amplification of the encoding gene. 443 Another tumor in this group had a level of c-myc gene expression similar to the one with amplification, but lacked both amplification and rearrangement. 443 In another interesting model system, hepatocellular carcinomas which develop spontaneously about 4 months after birth in the LEC strain of rats, after an apparent nonviral hepatitis, were found to have 7- to 30-fold increased expression of c-myc RNA. 444 There was no correlation of c-myc RNA level with mitotic activity. In fact, the preneoplastic hepatitis displayed a fairly high mitotic index and did not have elevated expression of c-myc RNA when both parameters were compared with livers from the parental strain (LEA), which did not have hepatitis or a tendency to liver cancer. 444 Several systems of hepatocarcinogenesis in rats have been worked out in which a number of preneoplastic states have been defined. These systems are a valuable resource for determining the molecular events in the development of liver cancer. One of the issues addressed in these studies concerns at which stage and cell type is there an alteration in myc expression. When hepatocellular carcinoma was induced by feeding a choline-deficient diet, all 13 induced tumors had increased expression and amplification of the c-myc gene. 445 In some animals c-myc amplification was also seen in nonmalignant portions of the liver. Thus, in this system, amplification of the c-myc gene may precede the last stages of tumorigenesis. 445A conflict is found in the literature on the issue of increased c-myc expression preceding tumorigenesis with 3'-methyl-4-dimethylaminoazobenzene: One group found that c-myc expression was high in the tumors, but not in the nontumorous parts of the liver, 446 while another found elevated c-myc expression in the entire liver during treatment with this carcinogen. 447 Carcinogenesis with diethylnitrosamine did not cause elevated c-myc expression in the whole liver or even in purified 7-glutamyl transpeptidase-positive preneoplastic cells, but some adenomatous nodules and most of the hepatocellular carci-

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nomas did have elevated expression. 442'448'449 The Solt-Farber carcinogenesis protocol, which involves the carcinogens diethylnitrosamine and 2-acetylaminofluorene, and partial hepatectomy for tumor promotion, resulted in increased c-myc expression preceding malignancy. 45~ This was detected in oval cells and basophilic foci by in situ hybridization, 45~ and in preneoplastic cells purified from the liver by the criteria of a lack of adherence to plates coated with asialofetuin. 451 In both cases the cells that had increased c-myc expression corresponded to the cell type from which the carcinomas are believed to arise. 450'451 The Solt-Farber protocol is usually done with male rats because of the faster carcinogenesis, and higher yield of tumors and preneoplastic nodules than in female rats. A corresponding gender difference was detected in the early response of the expression of c-myc RNA in the whole liver and in nodules removed at 8 months after initiation; expression was higher in the males. 452-454 The gender difference in c-myc expression and carcinogenesis has been associated with differences in growth hormone levels in male and female rats. 452'454However, even in the females the carcinomas still had elevated expression of c-myc RNA. 453'454 Liver carcinogenesis resulting from a choline-deficient diet, plus the carcinogen ethionine was also accompanied by increased expression of c-myc RNA in the preneoplastic oval cells. 455'456 One of the unanswered questions in most of these studies is the significance of elevated expression of the c-myc gene in carcinogentreated livers. Since it is known that elevated expression of c-myc is found in cells reentering a proliferative state after a period of quiescence, which will be discussed below for regenerating liver, how can we distinguish elevated expression due to a proliferative stimulus from the hepatocarcinogens from an intrinsic cellular alteration that contributes to transformation. One approach to this problem made use of an oval cell culture prepared from livers of animals getting the choline-deficient ethionine diet for 6 weeks. 456 These cells, when made quiescent by serum deprivation, responded to a proliferative stimulus from serum by transiently increasing their expression of c-myc RNA. However, a tumorigenic subline expressed high levels of c-myc RNA constitutively. 456 Thus, the early changes in expression of the c-myc gene during carcinogenesis may reflect the mitogenic effects of the carcinogenic protocol, but the later alterations in expression are probably due to an intrinsic alteration in regulation of gene expression. Spontaneous transformation of rat liver epithelial cells in culture can be induced by holding the cells for long periods at confluence. 457'458 Tumorigenic cell lines prepared in this fashion usually had elevated c-myc expression compared with sister cell lines that did not get transformed. 458 However, some exceptions have been noted. 457.458 Infection with hepatitis B virus is one of the most significant risk factors for hepatocellular carcinoma in humans. 459 A direct connection between activation of the c-myc oncogene and hepatitis B viruses has been found in several systems. In a survey of 9 hepatocellular carcinomas from woodchucks infected with woodchuck hepatitis virus (WHV), three had a rearrangement and overexpression of the

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c-myc gene. 46~ In two of these tumors there was an insertion of a piece of the viral DNA, which contained the viral enhancer, in the vicinity of the c-myc gene; one in the 3' untranslated region of exon 3, and the other 600 bp upstream of exon 1.461 These viral insertions, which are reminiscent of the ALV promoter insertions described earlier, resulted in an increase in expression of the gene. 46~ The c-myc rearrangement in the other tumor resulted in the discovery of the hcr gene, which was highly expressed exclusively in normal liver. 462'463 The rearrangement involved the insertion of the hcrpromoter and part of the hcr coding region into intron 1 of the c-myc gene, which caused a 50-fold increase in expression, relative to the expression of c-myc in normal liver, of an hcr-myc fusion gene. 462'463 It is unknown if the WHV had a direct role in causing the juxtaposition of the c-myc and her genes, or if the rearrangement was an independent event. Hepatocellular carcinoma in WHV infected animals has also been associated with elevated expression of the c-myc gene due to DNA amplification, and also elevated expression of one of the two N-myc genes in woodchuck due to viral integration. 464'465 Ground squirrel hepatitis virus is a much weaker carcinogen than WHV, but is associated frequently with elevated expression of the c-myc gene due to gene amplification in hepatocellular carcinomas from infected woodchucks 465 and ground squirrels. 466 In humans, the hepatitis B virus has not been found integrated near the c-myc gene. However, evidence has been presented which suggests that the human hepatitis B virus pX protein can trans-activate the transcription of the c-myc gene. 467 More work clearly needs to be done to determine if there is a connection between the myc family and carcinogenesis by the human hepatitis B virus.

Bioassay In a transgenic mouse experiment, a c-myc gene, linked to an albumin enhancer/promoter to direct expression to the liver, caused hepatic dysplasia in young mice and focal adenomas in some mice over 15 months old. 468 No malignancies were noted in the myc transgenics. However, when transgenic mice with more than one oncogenic transgene were made, the c-myc transgene had a significant carcinogenic effect. For example, SV40 T-antigen transgenic mice got liver tumors in 3-5 months, but with the addition of a c-myc transgene they got cancer in 3-6 weeks. 468 Similarly, using recombinant retrovirus vectors to insert oncogenes into a rat liver epithelial cell line, a v-myc retrovirus did not cause transformation, but infection with a v-raf/v-myc combination transformed 2- to 3-fold more efficiently than v-rafalone, and caused a hepatocellular carcinoma-like tumor when the cells were injected into nude mice or syngeneic rats, as opposed to the sarcoma-like malignancies caused by v-rafalone. 469 In this case, not only did myc increase the yield of transformants, but it changed the histology of the resulting tumors. 469 The myc does not seem to be as potent an oncogene in liver as SV40 T-antigen or raf, but it still has profound biological effects. 468'469

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The insertion of a construct that increases the expression of the c-myc gene product into rat liver epithelial cells resulted in a potentiation of cell response to the epidermal growth factor (EGF). 470'471 There was no change in the receptor or the dose response, just an increase in the amount of DNA synthesis that occurred in response to the added growth factor. Neither group noted the appearance of transformed foci. 470'471 When the myc construct was inserted with a selectable marker so that the cells which took up DNA could be isolated, a population of cells were prepared all of which presumably had elevated c-myc expression. 472 These cells were not morphologically transformed, but did grow to a higher cell density and produced 2.5-fold more colonies in a 14-day colony-forming assay than the parental cells. EGF enhanced the colony-forming ability of the myc transfectants, which is consistent with the above experiments. 472 In another set of experiments, 16 subclones of chemically transformed rat liver epithelial cells were prepared and evaluated for tumorigenicity and gene expression. 473 Tumorigenicity correlated most strongly with elevated expression of the c-myc gene. Interestingly, among the eight sublines with the highest expression of c-myc RNA, there was a correlation of tumorigenicity with increased expression of transforming growth factor ~ (TGF-o~).473 This growth factor is homologous with EGF and uses the same receptor. The increased response of cells with exogenous c-myc expression to EGF, described in the previous paragraph, has an echo in this tumorigenicity study. 473 Another example of the synergism between EGF/TGF-~ and c-myc was found in a transgenic mouse experiment in which a myc transgene potentiated the activity of a TGF-c~ transgene. 474The initial effects of the expression of these genes, seen at 3 weeks of age, included dysplasia and apoptosis. By 16 weeks, over 70% of the mice carried liver nodules of which 25% were hepatocellular carcinomas. Transgenic mice which expressed only the TGF-~ transgene took 10-15 months to get liver tumors. 474 Elevated c-myc expression is not always associated with the development of liver tumors. Two spontaneously immortalized rat liver epithelial cell lines had higher expression of the c-myc gene than their mortal parental cell type, but tumorigenic derivatives of these cell lines made by transfection of a mutationally activated ras gene (N-ras or Ha-ras), or chemical transformation using aflatoxin B l, did not cause any additional increase in c-myc expression. 475 This work suggests an immortalizing function for elevated expression of the c-myc gene. 475

Regulation Partial hepatectomy results in a regenerative regrowth of the liver. One of the early events in liver regeneration in both mice and rats was an increase in the expression of the c-myc gene. 476-484 In mice the increase was seen within the first hour and peaked between 1 and 6 hours at 10- to 100-fold over quiescent liver. 476-478 In rats, the increase in the expression of c-myc RNA was also early and transient. 480'483'484 In both species DNA synthesis did not begin for several hours after

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the early peak in c-myc expression. The partial hepatectomy systems confirm the association of an early increase in c-myc expression preceding the reentry of a quiescent cell into S phase, as described earlier for fibroblasts. Some groups have found an additional later increase in c-myc expression in rats after partial hepatectomy, which corresponded to around the beginning of DNA synthesis. 483'484 There are several other liver systems in which increased c-myc expression is an early event preceding proliferation. Insulin is a mitogen for the rat hepatoma cell lines Reuber H35 and H4IIE, in which it also caused a rapid, but transient, increase in the expression of c-myc RNA. 485'486 Epidermal growth factor and transforming growth factor-cz also caused an increase in expression of the c-myc gene in freshly isolated rat hepatocytes several hours before DNA synthesis started. 471'487-489 The effect of these two factors was rapid (less than 2 hours) and mediated through a pathway involving prostaglandins. Indomethacin, an inhibitor of prostaglandin synthesis, inhibited the TGF-c~ and EGF effects on myc expression and DNA synthesis, but addition of prostaglandins E2 and F2~ restored these effects. 487'489 When the hepatocytes were isolated from older rats, EGF still had the same stimulatory effect on the expression of c-myc RNA, but the DNA synthetic response was much less. 488 This last experiment brings up the possibility that increasing c-myc expression does not force proliferation in hepatocytes. In a number of other experimental systems elevated expression of the c-myc gene did not correlate with increased cellular proliferation in liver cells. A sham operation itself, without removing any liver tissues, caused an increase in c-myc expression in the liver of mice, rats, and humans, although the magnitude was usually less and the timing different from the response to partial hepatectomy. 433'478'484 Inflammation caused by the intraperitoneal injection of Freund's adjuvant led to an increase in the level of c-myc RNA in rat liver. 484 Another example of a lack of correspondence between increased c-myc expression and proliferation was found when freshly isolated hepatocytes were put into culture; there was an initial transient surge in expression of c-myc RNA, then a more sustained increase which was not followed by cell proliferation. 49~ Similarly, intraperitoneal injection of glycine in rats caused a rapid transient increase in c-myc expression which was not followed by increased DNA synthesis. 492 Deprivation of protein caused an increase in the expression of c-myc RNA slowly over several days, which was rapidly reversed by refeeding a protein rich diet. 493 DNA synthesis followed several hours after c-myc RNA levels fell to normal. 493 Thus, increased expression of the c-myc gene does not necessarily lead to proliferation. We speculate that in some of these systems Myc may be performing a function related to apoptosis. Earlier we noted an association between expression of c-myc and TGF-cz transgenes, and apoptosis in the liver. 474 The growth inhibitor and differentiation agent butyrate caused a decrease in expression of the c-myc gene in several rat hepatoma cell lines. 494'495 In the HTC line the effect of butyrate was very rapid regardless of the cell cycle phase the cells were in, and was not blocked by the protein synthesis inhibitor cycloheximide

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which indicates a direct effect of the agent that is not mediated by the synthesis of a new protein(s). 494 Postpubertal male rats had a twofold increased level of c-myc RNA in their livers compared with females. 496 When the male rats were castrated, c-myc expression equalized between the sexes. The gender differences were reestablished if the castrated rats were given testosterone. When intact males were given additional growth hormone by osmotic minipump for 1 week, their expression of c-myc RNA was decreased to the female level. 496 The evidence suggests that gender differences in the expression of growth hormone, which are caused by testosterone, may account for gender differences in the effectiveness of some carcinogenesis protocols (described above), and that the regulation of expression of the c-myc gene in the liver by growth hormone may be part of the mechanism. 452-454'496 However, another group found that growth hormone had the opposite effect of rapidly and transiently raising the level of expression of the c-myc gene in the livers of hypophysectomized rats in vivo. 419 These differences are more apparent than real since the experiments involved differences in the dose of growth hormone in both the controls and the experimental animals, strain of rats, and timing of growth hormone administration and harvesting of the liver for analysis. 419'496

J. Pancreas

Clinical Associations An early report on one adenocarcinoma of the pancreas showed a low level of c-myc RNA, 4~ while another group reported a pancreatic carcinoma with c-myc

amplification and elevated expression of the gene. 497 In adenomas and adenocarcinomas of the pancreas, induced in rats by azaserine, the expression of c-myc RNA was elevated 2- to 8-fold and 8- to 40-fold, respectively. 498 The level of myc RNA in the azaserine induced carcinomas was found to be greater than in regenerating nonmalignant rat pancreas, although both tissues had similar rates of proliferation. 498 Four cases of endocrine pancreatic cancer have been reported as having a level of c-myc RNA below that of normal pancreas. 499 Pancreatic cancer has been relatively neglected by those studying the structure and expression of the myc gene in clinical specimens.

Bioassay The biological impact on the pancreas of elevated expression of the c-myc gene has been studied in a number of ways. The beta cells of the fetal rat pancreas were stimulated to proliferate by electroporation of a construct that expressed the human c-myc gene under control of an insulin promoter, and the effect was more consistent when an activated Ha-ras gene was cotransfected. 5~176 However, continuously growing lines were not established. 5~176 The low level of expression in cancer of the

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endocrine cells, described above, and the lack of major biological activity of the c-myc gene in these cells, leads to the suspicion that this gene may not play a role in the rare cancers of the endocrine pancreas. Infection of newborn mice with a recombinant retrovirus containing the v-myc oncogene caused acinar carcinomas in 31% with a latency of 150 days. 5~ The latency and the fact that the tumors were clonal suggests that additional events were required to produce neoplasia. 5~ Early experiments in which transgenic mice expressed c-myc in the pancreas failed to develop neoplasia in that organ. 314'502 But when the transgene was expressed at a high enough level by using an elastase promoter/enhancer to stimulate transcription and direct it to the pancreas, and a growth hormone 3' untranslated region and polyadenylation site with the intent of stabilizing the usually very unstable c-myc RNA, 4~ carcinomas of the pancreas were induced between 2 and 7 months of age. 183 The initial effect of the c-myc transgene was to produce a dysplasia in the acinar cells, but the carcinomas were of both acinar and mixed acinar ductal histology. It seems that the ductal carcinomas arose from transformed acinar cells. The myc transgene influenced the histology of the resultant tumors, as similar transgenic mice with activated ras or SV40 T-antigen transgenes developed only acinar adenocarcinomas. 183'5~ The transgenic mice experiments implicate the c-myc gene as a potential element in the pathogenesis of ductal adenocarcinoma of the pancreas, which is the most common type of pancreatic cancer in man. 183 Clearly, overexpression of the myc oncogene can be a factor in pancreatic carcinogenesis; however the relative dearth of clinical studies does not allow a judgment about how frequently.

Regulation Regeneration of the rat pancreas in response to partial pancreatectomy was preceded by an increase in expression of c-myc RNA. 5~ myc RNA was up more than twofold at 12 hours after surgery, which was before the peak of DNA synthesis seen at 2 days. 5~ It will be interesting to look at earlier events in regeneration of the pancreas in view of the rapid changes in c-myc expression seen during liver regeneration (see Section I. on liver). Camostat, a low molecular weight proteinase inhibitor, has been shown to cause the secretion of cholecystokinin which then results in a wave of DNA synthesis in rat pancreas in vivo; a transient 14-fold increase in the level of c-myc RNA preceded DNA synthesis. 498 Several agents which stimulate secretion by pancreatic acinar cells, cholecystokinin, bombesin, and carbachol caused a small increase in the level of c-myc RNA in freshly isolated rat acinar cells, while other agents which stimulate secretion such as gastrin, secretin, or vasoactive intestinal peptide had no effect. 5~ Thus, elevated c-myc expression is not necessary for secretion in this tissue.

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K. Stomach and Esophagus

Clinical Associations Elevated expression of the c-myc oncogene has been shown to be rare in carcinoma of the esophagus. 5~176 However, some tumors with increased expression of c-myc RNA or protein have been found. 5~176 The expression of protein and RNA encoded by the c-myc gene was found elevated compared to nonmalignant gastric mucosa in about half of the primary stomach cancers that have been reported. 5~176 One group found that expression of c-Myc protein in the stromal cells of gastric carcinomas correlated with lower invasiveness and a better prognosis. 5~ In this study, staining of the tumor cells was not predictive of stage, metastatic ability, or prognosis. 5~ In inflammatory, metaplastic, and dysplastic lesions, increased expression of c-Myc protein was found in some cases. 5~176 However, an extremely high level of c-Myc protein in nonmalignant specimens was found only in some adenomatous polyps and dysplastic lesions. 511 Amplification of the c-myc gene was found to be more rare, occurring in 3 out of the 52 primary stomach carcinomas that have been reported, 286'5~2'513 and in 3 of 16 xenografts maintained in nude mice. 514 In a study that compared clinical specimens of gastric and colorectal carcinoma, the bowel malignancies had greater levels of c-myc RNA. 5~ The biological significance o f increased expression of the c-myc gene in gastric and esophageal cancer is unknown at this time.

Regulation In vitro, serum starved AGS gastric carcinoma cells responded to serum with an increase in proliferation and expression of c-myc RNA, effects which were diminished by simultaneous exposure to both vasoactive intestinal polypeptide and isobutylmethyl-xanthine. 515 These agents increase cAMP production and implicate this signaling pathway in the regulation of c-myc expression and growth in gastric epithelial cells. 515 The gastric carcinoma cell line TMK- 1 responded to the mitogen epidermal growth factor with a transient four-fold increase in expression of c-myc RNA at 1 hour, which preceded the increase in DNA synthesis. 516

L. Colon and Rectum Clinical Associations In a survey of 29 primary human adenocarcinomas of the large bowel, 72% had a 5- to 40-fold elevated level of c-myc RNA compared with nonmalignant mucosa. 517 Additional surveys have revealed similar frequencies of elevated expression of c-myc RNA. 407'505'518-526 Surveys of adenomatous polyps, which are considered premalignant lesions, showed about two-thirds had a modestly elevated

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level of c-myc RNA that was lower than in the carcinomas. 525'526 Therefore, overexpression of c-myc can occur early in the neoplastic process. Questions have been raised as to whether overexpression of the c-myc gene is a significant event in carcinogenesis or simply a marker of increased cellular proliferation. In one study enhanced expression of the c-myc gene in colorectal adenocarcinoma was paralleled by an increase in the expression of the cell-cycle genes 2A9, ornithine decarboxylase (ODC), and histone H3, compared to normal mucosa in five of the six tumors studied. 527 If the expression of the c-myc gene was the rate-limiting step in cellular proliferation, then we would expect such a result. However, it has been suggested that the increased expression of the c-myc gene in these tumors reflects their greater proliferative capacity, and that a "true" overexpression of the c-myc gene would result in an increase in the relative abundance of c-myc RNA compared to the expression of cell-cycle-dependent genes like the histones. 527 At this point, an examination of the kinetics of cellular proliferation in normal and malignant colonic mucosa might prove illuminating. Turnover of normal mucosa is faster than the potential doubling time of tumor cells assuming no cell loss occurs. 528-53~The S phase of the cell cycle is much longer in carcinoma cells than in normal cells. TM At any given time more cells are in S phase in tumor than in normal mucosa, and this probably accounts for the tumor-specific increase in expression of the histone genes, which are expressed only during S phase. 532 Thus, histone gene expression is probably not a good tool to evaluate the proliferation rate in tumors of the large bowel. The expression of the c-myc gene is not confined to the S phase of the cell cycle. 533-535 The growth of colon tumors appears to be due to a lack of maturation and loss of the mature cells in the lumen of the gut, which is the fate of normal colonocytes. Interestingly, even using the stringent criteria for overexpression of the c-myc gene that it has to be out of proportion to the expression of histone RNA, 56% of 25 tumors examined in one study fulfilled this criteria. 536 Other experiments have also confirmed a lack of correspondence between markers of cellular proliferation and expression of the c-myc gene. In a study of azoxymethane-induced colon carcinogenesis in F344 male rats, the expression of c-myc RNA was elevated in all of the adenomas and invasive carcinomas studied, compared to slight and variable increases for Ha-ras, ODC, and ~-actin. 537 In an immunohistochemical study, there was no relationship between Myc expression in human colorectal carcinomas and cellular proliferation" the latter being evaluated using the Ki-67 antibody. 538 Therefore, the level of expression of the c-myc gene does not seem to be an indicator of the rate of cellular proliferation in tumors of the large bowel. Using immunohistochemistry, a number of laboratories have studied the expression of Myc protein in normal and malignant colonic mucosa. This methodology allows the determination of the percentage and localization of cells expressing Myc. In normal colonic mucosa, embedded in paraffin, immunohistochemistry with the monoclonal antibody (Mab) 6El0 revealed maximal staining in the cytoplasm of

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cells in the maturation zone between the proliferating cells of the basal crypt and the mature surface epithelium. 539'54~However, staining of frozen tissue with several different anti-Myc antibodies differed with respect to the intracellular location and the location within the crypt: Myc was largely limited to the nuclei of cells in the basal crypt. 538'541 One study using paraffin-embedded tissue demonstrated cytoplasmic and nuclear staining in 40-50% of the cells in the lower half of the crypt using the Mab 6El0, compared with nuclear staining in 25-30% of cells in the lower third of the crypt using the Mabs H51C 116 and H8C 150. 538 Explanations for these discrepancies have been suggested: (1) nuclear localization of Myc may be lost after fixation, 538'539'542'543 and/or (2) cytoplasmic staining with 6E 10 may be due to cross-reactivity of the Mab. 540'541 Irt situ hybridization has been used to show that c-myc RNA is located in the base of the crypt. 519 Thus it is likely that Myc protein is also located in that region. Most recent studies agree that Myc is located in the nucleus of normal colonic mucosa. 538'541 Analysis of adenomatous polyps by immunohistochemistry revealed a broader distribution of positive staining cells extending from the basal crypts to the luminal surface. 538'539'544 In adenocarcinomas of the large bowel the intensity and number of staining cells increased even more dramatically. 538-54j'545 Thus, Myc protein is expressed at an elevated level in this malignancy consistent with the results from RNA studies. However, there is a great deal of heterogeneity in the staining patterns between tumors. Some adenocarcinomas expressed Myc in every cell, in contrast with others that had mixtures of both positively and negatively stained regions. 538"54~Two groups that found a nuclear location for Myc in normal mucosa found evidence for a cytoplasmic location in carcinomas. 538'543 This difference in localization may be due either to an artifact of fixation or it may suggest that Myc is less tightly associated with the nuclear matrix in tumor cells. 538'543 However, colon carcinoma cell lines stained exclusively in the nucleus. 538'546 Interestingly, histologically normal mucosa immediately adjacent to some adenocarcinomas gave a positive staining pattern identical to the tumors. 538 This phenomenon may be due to a preneoplastic change, or since it was not observed in adenomas, a factor secreted by adenocarcinomas that causes elevated expression of the c-myc gene in tumor tissue as well as in the adjacent normal mucosa. 538 Although elevated expression of the c-myc gene occurs in over 70% of primary colorectal carcinomas, gross amplification or rearrangement of the gene is pretty rare. Amplification of the c-myc gene was detected in only 4.7% of 232 patient samples ana]yzed. 517"520-522'524-526"547-549Very slight--less than twofold--amplification of the c-myc gene was found more frequently and was associated with rare, aggressive subtypes of colon cancer. 55~ In a survey of 13 mucinous and 7 poorly differentiated tumors, 50% revealed a low level of c-myc amplification, compared to less than 7% for 29 moderately and well differentiated tumors. The amplification in these tumors (with one exception) was not due to polysomy of the chromosome carrying the c-myc gene, since the c-mos gene, also located on chromosome 8,19

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was not similarly amplified. Slight amplification may be due to a subpopulation of cells within the tumor with a substantial amplification of the c-myc gene. 55~ Amplification of the c-myc gene has been demonstrated in 7 (COLO320, HT29, SW480, SW620, SW742, WiDr, NCI-H716) of 29 colon carcinoma cell lines analyzed.255,517,522,551-555 Amplification of the c-myc gene was also seen in a mouse colon cancer induced by dimethylhydrazine. 556 Cell lines and tumors with significant amplification of the c-myc gene had elevated levels of c-myc RNA 520,522,524526,546,553-556 with one exception. 525 These levels of c-myc expression were similar to tumors and cell lines without amplification of the c-myc gene. 522'546'553'555 Therefore, elevated expression of c-myc can occur with and without gene amplification. Rearrangement of the c-myc gene in colon cancer has only been reported in two cell lines, COLO320DM 557 and 5W480, 425'546 and one patient specimen. 547 In each case the rearrangement was associated with amplification of the gene, which may have contributed to the rearrangement by providing additional targets. The c-myc gene rearrangement in COLO320DM was not found in its sister cell line COLO320HSR, suggesting that the rearrangement may have taken place after establishment of these cells in culture. 554 Although gross rearrangements of the c-myc gene are rare, the possibility of more subtle genetic alterations cannot be ruled out. In endemic Burkitt's lymphoma, mutations in the exon l/intron 1 boundary region of the gene have been implicated in causing an increase in the expression of c-myc RNA. 558'559 DNA sequence analysis of this region in a group of colorectal carcinoma cell lines with elevated expression of c-myc RNA (HCT8, NCI-H498, NCI-H747, SW837, S W I l l 6 , WiDr) revealed no alterations in the sequence. 56~ Thus, an endemic Burkitt's lymphoma-like mutational event is unlikely to be responsible for the deregulation of c-myc expression seen in colorectal carcinoma. Methylation of DNA is a covalent alteration which is often correlated with decreased gene expression. 561 DNA from normal colonic mucosa possessed a high level of methylation of a site in the third exon of the c-myc gene, while adenomatous polyps and adenocarcinomas had significantly less methylation at this site. 562 Expression of the c-myc gene was not analyzed in this study so we do not know if the hypomethylated state had the expected impact on gene expression. 562 The rectal carcinoma cell line SW837 had both elevated expression of the c-myc gene and significant hypomethylation of the exon 3 site, while the WiDr cell line had elevated c-myc expression, but was completely methylated at the same site. 553 However, the WiDr cell line had an amplification of the c-myc gene which was probably responsible for the elevated level of c-myc RNA in these cells. The expression of c-myc RNA per gene copy was essentially normal in the WiDr cells, which is consistent with its normal methylation state. The hypomethylation of the c-myc gene in SW837, which did not have c-myc amplification, is consistent with demethylation being contributory to elevated c-myc expression. 553

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The potential clinical value of knowing about an abnormal structure and/or expression of the c-myc gene in a tumor is of great interest. Earlier we discussed the association of slight amplification with aggressive subtypes of colorectal cancer. On the expression side, several studies, including a total of 162 tumors, have not found a correlation of elevated c-myc expression with a number of clinical parameters, such as the histological type, stage, depth of invasion, recurrence of disease, or the survival rate. 5~8'523-525'551However, an interesting correlation has been found between elevated expression of c-myc RNA and the location of the tumor within the colon. 1e'563 Elevated expression of the c-myc gene occurs more frequently in tumors of the left side (distal) than in tumors of the right side (proximal). It has been argued that increased myc expression may be a marker for a genetically distinct form of colon cancer that is the sporadic (noninherited) version of the colorectal tumors that frequently occur in patients with the inherited disease adenomatous polyposis col i. 12,13,563-565 What causes the elevated expression of c-myc in those colorectal tumors that do not have amplification or rearrangement of the gene? The lack of any evidence for a cis alteration suggests that a trans mechanism is at work. In support of this, the overexpression of the c-myc gene and the tumorigenic phenotype were suppressed by fusion of colon carcinoma cells with cells that express low levels of c-myc RNA. 564"565Based on the association of elevated expression with adenocarcinomas of the distal large bowel, the A P C gene (adenomatous polyposis coli locus) was suggested as a potential regulator of the expression of the c-myc gene in colorectal mucosa. 12'13'563 This hypothesis is supported by the finding that elevated c-myc expression in colorectal adenocarcinoma correlated with a loss of heterozygosity for markers on human chromosome 5q, 564 which is the location of the A P C tumor suppressor gene. 566 Furthermore, transfer of a normal chromosome 5 into the DLD-I, S W I l l 6 , and COKFu colorectal carcinoma cell lines suppressed the overexpression of c-myc, as well as tumorigenicity in nude mice. 565'567 Abnormal regulation of expression of the c-myc gene may, therefore, be one of the consequences of loss of function of the A P C gene. Two experiments have provided evidence that an activated ras gene may be responsible for increasing the expression of the c-myc gene in colon cancer. In the first experiment, mutationally activated human Ha-ras gene, under the control of a MMTV promoter, was inserted into rat intestinal cells. 568 Expression of this construct, induced by dexamethasone, resulted in the elevated expression of c-myc RNA and an immortalized, but nontumorigenic, phenotype. 568 The second experiment involved the disruption of either the normal or mutated endogenous Ki-ras gene in DLD-I and HCTII6 colon carcinoma cells by homologous recombination. 569 While inactivation of the normal allele had no effect on either cell line, inactivation of the mutationally activated Ki-ras gene resulted in an altered morphology, a slowing of cell growth, an inhibition of growth in soft agar, a loss of tumorigenicity in nude mice, and a 10-fold decrease in expression of the c-myc gene in both lines. 569 Although there is evidence for both ras and A P C mutations being

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responsible for causing elevated c - m y c expression in colon cancer, the detailed mechanisms, and relative importance of ras activation, A P C inactivation, and other yet to be discovered events are unknown.

Bioassay The clinical studies provide evidence that c - m y c activation may be an important step in producing neoplasia of the large bowel. In support of this, a cause and effect relationship between elevated expression of the c - m y c gene and the proliferative potential of COLO320 cells has been established. 169 Treatment of COLO320 cells with a 15-base antisense oligonucleotide covering the translation initiation site located in exon 2 of the c - m y c gene resulted in a 40-75% decrease in the ability of the cells to form colonies in soft agar, which was dependent on the concentration of the antisense oligonucleotide. Oligonucleotides with a variant sequence, either sense or missense, were not effective. 169 The biological consequences of exogenous expression of the m y c gene and other oncogenes in rat colon has been studied using retroviral vectors. 57~ Fragments of rat fetal colon tissue, maintained in collagen gels, were infected with retroviral vectors containing combinations of the v - m y c , v-src or v - H a - r a s genes under control of strong promoters and evaluated for growth in culture. 57t Infection with a single oncogene resulted in a greater outgrowth of epithelial cells, which underwent senescence and death within 2 weeks just like uninfected fragments, but did not result in immortalized cell lines. Tissue infected with a combination of either v - m y c and v-Ha-ras, or v - m y c and v-src produced immortalized colonic epithelial cell lines in about 80% of the attempts. Cells transformed with v - m y c and Ha-ras displayed a classical epithelial morphology, sucrase isomaltase activity, and the expression of keratin filaments. Cells transformed with v - m y c and v-src were less adherent, more mesenchymal in morphology, possessed sucrase isomaltase activity, and did not express keratin filaments. 571 In a similar series of experiments, segments of descending colon and rectum, dissected from rat fetuses, were infected with retroviral vectors containing v-myc, v-src, or v - m y c and v-src together. 572 Infected colonic segments were transplanted subcutaneously into syngeneic rats and the established heterotopic implants were harvested after 60-100 days. Out of 16 transplants infected with a v - m y c retrovirus, 5 had focal areas of atypia or dysplasia and 1 displayed goblet cell hyperplasia. All 12 of the v-src-infected transplants had focal areas of atypia/dysplasia with one sarcoma. The combination of v - m y c and v-src produced one adenocarcinoma and 11 transplants with atypia and high-grade dysplasia out of 16 successful transplants. The combination of v - m y c and v-src resulted in a more severely dysplastic histology than v - m y c alone, and a more precarcinomatous histology than the metaplasia produced by v - s r c . 572 These experiments provide evidence for significant biological activity of the m y c gene in the colorectum. However, since tumorigenicity was not the endpoint of most of the

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experiments, we do not have as clear a picture as we would like of the potential role of the c-myc gene in carcinogenesis in this tissue.

Regulation Treatment of colon carcinoma cells with sodium butyrate may provide a model system for the early molecular events involved in differentiation of the epithelial cells of the colonic mucosa. Butyrate treatment of colon cancer cell lines caused altered morphology, a slowing of cell growth, and an inhibition of growth in soft agar. 169,573-577 The differentiation markers carcinoembryonic antigen and a placental-type alkaline phosphatase were also induced by butyrate. 147'560'573'574'576"578-580 A decrease in the expression of c-myc RNA has also been associated with exposure to sodium butyrate in several colon carcinoma cell lines. 147'169'577'581'582Treatment of the rectal carcinoma cell line SW837 with 2 mM of sodium butyrate caused a rapid decline in the expression ofc-myc RNA that was blocked by protein synthesis inhibitors, suggesting that butyrate caused the synthesis of a protein which has a negative effect on the abundance of c-myc RNA. 147 Treatment of SW837 cells with 2 mM butyrate for 6 hours caused a decrease in transcriptional elongation through the c-myc gene of sufficient magnitude to account for the 10-fold decrease in the level of c-myc RNA expression. 56~ This effect was accompanied by a change in promoter usage. 560 Therefore, the protein(s) induced by butyrate may inactivate or block the activity of a factor involved in transcriptional elongation, or it may have a direct role in limiting elongation. Conversely, butyrate induced a decrease in the abundance of c-myc RNA due to posttranscriptional alterations, CACO-2 cells. 582 Thus, there may be heterogeneity in the mechanism by which butyrate lowers c-myc expression. Agents other than butyrate have also been used to investigate the regulation of the c-myc gene. Treatment of COLO320 cells with 2-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase, depleted intracellular polyamines resulting in an inhibition of cell growth and a 90% decrease in the expression of c-rnyc RNA that was due to a decrease in the transcription rate of the c-myc gene. 583'584 These results suggest that polyamine metabolism is involved in regulating the transcription of the c-myc gene. Transcription of the c-myc gene was also inhibited (42% at 96 hours) in DLD-I Clone A cells treated with N-methylformamide. 149 This agent also caused a decrease in DNA synthesis, cell growth, and tumorigenicity in this human colon cancer cell line. 149 Exposure of DLD-1 Clone A cells to recombinant human interferon-[3 ser 17 (IFN-]~ ser 17) caused a dose dependent reduction in expression of the c-myc gene and an inhibition of cellular proliferation. 585 In this case, though, the stability of c-myc RNA was altered, with its half-life decreasing from 29 minutes to 15 minutes after a 4-day treatment with IFN-[3 ser 17. The evidence suggested that the activity of the 2',5' oligoadenylate synthetase/RNase L pathway, which is activated by IFN-]], may destabilize c-myc RNA. 585

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Treatment of the human colorectal carcinoma cell lines HCT116 and MOSER with the differentiation agent N,N-dimethylformamide (DMF) resulted in a reduction of c-myc expression, which did not require synthesis of new protein. 148 Proliferating MOSER cells also responded to the transforming growth factor [3 (TGF-131) with a decrease in the expression of c-myc RNA. 148'586The reduction of c-myc expression upon exposure to DMF or TGF-131 was associated with a decrease in cellular proliferation and a more benign phenotype. 148 Tumors are different from person to person, and even within an individual tumor there is heterogeneity. Atissue culture model system may be useful in understanding a tumor cell type, but an understanding of cancer must involve an appreciation of tumor cell heterogeneity. Colon carcinoma cell lines representing different tumor cell types have been developed and characterized. 587 These cell lines have been classified into groups based upon their state of differentiation in vitro. 587 Poorly differentiated cells (group I) grow in a monolayer and form poorly differentiated xenografts in nude mice. Well-differentiated cells (group III), which resemble normal epithelial cells, grow in three-dimensional patches and display brushborden microvilli and tight junctions. Compared to group III cells, group I cells are more tumorigenic in vivo and have higher plating efficiencies in soft agar. A series of investigations has explored differences in the way these cells respond to their environment with respect to growth and regulation of gene expression. In growing cell cultures, the expression of the c-myc gene did not differ appreciably between cell lines of the well- and poorly differentiated groups. 588 However, they did differ in the way the c-myc gene was regulated. Quiescent group III cells responded to the replenishment of nutrients and the growth factors epidermal growth factor, insulin, and transferrin (EIT) with an increase in both expression of c-myr RNA and DNA synthesis: When nutrients and EIT were added to quiescent cultures of group III cell lines, FET or CBS, the expression ofc-myc RNA increased fivefold after 4 hours of treatment and was back down to base line by 24 hours" DNA synthesis peaked at around 20 hours. 589'590 Both responses were inhibited by TGF-~I. 589'591 Interestingly, quiescent group I cells did not respond to EIT or TGF-I31, but did increase DNA synthesis when nutrients were replenished. 59~Group I HCT 116 cells did not respond to EIT with increased DNA synthesis or increased expression of c-myc RNA. 590'59~ When nutrients were replenished in the quiescent HCTI 16 cell cultures, DNA synthesis increased but the expression of c-myc RNA did not increase. 59~ Clearly, there is a big difference in the way these two different cell types respond to growth stimulation. They differ as well in response to growth inhibition using TGF-]3 or deprivation of growth factors. TGF-~I caused a decrease in expression of c-myc RNA in the group III cells but not in the group I cells. 591 The group I cell lines, HCT116 and RKO, increased their expression of c-myc RNA when deprived of growth factors, while the group III lines, FET and CBS, did not change appreciably.-588 The group III cells usually follow the classic pattern of responding to growth stimulation by increasing expression of the c-myc gene, and growth inhibition by decreasing expression. The group I cells respond in

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a seemingly inappropriate way or do not respond at all. An important lesson from these studies is the hazards of generalizing from results generated with one or a few cell lines. Not only is there tissue-specific differences in the regulation of the c-myc gene, but even in malignancies of the same tissue there is heterogeneity. The molecular mechanisms behind the differences in control of c-myc expression and the impact of these alterations on the other phenotypic differences will tell an interesting story. Animal models for the modulation of c-myc expression have also been established. All-trans retinoic acid significantly reduced the total number of aberrant crypt foci induced by azoxymethane (AOM) in Sprague-Dawley rats. 592 The number of aberrant crypts expressing c-myc RNA decreased from 82% in the AOM-treated rats to 25% in the rats treated with AOM and retinoic acid. 592 While retinoic acid decreases c-myc expression, estradiol may act to increase expression of the c-myc gene. Tumors, resulting from injection of MC-26 mouse colon carcinoma cells into Balb/c mice, were consistently larger (by weight) in females, suggesting that female gonadal hormones play a role in tumor growth. 593 Ovariectomized (OVX) mice injected with MC-26 cells presented significantly smaller tumors after 21 days than OVX mice treated with estradiol or sham-operated control mice. Increased doses of estradiol produced larger tumors in both male and female mice injected with MC-26 cells. The larger tumors induced by estrogen treatment had higher levels of expression of ODC and c-myc RNA. 593 A direct effect of estrogen on expression of the c-myc gene was not established in this study, as in estrogen-responsive breast cancer cell lines (see Section III.B). Radiation causes an increase in proliferation of cells in the colonic crypts in rats and mice, which compensates for the cells lost to the toxic insult. 594'595This increase in proliferation was accompanied by an increase in expression of the c-myc gene. The Bowman-Birk protease inhibitor (BBI), which has anticarcinogenic properties in several systems, inhibited the increase in expression of c-myc RNA seen in X-irradiated mouse colon and in y-irradiated rat colon at a 7-day timepoint. Interestingly, BBI had no effect on the proliferation of the crypt cells, or on the expression of c-myc RNA in unirradiated colon. 594'595 These results suggest that a protease may be involved in the pathway for elevating c-myc expression. 594 Since the anticarcinogenic property of the BBI was not tested in this system, we do not know if the inhibition of expression of the c-myc gene is associated with a reduction in carcinogenesis.

IV. PUTTING THINGS IN PERSPECTIVE: DOES MYC CAUSE CANCER EVERYWHERE? It is clear from the vast volume of work on the biological activity of the c-myc gene that it is a potentially carcinogenic agent in many tissues. One exception to this trend is the salivary gland. In the M M T V - m y c transgenic mouse strains developed

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by the Leder laboratory, the salivary gland was one of the tissues with the highest levels of myc expression, but not a single tumor was noted in this tissue. 312'314'315 MMTV-ras transgenic mice got salivary gland tumors, so there was no barrier to a MMTV-oncogene construct causing cancer in this organ. 315 The salivary gland appears resistant to the carcinogenic effects of elevated expression of the c-myc gene. The barrier may be the absence of some target or cofactor required for c-myc carcinogenicity. Cancer has long been considered the product of a number of independent events. 596-598 The evaluation of the biological potency of an activated c-myc gene in the systems described in this review reveal that in every case c-myc could not induce cancer by itself. Whenever it was evaluated, the tumors were monoclonal or oligoclonal proliferations in a tissue that had an activated c-myc gene in the nonmalignant cells. Activation of c-myc expression increased the probability of a cell becoming a tumor, but did not assure that result. Even in experiments in which an activated myc gene was combined with another activated oncogene, the tumors were clonal. Thus, activation of the c-myc oncogene is not sufficient f o r carcinogenesis. Another finding from the study of transgenic animals is that activated c-myc expression in many tissues is compatible with grossly normal developmen t. 89-91'312-314,385,415,468,474,599 Because of this, abnormal regulation of expression of the c-myc gene may be considered a possible facet in cancer predisposition. As described in the section on liver, there was a correlation between gender differences in regulation of the c-myc gene and susceptibility to hepatocellular carcinoma in a strain of rats. Polymorphisms in the regulation of myc expression may turn out to be significant in understanding genetic differences in susceptibility to malignancy. Not all cellular proliferation is associated with increased expression of the c-myc gene. Examples include the C3H10T1/2 mouse fibroblast cell line in which the proteinase inhibitor antipain caused a reduction in the expression of c-myc RNA, and a disappearance of the transient increase in expression which was seen when quiescent cells were stimulated to proliferate, without altering DNA synthesis, rate of proliferation, or saturation density. 6~176176 Similar findings have been made in vivo: During early development of the human placenta the highest level of c-myc expression corresponded to the cytotrophoblastic shell which is very proliferative, 6~ however in the fetus there was a low level of c-myc RNA despite rapid proliferation at 3-4 weeks postconception. 6~ At 6-10 weeks postconception highly proliferative epithelial layers had a high level of expression of c-myc RNA, but the cartilage cells of the rapidly growing limb bud and head had a low level of c-myc RNA. 6~ In mouse embryos the expression of c-myc RNA decreased sharply at 7.5 days in the most proliferative cells, the primitive ectoderm. 6~ Later in development of the mouse embryo there was a good correspondence between proliferation rate and c-myc expression. 6~176 As described earlier, adult mouse spermatogonia actively proliferate and have very low levels of c-myc RNA. 4~ The association

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between increased expression of the c-myc gene and cellular proliferation does not hold in many normal tissues, so elevated myc expression is not simply a marker of proliferation. Does carcinogenesis require activation of c-myc? As described earlier, the c-myc gene is overexpressed very frequently in colorectal cancer especially when it occurs in the distal colon, but not every adenocarcinoma of the large bowel has overexpression. On the other end of the spectrum, elevated c-myc expression was found much less frequently in leukemia, especially chronic lymphocytic leukemia in which elevated expression was rarely found. 607-609 As seen throughout this review, there are many examples of malignancy without elevated expression of the c-myc gene. Cancer does not require elevated c-myc expression. Even though a c-myc function appears to be a requirement for carcinogenesis in many experimental systems, there probably are ways to circumvent the need for overexpression of the c-myc gene itself. An example of this kind of carcinogenic complementation is found in the study of p53 mutations and mdm2 amplification6'0: The mdm2 gene product binds to and inhibits the tumor suppressor p53. Amplification and overexpression of mdm2 bypasses the need for a p53 mutation in carcinogenesis. Similarly, in some types of cancer an activated N-myc gene seems to substitute for c-myc activation. 1~'~12'6~'612 However, we do not yet have a clear picture of exactly what might qualify as a substitute for c-myc activation as a required step in carcinogenesis. Despite the extensive literature on the c-myc gene we still lack an understanding of how this gene causes cancer. However, a continuation of the rapid progress in the last few years on understanding the biochemical activities of Myc protein makes it likely that its role will become gradually clearer. The genes that are regulated by c-Myc and the rules that govern when the quantity of Myc protein is the critical variable in their regulation will be an important focus of future work. The events that result in activation of the c-myc gene are known for some cancers, and it is likely that additional mechanisms will be clear in the near future, perhaps involving the inactivation of tumor suppressor genes. There is still a mountain of work to be done before the myc gene is truly understood.

ACKNOWLEDGMENTS This work was supported by National Cancer Institute Grant CA50246 to P.G.R., and a Postdoctoral Fellowship to D.P.H. awarded by the Scientific Education Partnership, which is funded through the Marion Men'ell Dow Foundation. The laboratory was also supported by a grant from the Hall Family Foundations of Kansas City and the Patton Memorial Trust. We thank Darren Baker for help in gathering references. Despite our attempt to be comprehensive it is likely that we have omitted important references through oversight. For this and any unintentional misinterpretations we ask forgiveness.

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572. D'Emilia, J. C.; Mathey-Prevot, B.; Jaros, K.; Wolf, B.; Steele, G. Jr.; Summerhayes, I. C. Preneoplastic lesions induced by myc and src oncogenes in a heterotopic rat colon. Oncogene 1991, 6, 303-309. 573. Niles, R. M.; Wilhelm, S. A.; Thomas, P.; Zamcheck, N. The effect ofsodi um butyrate and retinoic acid on growth and CEA production in a series of human colorectai tumor cell lines representing different states of differentiation. Cancer Ira,est. 1988, 6, 39-45. 574. Chung, Y. S.; Song, I. S.; Erickson, R. H.; Sleisenger, M. H.; Kim, Y. S. Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines. Cancer Res. 1985, 45, 2976-2982. 575. Kim, Y. S.; Tsao, D.; Siddiqui, B.; et al. Effects of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells. Cancer 1980, 45, 1185-I 192. 576. Gum, J. R.; Kam, W. K.; Byrd, J. C.; Hicks, J. W.; Sleisenger, M. H.; Kim, Y. S. Effects of sodium butyrate on human colonic adenocarcinoma cells. J. Biol. Chem. 1987, 262, 1092-1097. 577. Barnard, J. A.; Warwick, G. Butyrate rapidly induces growth inhibition and differentation in HT-29 cells. Cell Growth & Differ. 1993, 4, 495-501. 578. Tsao, D.; Shi, Z.; Wong, A.; Kim, Y. S. Effect of sodium butyrate on carcinoembryonic antigen production by human colonic adenocarcinoma cells in culture. Cancer Res. 1983, 43, 12 ! 7-1222. 579. Deng, G.; Liu, G.; Hu, L.; Gum, J. R. Jr.; Kim, Y. S. Transcriptional regulation of the human placental-like alkaline phosphatase gene and mechanisms involved in its induction by sodium butyrate. Cancer Res. 1992, 52, 3378-3383. 580. Herz, F.; Schermer, A.; Halwer, M.: Bogart, L. H. Alkaline phosphatase in HT-29, a human colon cancer cell line: influence of sodium butyrate and hyperosmolality. Arch. Biochem. 1981, 210, 581-591. 581. Taylor, C. W.; Kim, Y. S.; Chiidress-Fields, K. E.; Yeoman, L. C. Sensitivity of nuclear c-mvc levels and induction to differentiation-inducing agents in human colon tumor cell lines. Cam'er Left. 1992, 62, 95- ! 05. 582. Souleimani, A.; Asselin, C. Regulation of c-myc expression by sodium butyrate in the colon carcinoma cell line Caco-2. FEBS Lett. 1993, 326, 45-50. 583. Celano, P.; Baylin, S. B.; Giardieilo, E M.; Nelkin, B. D.; Casero, R. A. Jr. Effect of polyamine depletion on c-m3z" expression in human colon carcinoma cells. J. Biol. Chem. 1988, 263, 549 i -5494. 584. Celano, P.; Baylin, S. B.; Casero, R. A. Jr. Polyamines differentially modulate the transcription of growth-associated genes in human colon carcinoma cells. J. Biol. Chem. 1989, 264, 89228927. 585. Chatterjee, D.; Savarese, T. M. Posttranscriptional regulation of c-myc proto-oncogene expression and growth inhibition by recombinant human interferon-[~ ser 17 in a human colon carcinoma cell line. Cancer Chemotherapy PharmacoL 1992, 30, 12-20. 586. Mulder, K. M.; Levine, A. E.; Hernandez, X.; McKnight, M. K.; Brattain, D. E.; Brattain, M. G. Modulation of c-myc by transforming growth factor-13 in human colon carcinoma cells. Biochem. Biophys. Res. Commtm. 1988, 150, 71 i-716. 587. Brattain, M. G.; Levine, A. E.; Chakrabarty, S.; Yeoman, L. C.; Willson, J. K. V.; Long, B. H. Heterogeneity of human colon carcinoma. Cancer Metastasis Rev. 1984, 3, 177-191. 588. Mulder, K. M. Differential regulation ofc-myc and transforming growth factor-~ messenger RNA expression in poorly differentiated and well-differentiated colon carcinoma cells during the establishment of a quiescent state. Cancer Res. 1991, 51, 2256-2262. 589. Mulder, K. M.; Humphrey, L. E.; Choi, H. G.; Childress-Fieids, K. E.; Brattain, M. G. Evidence for c-m~,c in the signaling pathway for TGF-13 in well-differentiated human colon carcinoma cells. J. Cell Physiol. 1990, 145, 501-507. 590. Mulder, K. M.; Brattain, M. G. Effects of growth stimulatory factors on mitogenicity and c-myc expression in poorly differentiated and well differentiated human colon carcinoma cells. Mol. Emiocrinol. 1989, 3, 1215-1222.

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591. Mulder, K. M.; Zhong, Q.; Choi, H. G.; Humphrey, L. E.; Brattain, M. G. Inhibitory effects of transforming growth factor 131 on mitogenic response, transforming growth factor o~, and c-myc in quiescent, well-differentiated colon carcinoma cells. Cancer Res. 1990, 50, 7581-7586. 592. Stopera, S. A.; Bird, R. P. Effects of all-trans retinoic acid as a potential chemopreventive agent on the formation of azoxymethane-induced aberrant crypt foci: differential expression of c-myc and c-fos mRNA and protein. Int. J. Cancer 1993, 53, 798-804. 593. Narayan, S.; Rajakumar, G.; Prouix, H.; Singh, P. Estradiol is trophic for colon cancer in mice: effect on ornithine decarboxylase and c-myc messenger RNA. Gastroemerol. 1992, 103, 18231832. 594. St. Clair, W. H.; St. Clair, D. K. Effect of the Bowman-Birk protease inhibitor on the expression of oncogenes in the irradiated rat colon. Cancer Res. 1991, 51, 4539-4543. 595. St. Clair, W. H.; Billings, P. C.; Kennedy, A. R. The effects of the Bowman-Birk protease inhibitor on c-myc expression and cell proliferation in the unirradiated and irradiated mouse colon. Cancer Lett. 1990, 52, 145-152. 596. Armitage, P.; Doll, R. The age distribution of cancer and a multistage theory of carcinogenesis. Bl: J. Cancer 1954, 8, 1-12. 597. Fisher, J. C. Multiple mutation theory of carcinogenesis. Nat,re 1958, 181,651-652. 598. Mooigavkar, S. H.; Knudson, A. G. Jr. Mutation and cancer: A model for human carcinogenesis. J. Natl. Cancer Inst. 1981, 66, 1037-1052. 599. Jackson, T.; Allard, M. E; Sreenan, C. M.; Doss, L. K.; Bishop, S. P.; Swain, J. L. The c-myc protooncogene regulates cardiac development in transgenic mice. Mol. Ceil. Biol. 1990, 10, 3709-37 i 6. 600. Chang, J. D.; Billings, P. C.; Kennedy, A. R. C-myc expression is reduced in antipain-treated proliferating C3H 10TI/2 cells. Biochem. Biophys. Res. Cortttntttt. 1985, 133, 830-835. 601. Chang, J. D.: Kennedy, A. R. Cell cycle progression of C3H 10Tl/2 and 3T3 cells in the absence of an increase in c-myc RNA levels. Carcinogenesis 1988, 9, 17-20. 602. Pfeifer-Ohlsson, S.; Goustin, A. S.; Rydnert, J.; et al. Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: implications for embryonic cell proliferation. Cell 1984, 38, 585-596. 603. Pfeifer-Ohlsson, S.; Rydnert, J.; Goustin, A. S.; Larsson, E.; Betsholtz, C.; Ohlsson, R. Celltypespecific pattern ofmyc protooncogene expression in developing human embryos. Proc. Natl. Acad. Sci. USA 1985, 82, 5050-5054. 604. Downs, K. M.; Martin, G. R.; Bishop, J. M. Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo. Genes De~: 1989, 3, 860-869. 605. Schmid, P.; Schulz, W. A.; Hameister, H. Dynamic expression pattern of the myc protooncogene in midgestation mouse embryos. Science 1989, 243, 226-229. 606. Himing, U.; Schmid, P.; Schulz, W. A.; Rettenberger, G.; Hameister, H. A comparative analysis of N-myc and c-myc expression and cellular proliferation in mouse organogenesis. Mechn. Dev. 1991, 33, 119-126. 607. Birnie, G. D.; Warnock, A. M.; Burns, J. H.; Clark, P. Expression of the myc gene locus in populations of leukocytes from leukaemia patients and normal individuals. Leukemia Res. 1986, 10, 515-526. 608. Rothberg, P. G.; Erisman, M. D.; Diehi, R. E.; Rovigatti, U. G.; Astrin, S. M. Structure and expression of the oncogene c-myc in fresh tumor material from patients with hematopoietic malignancies. Mol. Cell. Biol. 1984, 4, 1096-1103. 609. Ferrari, S.; Torelli, U.; Selleri, L.; et al. Study of the levels of expression of two oncogenes, c-myc and c-myb, in acute and chronic leukemias of both lymphoid and myeloid lineage. Leukemia Res. 1985, 9, 833-842. 610. Momand, J.; Zambeffi, 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.

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611. Mukherjee, B.; Morgenbesser. S. D.; DePinho, R. A. Myc family oncoproteins function through a common pathway to transform normal cells in culture: cross-interference by Max and transacting dominant mutants. Genes Dev. 1992, 6, 1480-1492. 612. van Lohuizen, M.; Verbeek, S.; Krimpenfort, P.; et al. Predisposition to lymphomagenesis in pim-I transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell 1989, 56, 673-682.

CYTOGENETIC A N D MOLECULAR STUDIES OF MALE GERM-CELL TUMORS

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

.

I!. 111. IV. V. VI. VII. VIII.

416 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karyotypic Profile of Oerm-Cell Tumors . . . . . . . . . . . . . . . . . . . . 416 Molecular Analysis of Oerm-Cell Tumors . . . . . . . . . . . . . . . . . . . . 418 Molecular Cytogenetics of Oerm-Cell Tumors . . . . . . . . . . . . . . . . . 419 Application of Cytogenetic Findings in the Diagnosis and Prognosis of Oerm-Cell Tumors . . . . . . . . . . . . . . . . . 423 Cytogenetic Basis of Malignant Transformation in Teratomatous Lesions . . . 423 Chromosome Abnormalities and Pathogenesis of Oerm-Cell Tumors . . . . . 424 425 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Genome Biology Volume 3B, pages 415--428. Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-835-8 415

416

E. RODRIGUEZ, C. SREEKANTAIAH, and R. S. K. CHAGANTI I.

INTRODUCTION

Adult male germ-cell tumors (GCTs) are a heterogeneous group of neoplasms that arise in premeiotic or early meiotic germ cells. They originate in gonadal or extragonadal sites (mediastinum, retroperitoneum, pineal) and histologically comprise two major groups: seminomas and nonseminomas. Seminomas are composed of neoplastic germ cells that mimic gametogenesis and act as immature spermatogenic cells. Nonseminomas present features of embryonic neoplastic germ cells and mimic histogenesis of the early embryo. Among nonseminomas, embryonal carcinoma is a pluripotential tumor which may progress along extraembryonic or trophoblastic lineages resulting in yolk sac tumor or choriocarcinoma, or along embryonic lineages resulting in teratoma. Individual tumors may present multiple histologies; mixed GCTs contain nonseminomatous components and combined GCTs present with seminomatous and nonseminomatous elements. Teratomatous lesions occasionally undergo malignant differentiation into tumors with other histologies such as sarcoma, carcinoma, neuronal or neuroectodermal tumor, or myeloid leukemia. The GCTs comprise a unique system for the study of the genetic basis of malignant transformation, differentiation, and drug sensitivity, The GCTs are characterized by a highly specific chromosomal abnormality, i(12p), which is present in 76% of the tumors. Other nonrandom cytogcnetic abnormalities are additional and are seen in lower frequencies. A subsct of GCTs do not exhibit the i(12p) marker; in these other abnormalities leading to multiple copies of 12p are usually present. A combination of cytogenetic analyses with molecular techniques such as fluorescent in-sit, hybridization and Southern blotting have resulted in a better resolution of some of the chromosomal changes and led to the identification of sites of candidate tumor suppressor genes in these tumors. This chapter provides a review of the current state of knowledge of cytogenctic and molccular cytogenetic studies of male GCTs and a correlation of these findings with the histological and clinical features of the tumors.

il.

KARYOTYPIC PROFILE OF GERM-CELL T U M O R S

Primary Chromosome Change. Atkin and Baker first (1982) described the association of testicular GCTs with a specific chromosomal abnormality, i(l 2p). Since then 225 tumors have been cytogenctically analyzed and an i(12p) has becn consistently reported in 76% of the tumors (Table 1). Despite the heterogeneity of the neoplasm, i(12p) has been characteristically observed in male GCTs of all histologies including seminomas, teratomas, embryonal carcinomas, choriocarcinomas, mixed and combined tumors from gonadal as well as extragonadal sites (Table 1). The data for Table 1 represent a combination of data from single case reports and cytogenetic series reported from different parts of the world; therefore, the incidence frequencies must be considered an approximation. The information,

417

Male Germ-Cell Tumors Table 1. Incidence of i(12p) in Histologic Subsets of GCTs Detected by

Cytogenetic Analysis 1'2'3

Gonadal Histologic Subset

with i(12p)

without i(12p)

Extragotladal with i(12p)

withottt i(12p)

Gonadal & Extragonadal with i(12p)

without i(12p)

Total

Seminoma

24(67)

12(33)

2(100)

0

26(68)

12(32)

38

Teratoma

61(88)

8(12)

4(100)

0

65(89)

8(11)

73

Embryonal carcinoma

27

20(77)

6(23)

0

1(100)

20(74)

7(26)

Choriocarcinoma

1(25)

3(75)

1(100)

0

2(40)

3(60)

5

Yolk sac tumor

3(100)

0

4(57)

3(43)

7(70)

3(30)

10

Non-seminoma with malignant transformation

6(100)

0

4(67)

2(33)

10(83)

2(17)

12

29(78)

8(22)

1(50)

1(50)

29(76)

9(24)

38

Combined GCT

Mixed GCT

5(42)

7(58)

I(100)

0

6(46)

7(54)

13

Undifferentiated Carcinoma

0

0

5(83)

1(17)

5(83)

1(17)

6

Pineal

0

0

0

3(100)

0

3(100)

3

Total

149(73)

44(27)

21(66)

11(34)

170(76)

52(24)

225

Notes." Ilncluding primary and metastatic lesions. -'Based on the data reported in the following publications and unpublished results oil 5 ! tumor biopsies from our laboratory: Albrecht et ai., 1993 ( i tumor biopsy); Atkin and Baker, i 983 (4 tumor biopsies); Atkin et al., 1993 (3 tumor biopsies); Casledo et al., 1988a,b, 1989a,b,c (42 tumor biopsies); Dal Cin et al., 1989 (! tumor biopsy); Dclozier-Blanchet, 1985 (4 tumor biopsies); Gibas et ai., 1984, 1986 (7 tumor biopsies); Haddad et al., 1988 (2 tumor biopsies); Hecht et al., 1984 (I tumor biopsy); Murty et al., 1990 (7 ceil lines); Oosterhuis et al., 1986, 1989, 1991 (4 tumor biopsies); P,'u'rington et al., 1987 (4 cell lines); Rodriguez et al., 1991, 1992a, 1993 (58 tumor biopsies); Samaniego et al., 1990 (24 tumor biopsies); Shen et al., 1990 (I tumor biopsy); Suijkerbuijk et al., 1993 (9 tumor biopsies, 1 cell line); Walt et al., 1986 (1 tumor biopsy). 3Numbers in parentheses indicate percentages.

nevertheless, is comparable to the findings on large series of tumors from single institutions in the published literature (de Jong et ai., 1990; Rodriguez et al., 1992a). Although not much is known about the exact nature and role of i(12p) in the development of GCTs, the frequent occurrence of this marker, clearly reflects the significance of i(12p)in the development of this neoplasm. AdditionaiNonrandom Chromosomal Changes. The modal chromosome number in GCTs is usually in the hyperdiploid to hypertetraploid range and numerous additional structural rearrangements are common. Based on the cytogenetic reports of the 225 tumors with clonal karyotypic abnormalities, the chromosomes most frequently rearranged, in addition to i(12p), were 1, 3, 6, 7, 9, 11, 12, and 17. A clustering of breakpoints to certain chromosomal sites and an association with histological or clinical features of tumors has been reported earlier (Rodriguez et al., 1992a). Thus, breaks at 1p32-36 and 7ql 1.2 were noted more frequently in

418

E. RODRIGUEZ, C. SREEKANTAIAH, and R. S. K. CHAGANTI

teratomas, while lp22 breaks were more frequent in yolk sac tumors. Breaks at 10p13, 7ql 1.2, and 12p l l-q13 were more frequently encountered in metastatic lesions compared to primary lesions and breaks involving lp36, 6q21, 7ql 3, and 12qll-I 3 were more frequent in posttreatment specimens compared to pretreatment specimens. Cytogenetic Evidence of Gene Amplification. Homogeneously staining regions (HSRs) and double minute chromosomes (dmins) represent chromosomal manifestations of gene amplification, a phenomenon commonly associated with resistance to treatment of cells in vitro or tumors in vivo with antineoplastic drugs (Schwab and Amler, 1990). In the case of GCTs, presence of HSRs and molecular evidence for DNA amplification in HSR-bearing tumors was first presented by Samaniego et al. (1990). HSRs or dmins have been identified in 13 cases of GCTs, all but one of which were metastatic lesions (Rodriguez et al., 1992a; Albrecht et al., 1993). In one of these tumors, although DNA amplification was confirmed by DNA in-gel renaturation, amplification of any gene previously shown to be amplified in multiple tumor systems was excluded by hybridization of tumor DNA with a large panel of probes (Samaniego et al., 1990). Thus, it appears that a novel gene(s) is uniquely amplified in association with malignant progression of GCTs. The isolation and characterization of such gene(s) should be of significance not only to GCTs but to other tumors as well.

!11. MOLECULAR ANALYSIS OF GERM-CELL TUMORS Molecular Methods for Detection of i(12p).

By conventional cytogenetic analysis, clonally abnormal karyotypes can be detected only in 50 to 70% of cases (Samanicgo et al., 1990; Rodriguez et al., 1992a). Thus, in a substantial proportion of the cases, the status of chromosome 12 remains undetermined. One method to determine the copy number of 12p and/or 12q was by Southern blot analysis of tumor DNA hybridized with probes for loci on the respective arms. In this assay, the ratio of the signal between the target gene (a gene on 12p) and a reference gene (a gene on any chromosome arm other than 12p) in tumor DNA is compared to the identical ratio obtained from analysis of normal DNA (control), such as placental DNA. Increase in copy number of a 12p locus in tumor DNA relative to control DNA would indicate presence of multiple copies of 12p and hence, probably, i(12p). Analysis of such DNA ratios in cytogenetically characterized tumors and tumor cell lines showed an excellent correlation between DNA and cytogenetic assays for i(12p) (Dmitrovsky et al., 1990; Samaniego et al., 1990).

Identification of Candidate Tumor Suppressor Genes from Deletions in 12q. The detection of specific chromosomal deletions has classically enabled the identification of tumor suppressor genes: e.g., RB, WT1, APC, and TP53 genes (Marshall, 1991). Cytogenetic studies in GCTs have identified deletions in 12q in up to 15% of GCTs, with or without simultaneous i(l 2p) (Castedo et al., 1989b; Murty

Male Germ-Cell Tumors

419

et al., 1990; Samaniego et al., 1990; Rodriguez et al., 1992a, 1993). In order to determine whether the cytogenetic deletions in 12q truly represent molecular deletions, a recent study undertook analysis of loss of heterozygosity (LOH) comparing germline and tumor genotypes at eight polymorphic loci mapped to 12q. These results showed high frequency of LOH (> 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

~D Q. v

_,'~o

0

0

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 <50 years, tumors involving midline structures, lung parenchyma, or lymph nodes), long-term survival with cisplatin-based chemotherapy was achieved (Richardson et al., 1981; Greco et al., 1982, 1986). In a recent study of nine patients with UEGCCS, four were shown to have abnormalities of chromosome 12 consistent with a diagnosis of GCT (Motzer et al., 1991). Therefore, patients with undifferentiated carcinomas of unknown primary, especially those involving the midline structures, must be subjected to cytogenetic analysis to rule out a diagnosis of GCT. Additionally, the use of powerful new techniques, such as those described above, now make it possible to determine precisely the number of 12p copies and verify the initial observations of Bosl et at. (1989) which suggested that the copy number of this chromosomal arm may predict survival.

VI. CYTOGENETIC BASIS OF M A L I G N A N T TRANSFORMATION IN TERATOMATOUS LESIONS A number of teratomatous lesions undergo malignant transformation presenting specific differentiation patterns of nongerm cell lineages such as sarcoma, adenocarcinoma, neuroepilthelioma, and myeloid leukemia; these cases have been shown to be accompanied by the appearance of specific chromosomal changes which have been previously characterized in de novo tumors of the same histology (Ulbright et al., 1984; Chaganti et al., 1989; Ladanyi et al., 1990; Rodriguez et al., 1991, 1992a). The origin of this transformation, in particular its clonal relationship to the teratomatous lesion, has been determined in a number of cases. One example is a patient in whom myeloid leukemia presented a mediastinal teratoma and yolk sac tumor in less than 2 years from the diagnosis. Both lesions were shown to be clonally related by detection of i(12p); the leukemia in addition acquired a deletion in 5q which is characteristic of de novo myeloid leukemias (Chaganti et al., 1989).

424

E. RODRIGUEZ, C. SREEKANTAIAH, and R. S. K. CHAGANTI

Similarly, malignant transformation of teratomatous lesions to embryonal rhabdomyosarcoma and neuroepilthelioma were shown to be associated with 2q37 and 1lq24 rearrangements, respectively--aberrations which are characteristic of de novo tumors with the same histologies (Rodriguez et al., 1991; 1992a). The elucidation of the cytogenetic basis of these transformations not only comprises additional support to the view that specific chromosome changes are etiologically relevant in tumorigenesis, but also provides new opportunities for isolation of genes at these sites.

VII. CHROMOSOME ABNORMALITIES A N D PATHOGENESIS OF GERM-CELL TUMORS Testicular seminomas as well as nonseminomas are suggested to develop from carcinoma in situ (CIS) (Skakkabaek et al., 1987), while primary mediastinal GCTs are considered to arise from primordial germ cells held behind in the midlinc during their embryonal migration from yolk sac to the gonadal ridge (Ashley, 1979; Gonzales-Crussi, 1982). However, due to the fact that mediastinal GCTs exhibit identical nonrandom chromosomal changes as gonadal GCTs, it has been proposed that all GCTs have a gonadal origin (Chaganti et al., 1993); occasional migration of GCT precursors early in their development to extragonadal sites would become established as primary mediastinal GCTs. Gonadal seminomas exhibit chromosome numbers in the hypertriploid to tctraploid ranges, and nonseminomas present chromosome numbers in the hypotriploid ranges (Atkin, 1973; de Jong et al., 1990). In addition, nonseminomas have been shown to be predominantly XXY in sex chromosome constitution (Atkin ct al., 1991 ). Based on these data, polyploizization has been suggested to be the initial cytogenetic change in the pathogenesis of this tumor (Vos et al., 1990; Atkin et al., 1991 ). de Jong et al (1990) proposed a modcl of pathogenesis which called for an initial polyploidization of the precursor cell resulting in CIS whose further evolution to seminomas and nonseminoma would be based on generation of the i(12p) marker and progressive and selective loss of chromosomes by nondisjunction. According to this model, the i(12p) formation would be a secondary event possibly related to progression. However, since 12p amplification has been shown to be present in i(12p)-negative tumors as well (Rodriguez et al., 1993; Suijkerbuijk et al., 1993), this suggests that this chromosomal change must be involved in the initiation process. A model for the genesis of GCTs has been proposed (Chaganti et al., 1993) in which a pachytene-diplotene spermatocyte is the precursor cell for all GCTs. If in an occasional pachytenediplotene cell the repair mechanism, necessary to complete crossingover, fail due to failure of induction of a key protein, the cell would be unable to progress further in meiosis and degenerates. A sporadic meiocyte with a defective repair mechanism may be rescued from death by initiation of a new program of mitotic division. Therefore, the precursor GCT cell could be a 4C cell with defective repair aberrant-

Male Germ-Cell Tumors

425

exchange events affecting the pericentromeric regions of 12p leading to i(12p), or tandem duplication of 12p segments providing the necessary extra copy number (and the increased product) of the key gene which initiates the new cycling event and rescues the cell from death. Initiation of one round of DNA replication in the 4C cell would lead to endoreduplication and a tetraploid cell with an i(12p) or increased 12p copies. Because of the initial defective repair, the cell may be genetically unstable and liable to undergo additional changes such as nondisjunction, deletion, and mutation. The potential significance of chromosome 12 alterations in GCT development has been alluded to above. The data comprise is: (1) i(12p) is common to all histologic subsets and anatomic presentations of GCTs, unrelated to ploidy levels of individual tumors; (2) most tumors with i(12p) [including those with 2 normal copies of chromosome 12 and one i(l 2p)] retain both paternal and maternal copies of chromosome 12, suggesting that i(12p) is generated by either exchanges in homologous nonsister chromatids as suggested by Mukherjee et al. (1991) or by unequal "misdivision" following a nondisjunctional gain of one of the chromosomes 12 in the precursor cell; and (3) deletion analysis provided evidence for two candidate tumor suppressor genes on 12q that are potentially unique to this tumor system (Murty et al., 1992). Therefore, chromosome 12 alterations are of critical significance in the development of these tumors.

VIII.

SUMMARY

Although the biological significance of GCTs to the study of malignancy and differentiation has been well recognized for some time, detailed genetic analysis based on fresh tumor biopsies has not been initiated until recently. The first stage of such studies, as with other, more extensively investigated tumor systems, has been cytogenetic analysis. To date, cytogenetic data on 225 tumors are available which, although small in number, have already yielded valuable insights into the biology of these tumors and a clinically useful marker. Thus, initial correlations between chromosome change and histology have been recorded, gene amplification associated with malignant progression has been identified, cytogenetic basis of malignant differentiation in teratomatous lesions has been clarified, and sites of candidate tumor suppressor genes unique to this system have been identified. The usefulness of i(12p) as a diagnostic marker, especially in tumors of uncertain histologies has been established. Because of the clinical usefulness of this marker, molecularly based methods for its detection, without the need for formal cytogenetic analysis, have been developed. Cytogenetic analysis of larger prospectively ascertained series than have been performed so far and analysis of large numbers of tumors utilizing molecular techniques can be expected to yield significant insights into the biology and clinical behavior of these tumors.

426

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INDEX

Abelson murine leukemia virus (AMuLV), 304 ABL gene, 304-307 (see also "BCR/ ABL oncogene...") Abortive transformation, 59 (see also "p53...") Acute myeloid leukemia (AML), 417 Adenovirus, 56, 61-63, 135 (see also

"p53...'~ ALL, 182, 183-186, 215 (see also "Chromosomes and cancer') hyperdiploidy in, 186 neoplasias with shared single recurrent chromosomal defect, 184-185 p53 mutations, 113 Philadelphia (Ph) chromosome, 186 T-cell acute lymphoblastic leukemia, 186 Alterations in genes as main cause of cancer, 158 AML, p53 mutations in, 113 Amplification, 337, 338 (see also "myc gene") in bladder cancer, 359 in brain cancer, 352-353 in breast cancer, 346-347 in cervical cancer, 350

in colorectal cancer, 369-370 in esophageal cancer, 367 in liver cancer, 359-362 in lung cancer, 344-345 in ovarian cancer, 351 in pancreatic cancer, 365 in prostate cancer, 354-355 in stomach cancer, 367 in uterine cancer, 351 Androgen, 355 Angiogenesis, 18, 22-24 (see also "Oncogenes... ") ANLL, 182-183, 187-188 (see also "Chromosomes and cancer") Antioncogenes, 19 (see also "Oncogenes... ") Antisense nucleic acids, 340, 341, 343, 349-350 apc gene, 277, 371 Apoptosis, 19, 35, 213-214 (see also "Oncogenes...") and myc gene, 340-341 (see also "myc gene") in liver cancer, 363 in prostate cancer, 355-356 Assessment methods for tumor clonality, 3 Ataxia-telangiectasia (AT), 197, 220 ATL, 220

429

430

B-cell malignancies, chromosome rearrangements in, 211-215 (see also "Chromosomal translocations... ") non-Hodgkin's lymphoma, 188189 Basic-helix-loop-helix (bHLH), 231, 243-245 (see also "Transcription...") leukemia, three additional proteins in, 244-245 bcll, 212-213 (see also "Chromosomal translocations...") bcl-2 gene, 19-20, 35, 213-214 and Epstein-Barr virus (EBV), 214 LMP, 214 bcl3, 214 BCL6, 214 BCR/ABL oncogene, role of in human leukemia, 299-329 abbreviations, common, 300 A BL gene, 304-307 Abelson murine leukemia virus (AMuLV), 304 FES, 307 Hardy-Zuckerman (HZ-2) virus, 304-306 transcripts and translation products, 306 BCR gene, 307-308 phi, 307 Rac, regulatory factor for, 307308 in blast crisis CML, 315-317 cytogenetics, 303-304 diagram, 305 experimental studies with, 318-319 fusions in acute leukemias, 314315 fusions in CML, 308-314 Southern blot analysis, 308, 309, 314 introduction, 299-300

INDEX

overview, 319-320 Philadelphia chromosome, 300-302 "blast crisis," 301 CML, 300-302, 303 hepatomegaly, 301 splenomegaly, 301 Beckwith-Wiedemann syndrome, embryonic tumors and, 5 (see also "Genetics...") Bladder cancer, 358-359 amplification, 359 bioassay, 359 clinical association, 358-359 DNA methylation, 358 Bloom's syndrome, 197 Bowman-Birk protease inhibitor (BBI), 375 Brain cancer, 352-354 amplification, 352-353 bioassay, 353-354 clinical associations, 352-353 differentiation, 353 from glioblastoma multiforme, 353 meningioma cell line, 353 p53 alterations, serological analysis of, 112 raf oncogenes, 353 rearrangement, 353 in transgenic experiment, 354 Breast cancer, 346-350 amplification, 346-348 antisense, 349-350 bioassay, 347-349 clinical associations, 346-347 estrogen, 349-350 fibrocystic disease, relationship to, 347 Ha-ras gene, 348 inheritance factor in, 291,347 Line-1 repetitive element, 347 p53 alterations, serological analysis of, 108-109

Index

progesterone, 350 prognostic indicators, search for, 347 rearrangement, 347 regulation, 349-350 tamoxifen, 350 transgenic mouse experiments, 348 btgl, 212 (see also "Chromosomal translocations...") Burkitt's lymphoma (BL), 104, 184186, 188-189, 209, 210, 211, 216, 370 (see also "Chromosomal translocations..." and "Chromosomes and cancer") basic helix-loop-helix oncogenes, 243-245 (see also "Transcription...") and Epstein-Barr virus (EBV), 214 and myc gene, 336 p53 alterations, serological analysis of, 113 Butyrate: in colon cancer, 373 in liver cancer, 364-365

c-myc gene, 332-408 (see also "myc gene") Calcium-binding proteins in tumor cell adhesion, 30 Carcinogenesis, 331-408 (see also "myc gene") Cancer, human, genetics of, 1-16, 331-408 (see also "Genetics..." and "myc gene") Cancer, transcription and, 227-272 (see also "Transcription...") CD44, 29-30 (see also "Oncogenes...") CDDP, 34-35 (see also "Oncogenes...") Cerebral tumors, p53 alterations and, 112

431

Cervical cancer, 350-352 amplification, clinical associations, 350-351 HPV, 111 p53 alterations, serological analysis of, 111-112 regulation, 351-352 CGH, 292 Chromosomal translocations in lymphoid malignancy, molecular genetics of, 205-225 B-ceU malignancies, rearrangements in, 211-215 ALL, 215 apoptosis, 213-214 bcll, 212-213 bcl2, 213-214 (see also "bcl2") bcl3, 214 BCL6, 214 btgl and myc, 212 centrocytic lymphoma, 213 CLL, 212 CML, 215, 300-302 cyclin D 1, 213 cyclins, 212-213 diffuse large cell lymphoma, 214 E2A gene, 215 EBV, 212 follicular lymphoma, 213 intermediate lymphocytic lymphoma, 213 myc, 211-212 nodular lymphoma, 213 notch homolog, 214 in parathyroid adenoma, 213 pbxl, 215 Philadelphia chromosome, 214215 (see also "Philadelphia...") pradl, 212-213 sporadic tumors, 212 t(1;19) fusion, 215 tanl, 214

432

conclusion, 220-221 features, general, 207-211 in B-cell malignancies, 208, 209210, 211-215 (see also "...Bcell...") Burkitt's lymphoma, 209, 210, 211 enhancers, 209-210 fusion proteins, 210 genes involved in, 207-209 karyotypes, abnormal, 207 mechanisms of, 210-211 mechanisms of deregulation, 209-210 Philadelphia chromosome fusion protein, 208, 210 Rag- 1 and Rag-2, 210 recombinase enzymes, 210-211 in T-cell malignancies, 209 topoisomerase, 211 introduction, 206-207 leukemias, acute and chronic, 206 molecular cytogenetics, 206 non-H odgkin's lymphomas (NHL), 206 T-cell leukemia, common characteristics of, 216-220 adult (ATL), 220 ataxia-telangiectasia (AT), 220 GATA-1,217 hox-11 gene, 219 LIM domains, 218 lyll, 218 myc locus, 216-217 notch, 219 RBTN 1 and 2, 218 rhombotin 1 and 2 loci, 218 TALl/SCL/TCL5, 217 TAN1, 219 TCL1 locus, 220 tcl3 gene, 219 Ttg-1,218

INDEX

Chromosome painting, 413-414 Chromosomes and cancer, 179-204 abnormalities, consistent, 180-181 banding techniques, 180 cloning of familial cancer genes, 181 neoplasia, specific, 180 tissue culture methods, 180 chronic myelogenous leukemia (CML), 181 consistent chromosomal abnormalities in, 181 hematologic disorder, most common, among myeloproliferative diseases, 181 Ph-chromosome, 181 conclusion, 198 human tumors, pattern in, 193197 retinoblastoma, 193 Wilms' tumor, 193 introduction, 180 leukemias, acute, 182-188 ALL, three FAB subtypes of, 182, 183-186 (see also "ALL") ANII, seven FAB subtypes of, 182-183, 187-188 (see also "ANLL") monoblastic (AMoL), 188 myeloblastic (AML-M2), 187 myelomonocytic (AMMoL), 187 neoplasias with shared single recurrent chromosomal defect, 184-185 promyelocytic (APL-M3), 187 lymphoproliferative disorders, 188-192 Burkitt's lymphoma, 188-189 non-Hodgkin's lymphoma, 188192 (see also "NonHodgkin's lymphoma")

Index

polycythemia Vera (PV), 190192 preleukemia, 192 refractory anemia, 192 neoplasms of chromosome breakage syndromes, 197-198 ataxia telangiectasia, 197 Bloom's syndrome, 197 Fanconi's anemia, 197 Kostmann's agranulocytosis, 197 Werner's syndrome, 197, 198 Xeroderma pigmentosum, 197, 198 Chronic myelogenous leukemia (CML), 97, 181,215, 300302, 303-304 (see also "Chromosomes and cancer") B C R / A B L fusions in, 308-314 in blast crisis CML, 315-317 p53 alterations, serological analysis of, 112-113 Cisplatinum (CDDP), 34-35, 343 (see also "Oncogenes...") CLL, 212 (see also "Chromosomal translocations...") p53 alterations, serological analysis of, 113 Colorectal cancer, studies on, 160161, 163, 287, 288, 291,367375 adenomatous polyps, 367-369 allelotyping, 287 amplification, 369-370, 371 antisense oligonucleotide, 372 A PC gene, 371 bioassay, 372-373 Bowman-Birk protease inhibitor (BBI), 375 butyrate, 373 clinical associations, 367-372 differentiation, 373-374

433

estrogen, 375 familial adenomatous polyposis coli, 287 interferon, 373 methylation, 370 as model of tumor progression, 96-102 p53 alterations, serological analysis of, 105-108 polyps, 287, 367 ras, 371-372 rearrangement, 370 regulation, 373-375 S phase, 368 TGF-/3 growth factor, 374 trans mechanism, 371 tumor cell heterogeneity, 374-375 Comparative genomic hybridization (CGH), 292 Constitutional heterozygosity, loss of in human cancer, 5-7 Cyclin D 1, 213 Cyclins, 76, 212-213 Cytogenetics, 303-304 DCC gene, 30, 277 (see also "Oncogenes...") in colorectal cancer studies, 161, 288 Deletion mapping, 289-290 Diffuse large cell lymphoma, 188189, 214 DNA methylation, 370 DNA binding protein, p53 as, 83-84 (see also "p53...") DNA tumor viruses, p53 and, 55-135 (see also "p53...") oncogenes, 57-58 E2A gene, 215 EBV, 214 Embryonal carcinoma, 410

434

Enhancers, 209-210 (see also "Chromosomal translocations... ") and Burkitt's lymphomas, 211 Epidermal growth factor (EGF) in liver cancer, 363, 364 Epithelial cells as primary cancer targe t , 344 Epstein-Barr virus (EBV), 214 erbB-2 gene, 20-21, 31, 38-40 ERK2, 169-171 Esophageal cancer, 367 amplification, 367 Barret's epithelium, 110 clinical associations, 367 epidermal growth factor, 367 p53 alterations, serological analysis of, 110-111 regulation, 367 Estrogen, 349-352, 357, 375 ets oncogene, 250-251 etsl protooncogene, 22 Familial adenomatous polyposis, 277 coli, 287 Familial cancer, role of genes in, 5 Fanconi's anemia, 197 FGF family, 23, 38 FISH technique, 292, 410, 413 Fluorescent in-situ hybridization (FISH), 410, 413 Follicular lymphoma, 213 fos, 34 Fusion proteins, chromosome translocation and, 210 (see also "Chromosomal translocations...") G6PD isoenzyme method of clonality assessment, 3, 320 Gardner's syndrome, 287

INDEX

Gastric carcinoma, p53 mutations and, 111 GATA-1,217 Gene amplification, 337 Gene therapy for neoplastic diseases, 14 Genetic disease, cancer as, 274 (see also "myc gene") retinoblastoma, 274 Genetics of human cancer:overview, 1-16 allele loss in tumors, 6-7 cancer predisposition and progression, 4 checkpoints in cell cycle, 3-4 p53, role of, 4-5 phases, five, 3 RB gene, 4 clonality of tumors, 2-3 assessment methods, 3 G6PD isoenzyme method, 3 RFLP, 3, 5 single cell, origin in, 2 TCR gene, 3 variable number of tandem repeat polymorphisms (VNTRP), 3 constitutional heterozygosity, loss of, 5-7 gene amplification, 13 gliomas, 13 medulloblastoma, 13 neuroblastoma, 13 gene therapy for neoplastic diseases, 14 genomic imprinting, 5-7 Beckwith-Wiedemann syndrome, 5 neuroblastoma, 7 osteosarcoma, 7 rhabdomyosarcoma, 5-7 RBI gene, 5 Wilms' tumor, 7

Index

heritable cancer, 4-5 p53, relationship of, 4-5 retinoblastoma, 4 introduction, 2 "predisposing" genes, 2 protooncogenes, 7-10, 12 amplification in human tumors, 12-13 molecular mechanism of carcinogenesis, 10 tumor suppressor genes, 10-13 Germ-cell tumors (GCTs), cytogenetic and molecular studies of, 409-422 chromosome abnormalities and pathogenesis of germ-cell tumors, 418-419 cytogenetic basis of malignant transformation in teratomatous lesions, 417-418 cytogenetics of, molecular, 413416 application of in diagnosis and prognosis of GCTs, 417 chromosome painting, 413-414 FISH technique, 413 UEGCCS, 417 introduction, 410 chromosomal abnormality, highly specific, 410 embryonal carcinoma, 410 fluorescent in-situ hybridization (FISH), 410, 413 i(12)(p 10) marker, 410, 417, 419 malignant transformation, basis for study of, 410 nonseminomas, 410 seminomas, 410 Southern blotting, 410, 412 teratoma, 410 karyotypic profile of, 410-412 chromosome changes, additional nonrandom, 411-412

435

chromosome change, primary, 410-411 gene amplification, cytogenetic evidence of, 412 molecular analysis of, 412-413 summary, 419 Gliomas, 13 deletion mapping, 289 genetic alterations in, 287-289 Greig's syndrome, 246 GTPase activating proteins, 165 Guanine nucleotide exchange factors (GNRF), p21 ~ and, 165, 168-169 Ha-ras gene in breast cancer, 348349 Hardy-Zuckerman (HZ-2) virus, 304-306 Hematologic malignancies, chromosomal basis of, 179-204 (see also "Chromosomes...") p53 alterations, serological analysis of, 112-113 AML, 113 in chronic myeloid leukemia (CML), 112-113 in lymphoid malignancies, 113 MDS, 113 Hepatocellular carcinoma, 360 p53 alterations, serological analysis of, 110 aflatoxin exposure area, 110 G---T transversions, 110 HER-2 gene, 20, 27, 35, 39 Heritable cancer, 4-5 p53 mutations in, 114-115 Heterozygosity, constitutional, loss of in human cancer, 273-297 conclusions, 291-292 CGH, 292 FISH, 292

436

deletion mapping, 289-290 11q 13 amplicon, 289 in familial breast carcinomas, 289 in gliomas, 289 in MEN 1-associated tumors, 289 genetic disease, cancer as, 274-275 familial aggregation of tumor types, 274 karyotype analysis, 275 Philadelphia chromosome translocation, 275 retinoblastoma (RB), 274 tumor suppressor genes, 275 xeroderma ~pigmentosum, 275 introduction, 274 cytogenetics, 274 genetic losses, screening for, 274 LOH, 274 protooncogenes, 274 loss of, principles of, 277-283 allele loss, 278-281 allelic imbalance, 281 CA-repeats, 278-279 chromosome abnormalities, examples of, 278 clonality of tumor cells, 283 DNA polymorphisms, 278-279 examples, 282 genetic alterations detectable by, 279-281 karyotyping, benefits and drawbacks of, 277-278, 280 micro-satellite markers, 278279, 281 mitotic recombination, 280 molecular genetic approach, 277-278 pitfalls, 281-283 restriction endonuclease, 278 RFLP, 278, 279, 284 Southern blot analysis, 279, 281

INDEX

studies, considerations in, 281283 VNTRs, 278-279 MEN examples, 284-286 in medullary thyroid carcinomas (MTC), 285 MEN 1 gene, 284 MEN 2A, 285-286 in parathyroid tumors, 285 in pheochromocytomas, 285 oncogenes, 275 retinoblastoma paradigm, 283-284 tumor progression, LOH in, 286289 colorectal tumor, 287, 288 (see also "Colorectal tumor...") genetic alterations, three types of, 286 glioma, 287-289 tumor suppressor genes, 275-277 dcc gene, 277 familial adenomatous polyposis (apc gene) Li-Fraumeni syndrome, 277 neurofibromatosis types, 277 p53 gene, 277 RB gene, 276-277 "two-mutation model," 276 Wilms'tumor, 277 Homeobox motif, 232-233 genes, 247-248 hox-2.4, 248 hoxl l, 248 pbx, 248 pre-B-ALL, 248 hox-I 1 gene, 219 HPV, 63-64 (see also "p53...") and cervical cancer, 111-112 Imprinting, genomic, 5-7 Beckwith-Wiedemann syndrome, 5 Inherited predisposition to cancer, 4

Index

int-2 oncogene, 23, 38-39 Interferon: in cervical, ovarian and uterine cancer, 352 in colorectal cancer, 373 in lung cancer, 346 Invasion, local, by malignant tumors, 18 Kaposi's sarcoma, 22-24 Karyotyping, 277-278 Kidney cancer, 357-358 bioassay, 358 clinical associations, 357 estrogen, 357 folic acid, 358 regulation, 358 in transgenic mouse experiment, 358 unilateral nephrectomy, 358 Kostmann's agranulocytosis, 197 Leucine zipper (LZ), 231-232 (see also "Transcription...") Leukemias, acute and chronic, 182188, 206 (see also "Chromosomes and cancer") BCR/ABL fusions in, 314-315 human, role of BCR/ABL oncogene in, 299-329 (see also "BCR/ ABL oncogene...") molecular basis of, 206 (see also "Chromosomal translocations... ") Li-Fraumeni syndrome (LFS), 114, 277, 343 LIM domains, 218 Liver cancer, 359-365 amplification, 359-362 apoptosis, 363 bioassay, 362-363 butyrate, 364-365

437

castration, 365 clinical associations, 359-362 epidermal growth factor (EGF), 363, 364 hepatectomy, partial, 363-364 hepatitis B virus, risk of, 361-362 hepatocellular carcinoma, 360 methylation site, 359-360 Morris hepatomas, 360 raf, 362 regulation, 363-365 Solt-Farber carcinogenesis protocol, 361 TGF-a growth factor, 363, 364 in transgenic mouse experiment, 363 LOH technique, 273-297 (see also "H eterozygosity... ") Lovastatin, 36-37 Lung cancer, 344-346 bioassay, 345-346 c-myc amplification, 344-345 clinical associations, 344 interferon, 346 p53 alterations, serological analysis of, 109-110 GC---TA transversions, 109 in radon-associated cancer, 110 regulation, 346 small cell lung cancer (SCLC), 345 lyl gene, 218 Lyl-i protein, 244 Lymphoid malignancies, molecular genetics of chromosomal translocations in, 205-225 p53 alterations, serological analysis of, 113 (see also "Chromosomal translocations...") Lymphomas, non-Hodgkin's (NHL), 206 (see also "Chromosomal translocations...")

438

Lymphoproliferative disorders, 188192 (see also "Chromosomes and cancer") MAP2 kinase, 169-170 Matrix metalloproteinase (MMP), 31 Max protein, 243-245, 341-342 mdm-2 gene, p53 alterations and, 115 mdrl, 33-34 (see also "Oncogenes... ") MDS, p53 mutations in, 113 Medulloblastoma, 13, 352-354 (see also "Brain cancer") Melanoma, 163-164 MEN 1 gene, 284 Metastasis, 24-29 (see also "Oncogenes... ") Methylation, 359 DNA, 370 Molecular cytogenetics of chromosomal translocations, 205225 (see also "Chromosomal translocations...") Molecular mechanism of carcinogenesis, 10 M onoblastic leukemia, acute (AMoL), 188 Morris hepatomas, 360 myb oncogene, 249-250 myc, translocations involving, 211212 (see also "Chromosomal translocations...") and btgl, 212 myc gene, 331-408 as cancer cause, 375-377 amplification, 377 Myc protein, 377 salivary gland as exception, 375-376 not required for cancer, 377 not sufficient alone, 376

INDEX

transgenic mouse strains, 375376 classic, 332-344 amplification, 338, 343 antisense nucleic acids, 340, 341, 343 apoptosis, 340-341,355-356 avian leukosis virus (ALV), 335336 basics, 332-333 biochemical aspects, 341-343 Burkitt's lymphoma, 336 cancer therapy and, 343-344 differentiation, 339-340, 343 DNA binding protein, 341 encoding gene, regulator of, 342 family, 338 in fibroblasts, 336 gene amplification, 337 Ha-ras gene, 337 history, 333-335 interferon, 341 L- and N-myc gene, 338, 345 Li-Fraumeni syndrome, 343 Max, 341-342 neu gene, 338 null mutants, 335 overexpression of, 335-337 PDGF, 338-339 proliferation of, 338-339 raf, 335, 346 ras, 335, 338, 340, 341,342-343 retroviruses, 335-336 RNAs, intgranuclear, stability of, 342 src, 335 trans mechanism, 337 as transcriptional activator, 342 in transgenic mice, 336, 341 v-myc, 333-335 from viral oncology, 333

Index

global, 344-375 (see also under particular head) animal models, 375 in bladder cancer, 358-359 in brain cancer, 352-354 in breast cancer, 346-350 in cervical cancer, 350-352 in colon cancer, 367-375 epithelial cells primary cancer target, 344 in esophageal cancer, 367 interferon, 346, 352 in kidney cancer, 357-358 in liver cancer, 359-365 in lung cancer, 344-346 in ovarian cancer, 350-352 in pancreatic cancer, 365-366 in prostate cancer, 354-356 in rectal cancer, 367-375 regulation of c-myc gene, 374375 salivary gland as exception, 375-376 in stomach cancer, 367 in testicular cancer, 356 tumor cell heterogeneity, 374 in uterine cancer, 350-352 introduction, 332 c-myc gene, role of in disease, 332 T-cell leukemia and, 216-217 basic helix-loop-helix oncogenes, 243-245 Myeloblastic leukemia, acute (AMLM2), 187 Myelodysplastic syndrome (MDS), studies on, 160-163 (see also "ras gene family") Myelomonocytic leukemia, acute (AMMoL), 187 Neoplasms: of chromosome breakage syndromes, 197-198

439

transcription and, 227-272 (see also "Transcription...") Nephroblastoma, 7, 142, 193, 245246 neu gene, 20, 27, 35, 39, 338 Neuroblastoma, 13 Neurofibromatosis type I gene as GTPase activating protein of p21 r~, 168 Neurofibromin, 168 NHL, 188-189 NIH3T3 cells, 26-28, 31-32 in identification of ras mutations, 158 nm23-H1 and -H2, 28-29 (see also "Oncogenes... ") Nodular lymphoma, 213 Non-Hodgkin's lymphoma (NHL), 188-189 B-cell, 188-189 Burkitt's lymphoma, 188-189 T-cell, 189-192 polycythemia Vera (PV), 190192 Nonseminomas, 410-419 (see also "Germ-cell tumors...") Oncogenes, role of in tumor progression, 7-10, 17-53, 275, 333335 and angiogenesis, 18, 22-24 etsl, 22 FGF family, 23 int-2, 23 Kaposi's sarcoma, 22-24 thrombospondin (TSP), 23-24 and cell adhesion, 29-30 calcium-binding proteins, 30 CD44, 29-30 cytoplasmic p H, 29 DCC gene, 30 P-cadherin, 30 ras; 29-30

440

and cell growth, 19-22 apoptosis, 19, 35 bcl-2 gene, 19-20, 35 erbB-2 gene, 20-21, 38-39 growth factor, 20 HER-2 gene, 20, 27, 35 HGF, 20 met hepatocyte growth factor, 20 neu gene, 20, 27, 35 TGF-et and/3 expressions, 21 trk, 20 conclusions, 41 definition, 18-19 and drug resistance, 33-35 cisplatinum (CDDP), 34 fos, 34 mdrl, 33-34 tamoxifen, 35 history, 333-335 and immune surveillance, 32-33 decreased major histocompatibility complex (MHC) class I antigens, 32 introduction, 17-18 angiogenesis, 18, 22-24 c-myc messenger, 19 effects of activation of, 18 growth of cells, effect on, 18 invasion, local, 18 mutations, 18 Philadelphia chromosome translocation, 18 ras gene, 19 and metastasis, 24-29 cooperation between oncogenes, 26-27 growth factor receptors, 27 NIH3T3 cells, 26-28, 31-32 nm23-H 1 and -H2, 28-29 ras mutation, 25-26 suramin, 28

INDEX

as outcome predictors, 38-40 in clinical area, 38 erbB-2, 39-40 PCNA, 38 as prognostic markers, 38-39 ras mutations, 40 and proteolysis, 31-32 cysteine proteases, 31 heparinase, 31-32 major excreted protein (MEP), 31 matrix metalloproteases (MMP), 31 NIH3T3, 31-32 and radiation resistance, 35-38 C-raf-1, 36 cyclin B expression, 38 G2 delay, 38 lovastatin, 36-37 retinoblastoma gene, 4, 19 transcription factor, 237-253 (see also "Transcription...") tumor suppressor genes, 10-13, 18 Ovarian cancer, 350-352 amplification, 351 clinical associations, 351 regulation, 351-352 vanadate, 352 P-cadherin, 30 (see also "Oncogenes... ") P-glycoprotein, 33 (see also "Oncogenes... '3 p21 ~, 157-177 (see also "ras...") p53 tumor suppressor gene, 55-135, 253-255, 277 activities, 83-86 as DNA binding protein, 83-84 as transactivating protein, 84 as transcriptional repressor, 84 wild-type p53, cellular genes regulated by, 85-86

Index

alteration in human cancer, 96-115 immunohistochemical analysis of p53 accumulation in tumor cells, 103-104 and LFS, 114 mdm-2 gene, 115 mutational events, 103 mutations, distribution of in molecule, 101-103 mutations in human cancer, frequency of, 96-102 neoplasias, other, 115 PCR technology, 97 ras gene mutations, 97-99 sarcomas, 115 serological analysis, 104-115 (refer alsoto particular cancer) in T-cell tumors, 113 in breast carcinoma, 255 cDNA cloning, 66 and cell cycle, 86-94 cell-cycle control, role in, 4-5, 138, 149 cell proliferation, role in, 86 cellular response to DNA damage, 92-94 in colorectal cancer studies, 161 conformational states, adopting, 90, 91 expression during, 86 signaling pathways, component of, 94 wild-type and mutant p53, 9092 wild-type as antiproliferative, 86-90 characteristics of from various species, 67 conclusions and perspectives, 115116 clinincal aspect of, 116 in differentiation, 94-95

441

and DNA tumor viruses, 58-66 abortive transformation, 59 adenoviruses, 61-63 HPV, 63-64 oncogenes, 57-58 p53, RB and DNA tumor virus oncogene products, 64-66 p53 protein, 60-61, 73 papillomavirus, 63-64 polyoma, 58-60 REF, 59-60 SV40, 58-61, 78-79 in embryogenesis, 94-95 in spermatogenesis, high expression in, 94-95 gene organization, 68-71 introduction, 56-58 and rbl gene, 58, 64 localization in cell, 71-73 distribution in normal and transformed cell, 71-72 nuclear localization signal, 72 in malignant mesothelioma, 255 mutations, 90-92, 96-115 (see also "...alteration...") protein, 60-61 adenovirus E 1B protein, interaction with, 79 cyclins, 76 domains, five, 73-74 functional domains of, 73 hsp70, interaction with, 80-81 mdm-2, interaction with, 81-82 organization, 75 and p53, interaction with, 81 p90, 81-82 E6 papilloma virus protein, interaction with, 79-80 phosphorylation, 75-77 regions, 74-75 SV40 large-T antigen, interaction with, 78-79

442

as tumor suppressor gene, 95-96 Ha-ras gene, 96 mutations in human cancers, 96 p 120GAP, 166-168 (see also "ras gene family") Pancreatic cancer, 365-366 amplification, 365 bioassay, 365-366 Camostat, 366 clinical associations, 365 ras gene, 365 regulation, 366 in transgenic mice, 366 Papillomavirus, 63-64 (see also "p53...") Papovavirus, 56-135 (see also "p53...") Parathyroid adenoma, bcll gene and, 213 pbxl, 215 PDGF, 338-339 Philadelphia chromosome translocation, 18, 208, 210, 275 in ALL, 186 (see also "ALL") fusion transcripts and proteins, 214-215 in human leukemia, 300-302 phi gene, 307-308 PKC and p21 r~ssignaling, 170-171 Polycystic kidney disease, 357-358 (see also "Kidney cancer') Polycythemia Vera (PV), 190-192 Polyoma virus, 58-60 (see also "p53...") POU, 233 (see also "Transcription...") Pradl , 212-213 "Predisposing" genes, 2 Preleukemia, 192 Progesterone, 350-352 Promyelocytic leukemia, acute (APL-M3), 187, 246-247

INDEX

Prostate cancer, 354-356 amplification, 354-355 androgen, 355 apoptosis, 355-356 bioassay, 355 castration, 355 ras, 355 regulation, 355-356 Protooncogenes, 7-10, 274, 275 amplification of in human tumors, 12-13 Rac, 307-308 Radiation resistance, oncogenes and, 35-39 (see also "Oncogenes... ") raf, 335, 338, 353 Rag- 1 and Rag-2, 210 (see also "Chromosomal translocations... ") ras gene family, |9, 60, 87, 335, 363, 365, 366, 368, 371-372 in breast cancer, 348-349 gene mutations in human tumors, 97-99 gene products, 164-165 ha-ras gene, 348-349 mutation, 25-26, 40, 157-177 alterations in genes as main cause of cancer, 158 in benign and malignant tumors, 160 colorectal cancer, studies on, 160-161,163 heterogeneity of, 163-164 history, 158 in human tumors, 158-160 in lung tumors, 159-160 introduction, 158 melanoma, 163-164 myelodysplastic syndrome (MDS), studies on, 160-163 seminomas, 163

Index

443

during tumor development, 160- Renal cell carcinomas, 357-358 (see 163 also "Kidney cancer") REP, 168-169 NIH3T3 as identifier, 158 p21 r~, 164-171 Retinoblastoma, 193 gene, 4, 19, 58-135, 148 (see also ERK2, 169-171 growth factors, activation by, "p53...") 166 hereditary characteristic of, 193,274 GTP and GDP, affinity for, paradigm, 283-284 164-165 RFLP, 3, 5, 142 GTPase activating proteins, 165 rGEF, 168-169 guanine nucleotide exchange Rhombotin 1 and 2, 218, 247 factors (GNRF), 165, 168169 Salivary gland, exception of to cmyc MAP2 kinase, 169 neurofibromatosis type 1 gene carcinogenic effect, 375-376 as GTPase activating proSarcomas, p53 mutations and, 115 tein of, 168 Seminomas, 163, 410-419 (see also p21 ~, ~nl7, 165 "ras gene family" and pl20GAP, 166-168 "Germ-cell tumors...") PKC, 170-171 Serine/threonine kinases, p21 ~ and, REP, 168-169 169-171 rGEF, 168-169 Signal transduction, functions of p21 r~ in, 165-166, 170 and serine/threonine kinases, 169 tyrosine kinases, role of, 166 signal transduction, functions ski oncogene, 251 Skin carcinoma: in, 165-166, 170-171 tyrosine kinases, role of, 166p53 alterations, serological analy168, 171 sis of, 111 in prostate cancer, 355 Small cell lung cancer (SCLC), 344three in human genome, 158 346 (see also "Lung cancer") as "transforming" gene, 158 Solt-Farber carcinogenesis protocol, RB gene as prototype tumor sup361 pressor gene, 4, 138, 283-284 Southern blotting, 279, 281,307, RBI gene, 5 (see also "Genetics...") 308, 314, 410 rbl gene, 58-135 (see also "p53...") src cells, 335 RBTN 1 and 2, 218 Stomach cancer, 367 Recombinase enzymes, 210 (see also amplification, 367 "Chromosomal clinical associations, 367 translocations...") epidermal growth factor, 367 Rectal cancer, 367-375 (see also p53 alterations, serological analy"Colorectal cancers...") sis of, 111 rel oncogene and relations, 248-249 regulation, 367 Refractory anemia (RA), 192 SV40 virus, 58-61 (see also "p53...")

444

t(l;19) fusion, 215 T-cell acute lymphoblastic leukemia, 186 T-cell leukemia, common characteristics of, 216-220 (see also "Chromosomal translocations...") non-Hodgkin's lymphoma, 189192 (see also "NonHodgkin's lymphoma") tal-1 gene, 244 TAL 1/ SCL/TCL5, 217 GATA-1,217 TAN 1 translocation, 219 Tamoxifen, 35 in breast cancer studies, 350 TCL1 locus, 220 tcl3 gene, 219 TCR gene, 3 Teratoma, 410 Testicular cancer, 356 bioassay, 356 clinical association, 356 regulation, 356 transgenic mouse experiment, 356 TGF-a growth factor, 363 -/3, 374 Thrombospondin (TSP), 23-24 (see also "Oncogenes...") Topoisomerase, 211 Transcription and cancer, 227-272 conclusion, 253-255 T-cell leukemias, 254 eukaryotic transcriptional regulation, 228-239 basic-helix-loop-helix (bHLH), 231 control, 231-239 deregulation and neoplasia, 237-239 differentiation and development, 236-237 factors, basic, 229-231

INDEX

factors, oncogenic, and human tumors, 239 factors, oncogenic, in transforming retroviruses, 237238 factors, structural features of, 231-234 homeobox, 232-233, 236 homeotic selector genes, 236237 inducible gene expression, 234236 leucine zipper (LZ), 231-232 ligand-mediated activation, 235 machinery, general, 228-231 myc proteins, 232 phosphorylation, 235 Pit- 1,234 POU, 233, 237 RNA polymerase II, 228-229 in segmentation, 236 TBP-associated factors, 230 tissue-specific gene expression, 234 zinc finger (ZF) motif, 232, 245247 introduction, 227-228 definition, 228 oncogenes, factors as, 237-253 basic helix-loop-helix, 243-245 bcl-3, 249 bmi-1, 247 bZip-family oncoproteins, 240242 evi-1, 246 fli-1, 251 glil, 246 HLF, 242 homeobox genes, 247-248 (see also "Homeobox...") Lyl-1 protein, 244 lyt-lO, 248 Max protein and Mad, 243-245

Index

mel-18, 247 p53 gene, 253 pml, 247 pRb product, 252 rb, 252 rel and relations, 248-249 retinoblastoma gene product, 252-253 retinoic acid receptor c~-gene (RARA), 247 rhom-1 and-2, 247 ski, 251 spi-l, 251 steroid receptor family, 246-247 Tal-1,244 tumor suppressor genes, 252-253 v-ErbA, 246-247 v-ets, 250-251 v-fos, 241-242 v-jun, 240 v-maf, 242 v-myb, 249-250 v-myc, 243 Wilms' tumor gene (WT), 245246 zinc-binding factors as cancer genes, 245-247 trk protooncogene, 20 Ttg-1,218 Tumor cell heterogeneity, 374 Tumor development, ras gene mutations in, 160-163 (see also "ras gene family") Tumor metastasis, 24-29 (see also "Oncogenes...") Tumor necrosis factor (TNF), 352 Tumor suppressor genes, 10-13, 137156, 275-277 genetic aspects of, 137-156 abstract, 138 amplification of gene, 147 cell fusion techniques, 138-151 cell hybrids, 141-142

445

cellular immortality, 144-147 cellular senescence, 144-147 complementation analyses, 141142 conclusions, 150-151 gene amplification, 147 history, 138-139 hybrid cells, control of tumorigenicity in, 139-141 imprinting, genomic, 146 introduction, 138-139 lymphoid cells, tumorigenicity in, 140-141 mechanisms, 150 metastatic progression, 147-148 methylation, 146 microcell hybridization procedure, 142 monochromosome transfer, 142-143, 148 p53, 138, 149 phenotypes, transformed, other, 144-148 rb, 138 retinoblastoma gene, 148 and somatic cell genetics, correlation of, 138-139, 148-149 terminal differentiation, induction of, 151 tumorigenicity, definition, 139 viraUy transformed cells, tumorigenicity in, 141 Tumors, solid human, chromosome pattern in, 193-197 retinoblastoma, 193 Tyrosine kinases, p21 ~ and, 166-168, 171 (see also "ras gene family") UEGCCS, 417 Uterine cancer, 350-352 amplification, 351 clinical associations, 351 regulation, 351-352

INDEX

446

v-myc gene, 333-335 (see also "myc gene'3 Vanadate, 352 Variable number of tandem repeat polymorphisms (VNTRP), 3

WAGR syndrome, 245 Werner's syndrome, 197, 198

Wilms' tumor (WT), 7, 142, 193, 245-246, 277 Xeroderma pigmentosum, 197, 198, 275 Zinc finger (ZF) motif, 232

Advances in Developmental Biology Edited by Paul Wassarman, Department of Ceil and Developmental Biology, Roche Institute of Molecular Biology Volume 1, 1992, 192 pp. ISBN 1-55938-348-8

$97.50

CONTENTS: Introduction. Y Chromosome Function in Mammalian Development, Paul S. Burgoyne. A Super Family of Putative Developmental Signalling Molecules Related to the Proto-Oncogene Wnt- 1/int-1, Andrew P. McMahon and Kenneth R. Howard. Pattern Formation in Caenorhabditis Elegans, Min Han and Paul W. Sternberg. Gap Junctional Communication During Mouse Development, Norton B. Gilula, Miyuki Nishi, and Nalin Kumar. Lens Differentiation and Oncogenesis in Transgenic Mice, Heiner Westpha/. Subject Index.

Volume 2, 1993, 195 pp. ISBN 1-55938-582-0

$97.50

CONTENTS: Preface, Paul M. Wassarman. The Sry Gene and Sex Determination in Mammals, Blanche Cape/and Robin Lovel/-Badge. Molecular and Genetic Studies of Human X Chromosome Inactivation, Carolyn J. Brown and Huntington F. Wi//ard. Genomic Imprinting and Regulation of Mammalian Development, Colin L. Stewart. Cell Interactions in Neural Crest Cell Migration, Marianne Bronner-Fraser. Enzymes and Morphogenesis: Alkaline Phosphatase and Control of Cell Migration, Saul L. Zackson. Subject Index.

Volume 3, 1994, 194 pp. ISBN 1-55938-865-X

$97.50

CONTENTS: Preface, Paul M. Wassarman. Mechanisms of Neurogenesis in Drosophila Melanogaster, Jose A. CamposOrtega. The Role of Growth Factors in Mammalian Pregastrulation Development, Daniel A. Rappolee and Zena Werb. Retinoid Signaling in Mouse Embryos, Elwood Linney and Anthony-Samuel LaMantia. RNA Localization During Oogenesis in Drosophila, Elizabeth Ft. Gavis and Ruth Lehmann. Actin as a Tissue-Specific Marker in Studies of Ascidian Development and Evolution, William R. Jeffery. Index.

.1 A [ P R E S S

J A l P R E S S

Advances in

Developmental Biochemistry

Edited by Paul Wassarman, Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Volume 1, 1992, 256 pp. ISBN 1-55938-347-X

$97.50

CONTENTS: Introduction. Organelle Assembly and Function

in the Amphibian Germinal Vesicle, Joseph G. Gall. DNA Replication and the Role of Transcriptional Elements During Animal Development, Melvin L. DePamphilis. Transcriptional Regulation During Early Drosophila Development, K. Prakash, Joanne Topoi. C.R. Dearolf, and Carl S. Parker. Translational Regulation of Maternal Messenger RNA, L. Dennis Smith. Gut Esterase Expression in the Nematode Caenorhabditis Elegans, James D. McGhee. Transcriptional Regulation of Crystallin Genes: Cis Elements, Trans-factors and Signal Transduction Systems in the Lens, Joram Piatigorksy and Peggy S. Zelenka. Subject Index. Volume 2, 1993, 237 pp. ISBN 1-55938-609-6

$97.50

CONTENTS: Preface, Paul M. Wassarman. Drosophila Ho-

meobox Genes, William McGinnis. Structural and Functional Aspects of Mammalian HOX Genes, Denis Duboule. Developmental Control Genes in Myogenesis of Vertebrates, Hans Henning-Arnold. Mammalian Fertilization: Sperm Receptor Genes and Glycoproteins, Paul M. Wassarman. The Fertilization Calcium Signal and How It Is Triggered, Michael Whitaker. Subject Index. Volume 3, 1994, 188 pp. ISBN 1-55938-853-6

$97.50

CONTENTS: Preface, Paul M. Wassarman. Expression and

Function of Protein Kinases During Mammalian Gametogenesis, Deborah L. Chapman and Debra J. Wolgemuth. Regulation of the Dopa Decarboxylase Gene During Drosophila Development, Martha J. Lundell and Jay Hirsh. Transcription Factors in Mammalian Development: Murine Homeobox Genes, S. Steven Potter. Expression and Function of C-Mos in Mammalian Germ Cells, Geoffrey M. Cooper. Regulation of Pigmentation During Mammalian Development, Friedrich Beermann, Ruth Ganss, and Gunther Schutz. Index.